Forensic Science International: Genetics Supplement Series

Editorial Board

2009-12-01

Supplement Progress in Forensic Genetics 13

2009-12-01

Proceedings of the 23rd International ISFG Congress

Niels Morling — 2009-12-01

The 23rd International Congress of the International Society for Forensic Genetics was held from 15 to 18 September 2009 in Buenos Aires, Argentina. The president of the congress was Professor Eduardo Raimondi, Favaloro Foundation Immunogenetics (PRICAI), Buenos Aires, Argentina. Before the congress, educational workshops were held 14–15 August 2009.

PowerPlex® ESX and ESI Systems: A suite of new STR systems designed to meet the changing needs of the DNA-typing community

2009-10-16

Abstract: With over 6 million profiles currently stored in European databases and the number expected to increase with cross-border data sharing, the likelihood of random matches will undoubtedly increase as well. To improve the overall power of discrimination as well as provide standardization across Europe, the ENFSI and EDNAP committees have made a recommendation to extend the current European Standard Set (ESS) for STR systems. The five-color PowerPlex® ESX and ESI Systems allow co-amplification and detection of the current commonly tested loci, plus the five new recommended loci. These kits will be offered in multiple formats, including one to detect SE33, to accommodate various requirements or preferences. Additionally, the kits have increased tolerance to common inhibitors and increased sensitivity to obtain full profiles from low-level DNA, and are robust enough to genotype degraded DNA samples through the use of mini STR loci. In this manuscript we present an overview of these systems and data on performance, including sensitivity and resistance to inhibitors. The PowerPlex® ESX and ESI Systems are useful tools in database sharing and standardization throughout Europe.

A review of low template STR analysis in casework using the DNA SenCE post-PCR purification technique

T. Gross, J. Thomson, S. Kutranov — 2009-11-02

Abstract: LGC has developed a method for analysing low-level DNA samples called DNA SenCE (Sensitive Capillary Electrophoresis) based on post-PCR treatment of standard 28-cycle SGMplus PCR product and demonstrated to be equally effective at enhancing profiles as 34-cycle PCR. The method has been validated and accredited and used in casework since July 2007. Inherent in the method is the initial generation of a standard 28-cycle SGMplus profile so a direct comparison of standard and DNA SenCE results for all casework is possible. Here we review DNA SenCE casework, reporting the magnitude of peak enhancement and stochastic effects seen in the DNA SenCE profiles.

Evaluation of reliability of STR typing in human colon carcinomas tissues used for identification purpose

Chengtao Li, Shumin Zhao, Jianxin Fang, Yan Liu, Li Li — 2009-09-23

Abstract: The short tandem repeats (STRs) have become an important and widely used tool in forensic casework. Clinical tissue samples are not usually employed in forensic casework, but sometimes, malignant tissue samples may be the only source of biological material for forensic investigations. However, in use of such samples, uncertainties due to microsatellite instability (MSI) and loss of heterozygosity (LOH) may be encountered. In our study of 77 human colon carcinomas tissue with the AmpFlSTR Identifiler Kit comprising 15 STR loci and the amelogenin gene, we detected four kinds of changes between normal tissue and tumor tissue including pLOH, LOH, occurrence of an additional allele (Add) and occurrence of a new allele (New) instead of that found in normal tissue. The overall variation detectable rate was 11.28%, of which pLOH was 79.1%, LOH was 7.9%, Add was 7.9% and New was 5%. Of the above four changes, the incidence rate of pLOH, LOH, Add and New was respectively 8.93%, 0.89%, 0.89% and 0.57%. The STRs mostly affected were D18S51, D5S818, FGA and D19S433. Only pLOH was found at five loci including vWA, TPOX, TH01, D13S317 and amelogenin gene. Our results demonstrate that great care should be taken in the evaluation of typing results obtained from clinical tissue specimens, in particular when no reference samples are available, because genetic instability is a very common event observed in different tumors and the STRs used for individual identification could sometimes be affected.

Effect of low-dose radiation on mutation rates of STR loci commonly used in forensic casework

V.A. Orekhov, G.O. Shaikhaev, A.V. Aghajanyan, M.V. Zakharenko, G.P. Snigiryova — 2009-11-16

Abstract: The possible effect of low-dose radiation on STR markers in people exposed to radiation during their professional activities was estimated in this study. We evaluated mutation rates in 17 forensic STR loci typed by the COrDIS-18 kit (CSF1PO, D10S1248, D12S391, D13S317, D16S539, D18S51, D21S11, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX, and vWA) in 78 families (father–mother–child) with one parent exposed to low-dose radiation before fertilization. Five mutations were observed. In two cases, the new alleles were delivered from the non-exposed parent. The calculated mutation rate for the 17 studied STR loci in families appeared to be in good concordance with data published for normal populations. No evidence for an elevated mutation rate in STR markers after low-dose radiation was found.

Allelic alterations of STRs in archival paraffin embedded tissue as DNA source for paternity testing

Yan Liu, Li Li, Chengtao Li, Zhenmin Zhao — 2009-09-23

Abstract: Owing to a wrong name registered on ID card, the identity of a businessman who had been dead and cremated was suspected, which led his son failed to get legacy. In order to prove the parenthood, the son submitted the gastric cancer tissues surgically removed and embedded in a paraffin block as DNA source for paternity test. After extracting DNA with QIAamp DNA Blood Mini Kit, the 16 STR loci was amplified by two commercial kits of Sinofiler® (ABI)and Powerplex 16 (Promega), respectively. Both of the STR profiles were similarly showing allelic imbalance pattern at some loci and an additional allele at locus D18S51. The cancerous tissues and adjacent normal tissues were then partitioned off from each other by microscopic analysis of H.E. stained sections and followed by DNA extracting and STR typing, respectively. The allelic alteration could not be found in normal tissues whereas it did in cancerous tissues whose STR profile showed complete loss of one allele (LOH) at loci D13S317 (allele 11 was lost), partial loss of one allele (pLOH) at loci D21S11, D7S820, D19S433, vWA, D12S391 and Amelogein and occurrence of an additional allele (allele 20 was added) at locus D18S51. The results demonstrated that the Paraffin Embedded cancer Tissue used as DNA source for forensic identification is possibly questionable because of their microsatellite instability (MSI) or loss of heterozygosity. It was suggested to partition the normal tissues from the cancer tissues by microscopic evaluation first and then analyzing DNA separately. Comparing the STRs profile of normal tissue with the son's blood sample, the final conclusion was acquired that the donor of the paraffin embedded tissues is the biological father of the son.

Forensic STR analysis reveals DNA contamination previously undetected during clinical analysis of chronically inflamed tissues

G.L. Axler-DiPerte, E. Wurmbach, Z.M. Budimlija, B. Jian, F. Fogt, M. Prinz — 2009-12-01

Abstract: While investigating the potential for genetic instability in chronic inflammatory disease, using ulcerative colitis (UC) as a model, we analyzed microsatellite DNA of both pre- and post-surgical affected and histologically normal tissues. These samples were also characterized using the forensic Identifiler® Multiplex System from ABI. Apparent instability was found in the majority of patients using the clinical panel. This panel assumed all samples were single source, whereas the forensic panel revealed that 57% of samples tested with Identifiler® were mixtures of more than one contributor. It is likely that DNA contamination occurred during routine histological processing. This contamination could lead to erroneous assessments of instability. Microsatellite analysis is used in tumor characterization and therapeutic determinations. Incorrect determinations could affect patient care. Given the sensitivity and widespread use of molecular tests on biopsies and preserved post-surgical tissues, we recommend that an STR multiplex used for forensic individualization be used prior to diagnostic tests to ensure the sample is from a single source.

Validation of the MiniFiler™ Kit in archaeological samples

2009-09-10

Abstract: We evaluate the usefulness of MiniFiler™ Kit in the field of ancient DNA. A set of samples belonging to different locations from Iberian Peninsula, with ages ranging from Neolithic to XVII century, was tested. Results could be replicated in only one burial site, probably due to the taphonomic conditions. Other cases could only produce partial or none genetic profiles.

Analysis of AmpFlSTR® MiniFiler™ loci and its forensic application

2009-10-15

Abstract: The allele frequencies of eight MiniFiler™ loci have been analyzed in 101 Japanese individuals living in Kanagawa with informed consent by means of ABI 310 Genetic Analyzer. A total of 7 alleles for D13S317, 8 alleles for D7S820, 11 alleles for D2S1338, 11 alleles for D21S11, 5 alleles for D16S539, 14 alleles for D18S51, 8 alleles for CSF1PO, and 13 alleles for FGA were observed. The polymorphic profiles of these MiniFiler™ loci in the present study were essentially the same as those obtained by using the AmpFlSTR® Identifiler® PCR Amplification kit. The combined matching probability of eight MiniFiler™ loci and cumulative probability of paternity exclusion were estimated as 1.97×10−10 and 0.9996, respectively. The MiniFiler™ kit was useful for individual identification in forensic analysis.

Development of two new Mini-STR multiplex assay for typing archival Bouin's fluid-fixed paraffin-embedded tissues

Stefania Turrina, Giulia Filippini, Luciana Caenazzo, Domenico De Leo — 2009-10-05

Abstract: Short amplicon autosomal short tandem repeat (Mini-STR) assay has proved to be a highly useful tool in forensic applications, especially for highly degraded DNA samples that typically result in partial profiles and total loss of information from regular STR amplicons.In this study two new quadruplex systems were designed to get nuclear DNA profile from degraded forensic casework samples. In order to obtain PCR products less than 120bp in size, primer pairs of eight STR markers, included in available commercially multiplex PCR kits, were redesigned and assembled in two PCR-multiplexes: D8S1179, D3S1358, TPOX, D16S539 and CSF1P0, TH01, D13S317, D5S818.After validation, these two Mini-STR quadruplex were employed in paternity testing case that involved DNA extraction from archival postmortem Bouin's fluid-fixed paraffin-embedded tissue where commercial kit yielded low success. The results obtained with the present Mini-STR PCR-multiplexes proved clearly demonstrating their usefulness in analyzing degraded DNA samples.

The single most polymorphic STR Locus: SE33 performance in U.S. populations

2009-10-15

Abstract: The STR locus SE33 (ACTBP2) located on chromosome 6 (6q14) is arguably the most polymorphic marker examined thus far by the forensic community with a heterozygosity of >0.95 in some populations. Three different primer sets were utilized in this study in order to assess the possibilities of primer binding site mutations. Population variation was measured in 460 U.S. Caucasian, 445 African American, 336 Hispanic, and 202 Asian samples along with mutation rates from almost 400 father–son pairs. In addition, the 10 genomic DNA components in NIST Standard Reference Material SRM 2391b were sequenced and found to exhibit a variety of additional base changes, insertions, and deletions outside of the SE33 repeat region.

Development and validation of a next generation STR ESS-pentaplex

2009-10-12

Abstract: We constructed a simple STR pentaplex of new loci recommended as next generation markers for the European Standard Set (ESS) comprising normal-amplicon STRs: D12S391 and D1S1656, plus mini-amplicon STRs: D2S441, D10S1248 and D22S1045. Validation of the pentaplex included evaluation of its ability to amplify DNA from a variety of degraded forensic casework samples. Although the ESS-pentaplex was designed in the first instance to generate allele frequency data to supplement existing databases of established STRs, the multiplex proved to be a valuable tool for the analysis of challenging DNA when certain markers of Identifiler or MiniFiler occasionally failed.

Validation of the AmpFℓSTR® SEfiler Plus™ PCR Amplification kit for forensic STR analysis

Stine Frisk Fredslund, Helle Smidt Mogensen, Niels Morling — 2009-10-12

Abstract: Validation of the AmpFℓSTR® SEfiler Plus™ PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpFℓSTR® SGM Plus™ kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but showed more stutters, drop-ins, drop-outs and allelic imbalances.

Uses of the NIST 26plex STR assay for human identity testing

2009-10-15

Abstract: Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.

Construction and application of four fluorescence labeled multiplex typing system for 3 miniSTR loci

Shujin Li, Ning Liu, Xue Bai, Jianli Gu, Zhiping Hou, Bin Cong — 2009-10-19

Abstract: We constructed a multiplex PCR system for 3 miniSTR loci D20S482, D3S3053, D6S474. This typing system showed high stability and sensitivity (0.05ng). Population data investigated in 120 healthy unrelated Chinese Han individuals showed higher genetic polymorphism, with the combined power of discrimination and power of exclusion being 0.998 and 0.84. The amplification product length ranged from 88bp to 127bp for all three loci. The successful rate of typing highly degraded samples using this miniSTR multiplex PCR system was significantly higher than using identifiler kit, indicating the multiplex set represents a useful tool in Chinese forensic practice, especially for the highly degraded DNA sample.

PowerPlex® 16 HS: Internal validation of a new tool for genetic analysis of forensic and parentage testing

Mary Acosta Loyo, Gerson Caraballo, Karen Sánchez, Howard Takiff — 2009-11-16

Abstract: Forensic human identification requires powerful and efficient tools to obtain useful results in a minimum timeframe. In this study several forensic and parentage samples that could not be analyzed with others kits were studied using the recently available PowerPlex® 16 HS kit (Promega). DNA was extracted using four different methods, depending upon the particular sample, and the PCR products were run on an ABI 3130XL Sequencer. The resultant DNA profiles were analyzed using Gene Mapper ID v 3.2 Analysis Software (ABI). Of 30 samples processed with the PowerPlex® 16 HS system, genetic analysis was successful in 18 (60%). The results obtained show that the PowerPlex® 16 HS is a valuable tool for forensic identification and parentage testing that is particularly useful for difficult samples that have not yielded adequate results with other methods.

Integration of the AmpFlSTR Identifiler PCR Amplification Kit with SRY-specific primers for gender identification

S. Inturri, C. Robino, S. Gino, S. Caratti, C. Torre — 2009-10-23

Abstract: Dropout of the amelogenin Y-specific allele due to an interstitial deletion of the Yp involving the amelogenin Y locus (AMELY) can cause misidentification of sex genotype with potentially serious consequences in personal identification processes and criminal investigations. Inclusion of additional sex-defining markers in forensic DNA typing kits is therefore advisable. In this study, the co-amplification of the sex-determining region Y (SRY) gene and 16 STR loci included in the AmpFlSTR Identifiler PCR Amplification Kit was evaluated. Combination of SRY and Identifiler primers did not compromise the amplification outcome and generated a 90bp male-specific SRY fragment, showing a reproducible peak height ratio in comparison with the AMELY peak. The SRY peak was detectable in presence of amounts of template DNA as low as 125pg, and in mixed samples with a male/female DNA ratio of 1:100.

Observation of tri-allelic patterns in autosomal STRs during routine casework

2009-09-01

Abstract: We report three cases of tri-allelic patterns observed during routine forensic casework on 5964 Belgian residents. These individuals had been typed for the following 15 autosomal STRs: CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, vWA, FGA, TH01, TPOX, D2S1338 and D19S433.The first example of a tri-allelic pattern had the genotype 13;15;16 for the D8S1179 locus. In the second observation there was 16;21;22 pattern for the D18S51 locus. The third case had the alleles 10;11;13 also for D18S51.All cases belonged to the Type I tri-allelic pattern, with three uneven peaks, the sum of the heights of both smaller peaks equalling the height of the tallest peak.Three cases in 5964 typed individuals is a frequency for tri-allelic patterns in autosomal STRs of 0.05%.

Further allelic variation at the STR-loci ACTBP2 (SE33), D3S1358, D8S1132, D18S51 and D21S11

E.M. Dauber, E.M. Schwartz-Jungl, S. Wenda, G. Dorner, B. Glock, W.R. Mayr — 2009-10-05

Abstract: ACTBP2 (SE33), D3S1358, D8S1132, D18S51 and D21S11 are frequently used STR-loci in the forensic field. This study reports sequence data of further new or rare alleles at these loci, varying in length or in sequence, which were detected in course of investigations for various purposes.

Characterisation of 12 new alleles in the STR system D18S51

A. Morales-Valverde, S. Silva-De La Fuente, G. Nuñez-Rivas, M. Espinoza-Esquivel — 2009-10-12

Abstract: D18S51 alleles 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 and 40 were detected and sequenced; these new alleles are mistyped with commercial STR kits, causing presumptive null alleles or false exclusions when paternity testing.

A X-chromosome STR hexaplex as a powerful tool in deficiency paternity cases

Juliana Aquino, Carla Peixe, Dayse Silva, Celso Tavares, Elizeu F. de Carvalho — 2009-10-12

Abstract: The X-chromosome short tandem repeat (STR) markers have been described as very adequate tools for solving deficiency paternity cases and kinship tests when women are involved. In the absence of the alleged father, presumed paternal relationship can be more efficiently investigated by using a set of six to ten X-STR markers compared to fifteen autosomal STR. For this study, we compared the usefulness of a X-STR hexaplex developed in our laboratory (DXS7133, DXS7424, DXS8378, DXS6807, DXS7423 and DXS8377) and the commercial kit Identifiler in solving deficiency paternities. We have worked on distinct groups of caseworks involving daughters, their mothers and presumed paternal grandmothers or putative half sisters and their respective mothers. The PCR products were separated by capillary electrophoresis and detected in an ABI Prism 3100. In the majority of the caseworks (>90%), the likelihood ratio (LR) obtained by using the X-STR hexaplex was higher than the LR value observed when the Identifiler kit was used for genotyping. The combination of the two STR typing systems was able to solve all the cases.

Optimization and validation studies of the Mentype® Argus X-8 kit for paternity cases

2009-10-12

Abstract: The use of ChrX-STRs is enormous in forensic case as these have proven to be powerful tools, mainly in deficiency paternity cases when the disputed child is female, and also some special cases involving blood relatives, incest cases, fetal typing in abortion material. The Mentype® Argus X-8 kit is a commercial multiplex system which contains Amelogenin for gender determination as well as gonosomal STR markers (DXS8378, HPRTB, DXS7423, DXS7132, DXS10134, DXS10074, DXS10101 and DXS10135). Validation studies were being performed on blood obtained from the volunteers in Turkish population. In this study, some parameters were taken under consideration for validation like DNA extraction using different protocols, quantitated by using commercially available Invitrogen Qubit Fluorometer, reaction volume validation of Master Mix and the analysis of female/male, female/female and male/male mixtures were performed. The conditions were optimized and validated using GenAmp 9700 and reducing reaction volume from 25μl to 12.5μl and 6.5μl. After reducing the total volume of the reaction, the results were same and there was no effect on peak height and quality when analyzed on ABI 310 genetic analyzer. 2 paternity cases were also performed which gave the same power of discrimination as has been mentioned in Mentype® Argus X-8 kit.

Sequence polymorphisms at the DXS6789, DXS8377 and DXS101 loci in three Asian populations

A. Nagai, M. Hara, A. Kido, A. Takada, K. Saito, Y. Bunai — 2009-10-12

Abstract: Sequence analyses of X-chromosomal short tandem repeats, DXS6789, DXS8377 and DXS101 were performed for representatives of 3 Asian populations: 130 Japanese, 61 Bangladeshi and 89 Indonesian males. At DXS6789, the sequence polymorphism was found in 7 alleles in the Japanese, 3 in the Bangladeshis and 3 in the Indonesians. At DXS8377, the sequence polymorphism was found in 13 alleles in the Japanese, 9 in the Bangladeshis and in all alleles identified in the Indonesians. At DXS101, the sequence polymorphism was found in 7 alleles in the Japanese, 9 in the Bangladeshis and 8 in the Indonesians. Because sequence polymorphisms were found in most of the alleles at the DXS6789, DXS8377 and DXS101 loci, it was concluded that sequencing was essential for identifying the alleles at these loci in all 3 Asian populations.

Updated allelic structures of the DXS10135 and DXS10078 STR loci

D.R. Sumita, M.R. Whittle — 2009-09-23

Abstract: DXS10135 and DXS10078 are two highly polymorphic STR loci situated in two different linkage groups on the short arm of the human X chromosome. Both loci comprise complex tetrameric repeat units which may partially explain their high degree of polymorphism. DXS10135 is relatively well characterized and is included in a commercially available kit, while DXS10078 has not been well described. We sequenced a large number of alleles of both loci to try and understand the allelic variation and as a prelude to construct allelic ladders from cloned alleles. Our data show interesting features and should encourage other workers to use these loci in forensic genetic investigations.

STR analysis of degraded DNA using a miniplex

M. Nastainczyk, S. Schulz, M. Kleiber, U.D. Immel — 2009-10-05

Abstract: Analysis of short tandem repeat (STR) markers currently represents the most useful instrument in the field of forensic genetics. The problem with forensic material is the degradation of the sample material. In recent years, several papers have demonstrated that short amplicon STR (miniSTR) represents one of the most useful tools for analyzing degraded DNA samples.In the present study, we attempted to develop a short amplicon STR multiplex system (autosomal and y-chromosomal) for analyzing degraded DNA using some newly designed primer sets for a multiplex polymerase chain reaction (PCR) systems for typing.An assay of degraded DNA samples using the designed multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. Comparing the multiplex with commercial kits, first results show a well success rate.

Unexpected patterns in Y-STR analyses and implications for profile identification

Ilaria Carboni, Ugo Ricci — 2009-09-28

Abstract: Y-STR analysis is widely used in many fields, such as paternity testing, genealogy studies and in male/female mixtures. In many rape cases, Y-STRs are also useful for the determination of contributors’ number. Here we described a father/son pair with double peaks at DYS439 and DYS635 loci. This case should focus the attention on forensic interpretation of Y-haplotype profiles, because multiple alleles at various loci do not forcibly indicate that the sample originates from a mixture.We also report a case of two half-brothers with null allele at DYS448.Since DYS439 and DYS635 loci are located in the AZFa region and DYS448 locus in the AZFc region, we performed a molecular genetics study of these regions to evaluate a possible correlation between Y-STR profiles and Y chromosome deletions involved in infertility.

Development and evaluation of multiplex Y-STR assays for application in molecular genealogy

2009-10-05

Abstract: Three multiplex PCRs were developed for the analysis of 14 single-copy and 4 multi-copy Y chromosome Short Tandem Repeat (STR) loci routinely used by several public genealogical databases. These assays were used in addition to PowerPlex® Y for the analysis of 245 DNA samples from a genealogical project. In total 244 different haplotypes composed of 37–40 alleles were identified with one haplotype identical between two males with the same surname. The multi-copy loci DYS464 and DYS724 were the most polymorphic with a gene diversity of at least 0.964. The use of DYS454 and DYS455 can be questioned as these loci had the lowest gene diversity (0.039 and 0.269, respectively).

Y-STR mutational rates determination in South Portugal Caucasian population

C. Vieira-Silva, P. Dario, T. Ribeiro, I. Lucas, H. Geada, R. Espinheira — 2009-10-21

Abstract: Y-STR mutational rate estimation is very important for the correct evaluation of typing results in forensic casework and specially kinship genetic studies. In this work we studied 95 Southern Portuguese Caucasian father/son pairs in order to estimate mutational rates for the 17 Y-STRs multiplex used in routine casework. In a total of 1615 allele transfers three single step mutations were detected in DYS385a, DYS439, and DYS448, with an estimated mutation rate of 10,526×10−3 (95%CI 0.265×10−3 to 20.788×10−3). The estimated average mutation rate is 1.858×10−3 (95%CI 8.08×10−4 to 2.908×10−3). It would be important to characterize more father/son pairs in order to estimate more reliable allele specific mutation rates for the most widely used Y-STRs markers in forensic genetics.

Adaptation and evaluation of the PrepFiler™ DNA extraction technology in an automated forensic DNA analysis process with emphasis on DNA yield, inhibitor removal and contamination security

2009-10-12

Abstract: Within the initial step of the forensic DNA analysis process, the DNA extraction efficiency and especially the removal of potential PCR inhibitors is crucial for subsequent steps, e.g. quantification by real-time PCR and amplification of short tandem repeats (STRs). The protocol of the PrepFiler™ Forensic DNA Extraction Kit was optimized for the application on a Tecan liquid handling workstation Freedom EVO® 150. This modified application of the PrepFiler™ technology was compared with respect to DNA yield, sensitivity and the ability to remove potential PCR inhibitors to an established routine method working on the same liquid handling workstation based on ChargeSwitch® Technology (CST) from Invitrogen.

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

2009-12-01

Abstract: Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR® Idenitfiler® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.

Automated Quantifiler® quantitative PCR setup, template normalization and PCR setup using HID EVOlution™ qPCR/STR setup on trace evidence samples

2009-09-23

Abstract: We have implemented and validated customized protocols for automated Quantifiler® setup, template normalization and PCR setup using the Tecan HID EVOlution™ qPCR/STR setup. The protocols were validated for the Quantifiler® human DNA quantification, AmpFℓSTR® SGM Plus® and SEfiler Plus™ PCR Amplification Kits (Applied Biosystems) according to EN/ISO 17025.

Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

Michael Stangegaard, Mads Jørgensen, Anders J. Hansen, Niels Morling — 2009-09-01

Abstract: We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs, and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpFℓSTR® Identifiler® and AmpFℓSTR® Yfiler® (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025.

Automated washing of FTA Card punches and PCR setup for reference samples using a LIMS-controlled Sias Xantus automated liquid handler

2009-09-01

Abstract: We have implemented and validated automated methods for washing FTA Card punches containing buccal samples and subsequent PCR setup using a Sias Xantus automated liquid handler. The automated methods were controlled by worklists generated by our LabWare Laboratory Information Management System (LIMS). The protocols were validated according to EN/ISO 17025 for use with STR amplifications kits AmpFℓSTR® Identifiler® and Y-filer® (Applied Biosystems).

Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

2009-09-01

Abstract: We have implemented and validated automated methods for DNA extraction and PCR setup developed for a Tecan Freedom EVO® liquid handler mounted with a Te-MagS™ magnetic separation device. The DNA was extracted using the Qiagen MagAttract® DNA Mini M48 kit. The DNA was amplified using AmpFℓSTR® Identifiler®, Y-filer® (Applied Biosystems), GenePrint® FFFL and PowerPlex® Y (Promega). The methods were validated for fresh whole blood and blood from deceased according to EN/ISO 17025.

Customizing a commercial laboratory information management system for a forensic genetic laboratory

2009-09-23

Abstract: The need for high-throughput laboratories to comply with regulatory requirements makes data management an important aspect of forensic genetics. A Laboratory Information Management System (LIMS) enables efficient workflows and ensures traceability if designed and implemented properly. We customized a commercial LIMS to support STR typing of reference samples according to in-house defined requirements. The customization focused on data validity, traceability and automated solutions.

A production system to generate reference genetic profiles from buccal swab cells on FTA® cards

N. Scaramozzino, P. Terrenoire, L. Baron — 2009-12-01

Abstract: To generate the large quantity of genetic profiles that continuously feeds the French Reference Sample Database (F.N.A.E.G.), a secure and robust process is required. A complete automated production chain has been developed by Hamilton Robotics and the French Police Scientifique in Lyon to produce genetic profiles from buccal swab cells on FTA® cards. The activities have been divided between Pre- and Post-PCR. The samples on FTA® cards are punched directly into PCR plates. The plates are then transferred onto the Hamilton Microlab® STAR liquid handling instruments. Air displacement pipetting coupled with Hamilton's CO-RE Technology allows a highly secure and contamination free pipetting for FTA® wash and STR® Identifiler® PCR reaction set-up. Plates are sealed and transferred to the Post-PCR system for pooling into 384 format and denaturation. Throughout the process steps a complete sample tracking is performed and sample information is transferred to the LIMS system. This poster describes in detail this production system with an actual throughput of 40,000samples/month, which is in production since 2006.

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot® EZ1 Workstation

2009-10-05

Abstract: FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpFℓSTR® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.

Automated DNA extraction using the QIAsymphony platform: Estimation of DNA recovery from simulated forensic stains

C. Gehrig, D. Kummer, V. Castella — 2009-09-22

Abstract: The aim of this study was to evaluate the forensic protocol recently developed by Qiagen for the QIAsymphony automated DNA extraction platform. Samples containing low amounts of DNA were specifically considered, since they represent the majority of samples processed in our laboratory. The analysis of simulated blood and saliva traces showed that the highest DNA yields were obtained with the maximal elution volume available for the forensic protocol, that is 200μl. Resulting DNA extracts were too diluted for successful DNA profiling and required a concentration. This additional step is time consuming and potentially increases inversion and contamination risks. The 200μl DNA extracts were concentrated to 25μl, and the DNA recovery estimated with real-time PCR as well as with the percentage of SGM Plus alleles detected. Results using our manual protocol, based on the QIAamp DNA mini kit, and the automated protocol were comparable. Further tests will be conducted to determine more precisely DNA recovery, contamination risk and PCR inhibitors removal, once a definitive procedure, allowing the concentration of DNA extracts from low yield samples, will be available for the QIAsymphony.

Efficiency of DNA IQ System® in recovering semen DNA from cotton swabs

A. Colussi, M. Viegas, J. Beltramo, M. Lojo — 2009-11-02

Abstract: With the aim to asses the efficiency of the DNA IQ System in the recovery of DNA from semen samples, cotton swabs were prepared from 1/5 serial dilutions of semen. Each swab was fractionated in four equivalent quarters and the DNA was further extracted following the differential lysis protocol. The recovered DNA was quantified by means of real time PCR and the average DNA yield was used to compare results. Direct extractions of equivalent aliquots of each semen dilution were used as reference samples. Even though a high percentage of the starting material was lost during the process of transfer to/recover from the solid support, our experimental results demonstrated that the DNA IQ system was able to detect around 103 sperm cells in the starting material, enabling to obtain a complete DNA profile with AmpFl STR IdentiFiler PCR Amplification Kit (Applied Biosystems).

A novel platform for the modular integration of forensic assay setup and medium- to high-throughput purification of nucleic acids

2009-10-12

Abstract: With its extraction and assay setup modules, the QIAsymphony® provides a highly flexible solution for processing forensic samples in a medium- to high-throughput scale. We tested the sensitivity of extraction, precision of sample processing, and accuracy of automated assay setup of this integrated system. Results attest to QIAsymphony's ability to isolate DNA from a spectrum of common forensic samples and process these samples without cross-contamination. Furthermore, accurate assay setup for downstream applications, like PCR, make this system highly suited for enhancing laboratory workflow.

Results of the 2009 Paternity Testing Workshop of the English Speaking Working Group of the International Society for Forensic Genetics

Susanne Lunøe Friis, Charlotte Hallenberg, Bo Thisted Simonsen, Niels Morling — 2009-10-05

Abstract: Here we present the results of the 2009 Paternity Testing Workshop of the English Speaking Working Group of the International Society for Forensic Genetics. The exercise included paternity testing of blood samples from a mother, a child and two alleged fathers. The laboratories were encouraged to answer questions concerning their laboratory routines and a paper challenge was distributed in order to compare statistical calculations. A total of 62 laboratories participated. The laboratories used a total of 49 autosomal STRs and PCR-investigated VNTRs, 19 Y-chromosomal STRs, 8 X-chromosomal STRs, 7 VNTR systems investigated with RFLP, 49 autosomal SNPs and 11 mtDNA SNPs. The rate of typing and reporting errors was 0.1%.

Results of the 2008 Colombian paternity testing quality control exercise

2009-10-15

Abstract: The Reference National Laboratory, Genes Ltda, designated by the Commission of Accreditation and Alertness created by Law 721 of 2001 of Republic of Colombia, organized and coordinated the Quality Control Exercise of 2008 for laboratories undertaking paternity and maternity tests with DNA markers. The Quality Control Exercise included both practical and theoretical exercises. For the practical exercise, three blood samples in FTA Classic Card were sent to each participating laboratory to be genotyped for DNA markers using the routine methodologies in their laboratories. For the theoretical exercise, it was asked to the participating laboratories to calculate the partial and final paternity indexes based on two genetic profiles of an alleged biological father and his son. Allele frequencies were made available to the participants, as well as Y chromosome haplotype database. A total of 12 laboratories have participated with data from 57 STRs, including autosomal and sex chromosome markers. Consensus was found in 37 STRs, 21 in autosomes and 16 Y chromosome linked. The rate of reporting errors was 3.1% (concentrated in just one laboratory). The theoretical exercise had consensus.

Sequencing of mitochondrial DNA and the problem of human specificity

2009-10-12

Abstract: When analysing trace materials and degraded DNA the issue of human specificity is highly important. Especially when it comes down to the analysis of mitochondrial DNA which is extremely susceptible to contamination authenticity is the main question. Therefore in the presented study mitochondrial primers were tested on their human specificity. In all cases it was possible to amplify DNA of animals with human mt-primers. These unintentional amplifications could only be decreased by choosing austere PCR parameters. The study implies the importance of comprehensive evaluation of primers, chemicals and PCR parameters.

A new technology in mtDNA sequencing: Success rates vs time

2009-09-14

Abstract: Study of mitochondrial DNA (mtDNA) control region is a current practice in forensic genetics. In our service, mtDNA analysis is performed in many evidentiary specimens. Evaluation of this methodology is important to improve quality, increase efficiency and decrease artefacts, in order to reduce costs and time consuming.A case with 12 reference samples (bucal swabs) and 190 telogenic hair specimens extracted with DNA IQ™ System Tissue and Hair Extraction Kit (Promega) is reported. HVS-1 and HVS-2 control regions were sequenced with BigDye® Terminator v1.1 Kit (Applied Biosystems), using BetterBuffer (Microzone Limited), followed by a simple bead purification method (XTerminator) to remove unincorporated terminators. Application of this procedure had success in 180 hair samples within a very short time comparing to dRhodamine/ethanolic precipitation sequencing strategy and also demonstrated that better results are achieved with clean sequence data closer to the primer.The quality of data produced by the BigDye/BetterBuffer/XTerminator (BDX) procedure has been demonstrated to be very high. Besides that the BDX procedure can significantly reduce overall processing time and cost per reaction. This new methodology has additional advantages like fewer reagent transfers and smaller amounts of DNA.

Optimization and validation of 10 mitochondrial DNA SNPs using SNaPshot Kit

D. Argac, O. Bulbul, M.S. Shahzad, E. Acar, H. Altuncul, G. Filoglu — 2009-10-12

Abstract: In some forensic cases, nuclear DNA is degraded and cannot be analyzed. In such a case mitochondrial DNA (mtDNA) is usually used in forensic cases for identification because of its special features as high number of copies per cell, maternal inheritance and high mutation rate. Single nucleotide polymorphisms (SNPs) represent the most abundant class of human polymorphisms. The aim of this study was optimization of 10 mtDNA SNPs by using SNaPshot minisequencing technique on ABI310 genetic analyser in forensic molecular genetics laboratory. At the end of this study, the optimization of minisequencing technique was done by changing some assay parameters. Also, during the optimization of 10 mtSNP loci in our laboratory.

MTexpert, an automated software system for forensic mitochondrial DNA data analysis

Bobi K. Den Hartog, John W. Elling, Russell B. Kepler — 2009-10-19

Abstract: Advances in automation have led to the ability to rapidly sequence large numbers of samples. However, the large number of samples requires time consuming manual analysis and review of the data in order to produce a validated result. An automated software system for mitochondrial DNA (mtDNA) data analysis, developed under contract with the United States Federal Bureau of Investigation DNA Analysis Unit Two, allows forensic analysts to get consistent, accurate results more quickly.In MTexpert™, ab1 trace files are assembled into a consensus sequence and the difference report “mitotype” is generated using the standardized Mitotyper nomenclature rules. Expert system rules and procedures are incorporated to automate routine trace and assembly edits. The analysis of quality control samples is also automated. Every step in the data analysis process, both automatic and manual, is tracked and logged. Project files that archive the data, the analysis process log, the assembled sample consensus sequence, and the type description are automatically generated.Currently, two examiners are required to read and edit mtDNA sequences. This can be time consuming and laborious. The MTexpert software completely automates the analysis of high quality data sets, replacing the need for one of the two forensic analysts in a two-reviewer process. When this is not possible, the software efficiently directs the analysts’ attention to the issues that prevented automated completion of a project.

Comparison of the Plexor® HY System, Quantifiler® and Quantifiler Duo® kits using the Roche LightCycler 480 System and the ABI 7900 real time PCR instrument

Nicole Bulander, Burkhard Rolf — 2009-10-12

Abstract: In this study, we used two real time PCR platforms (Roche LightCycler 480 System and the ABI 7900 real time PCR instrument) to compare three commercial kits for DNA quantification. Special emphasis was put on PCR efficiency, detection limit and detection range. Furthermore, we tested the influence of the calibrator DNA included in the different kits on the absolute values. 40 artificial stain samples as well as 40 reference saliva samples were tested and compared. Two main observations could be made: the kits had a strong influence on the amount of DNA determined (Quantifiler®

Quantifiler Human DNA Quantification Kit (Applied Biosystems) as a screening kit for DNA profiling

A. Kremser, B. Bayer, S. Jung, K. Anslinger — 2009-10-05

Abstract: This study investigates the connection between the results of DNA quantification with Quantifiler Human DNA Quantification Kit (AB) and DNA profiling. For this purpose the DNA concentration of 3.068 routine casework samples was determined and DNA profiling was carried out. For discussion, depending on the specific DNA concentration, the samples were divided into four groups (0–5, 5–10, 10–30 and more then 30pg/μl DNA) and the obtained number of full or partial profiles and the negative typing results was listed. Moreover group 1 (0–5pg/μl DNA) results were subdivided and analysed more precisely. Based on the amount of 4% positive typing results in group 1 we decided to analyse every sample with quantification results >0pg/μl DNA. A real cut-off no longer exists. Only samples showing 0pg/μl on each result were sorted out.

Fast PCR amplification using AmpFlSTR Identifiler: Second report

Kazuhiko Tsukada, Yuta Harayama, Yoshinobu Kurasawa, Koichi Kasahara — 2009-10-09

Abstract: At ISFG2007, an earlier conference, we presented reports on two fast PCR cycling methods using AmpFlSTR Identifiler. In our current study, which involved PCR amplification using AmpliTaq Gold Fast PCR Master Mix, UP, and a standard PCR thermal cycler rather than the Fast PCR thermal cycler, we succeeded in allele typing while reducing PCR running times by half (to approximately 90min).

Rapid amplification of commercial STR typing kits

Peter M. Vallone, Carolyn R. Hill, Daniele Podini, John M. Butler — 2009-10-12

Abstract: Forensic DNA typing is currently conducted in approximately 8–10h. The process includes DNA extraction, quantitation, multiplex PCR amplification, and fragment length detection. Today's commercial multiplex short tandem repeat (STR) typing kits are not optimized for rapid PCR thermal cycling. Current protocols require approximately 3h for amplifying a multiplex containing 15 STR loci plus amelogenin. With the continuing development of miniaturization technologies such as microfluidic and micro-capillary devices, there is a desire to reduce the overall time required to type DNA samples. Such miniature devices could be used for initial screening at a crime scene, at a border, and at airports. There is also the benefit of reducing the required PCR amplification time for labs typing single-source reference samples. Surveys of fast processing polymerases working in combination with rapid cycling protocols have resulted in the development of a ‘rapid’ PCR amplification protocol. Results are obtained in less than 36min run on a standard peltier-based thermal cycler employing a heating rate of 4°C/s. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci, strong signal intensity and minor adenylation artifacts. Genotyping results are concordant with standard amplification conditions utilizing a standard 3h (non-rapid) thermal cycling procedure. The rapid assay conditions are robust enough to routinely amplify 0.5ng of template DNA (with 28 cycles).

Direct amplification of STRs from blood or buccal cell samples

Dennis Y. Wang, Chien-Wei Chang, Nicola J. Oldroyd, Lori K. Hennessy — 2009-09-25

Abstract: Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA® paper. Prior to PCR amplification of the STRs, the FTA® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA® paper. We evaluated the effect of various factors on the DNA profile including: FTA® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.

Rapid STR analysis of single source DNA samples in 2h

Dennis Y. Wang, Chien-Wei Chang, Lori K. Hennessy — 2009-10-13

Abstract: The establishment of offender DNA databases is critical to future crime prevention. Many countries have established databases or are in the process of passing database legislation. With new legislation the number of samples that will be collected could begin to exceed the testing capacity of many labs leading to backlogs.Two bottlenecks in the workflow that can contribute to a backlog of samples are DNA purification and PCR cycling time. The average purification time is approximately 2h and the average cycling time of current STR kits is approximately 3h. To address the second problem we investigated alternative DNA enzymes to decrease PCR cycling time. It was necessary to balance the increase in time to result against the need to address factors which can impact interpretation of a DNA profile such as: generation of stutter products, non-template addition, intra-locus balance, accuracy, and species specificity.Initial feasibility studies demonstrate that alternative enzymes can decrease PCR cycling time. The data show that this assay can increase throughput, providing results in less than 2h. However, decreasing PCR cycling time will have an affect on multiplex STR performance.

Preliminary trials of low volume AmpFlSTR® Profiler Plus® amplification using AmpliGrid (AG480F) slides

Runa Daniel, Adam Poy, Natalie Pedersen, Skye Thorpe, Roland A.H. van Oorschot — 2009-09-25

Abstract: Low volume PCR using the AmpliGrid (480F) slide system can potentially enhance the generation of more complete profiles from trace samples, in addition to providing a more cost-effective alternative for typing standard samples. Based on our preliminary results, implementation will require a reasonable investment in optimisation and validation for the intended purpose.

Validation of a microchip electrophoresis system as a DNA amplification control

2009-10-05

Abstract: As part of the normal procedure in a forensic DNA laboratory, a quality control step of the amplified DNA is often implemented to ensure the correct amplification of the sample before it is analysed in downstream applications. A validation study was undertaken to investigate a new microchip electrophoresis system (MultiNa, Shimadzu Corporation) claiming high resolution and sensitivity compared to routine polyacrylamide gel electrophoresis (PAGE). An array of STR multiplexes (AmpFISTR™ SGM+, GenePrint® FFFL, PowerPlex™ 16, PowerPlex™ Y, an in-house Y-STR multiplex and AmpFISTR™ Profiler) was tested under both standard and low copy number PCR parameters to evaluate the accuracy, reproducibility and sensitivity of this technique. These tests showed that the microchip system did not have improved sensitivity compared to PAGE though had increased resolution and high reproducibility between samples.

Mini-STRs: A powerful tool to identify genetic profiles in samples with small amounts of DNA

2009-09-24

Abstract: Degraded human remains and crime scene evidences with small amounts of DNA typically reveal incomplete or null genetic profiles when using standard (large) STR amplicons. The technology of mini-STRs, using reduced-size STR amplicons, can help to recover information from these samples. In our Forensic Genetic Service several genetic profiles were obtained or completed using MiniFiler kit (Applied Biosystems) increasing the success rate in sample typing. In all studied cases no inconsistencies were found between profiles obtained with MiniFiler and Identifiler, suggesting that this mini-STR kit can be used to include low copy number (LCN) evidence profiles in STR databases.

Increased sensitivity for amplified STR alleles on capillary sequencers with BigDye® XTerminator™

2009-10-05

Abstract: Free fluorescent dyes from PCR primers or amplification products can interfere with the interpretation of STR alleles in an electropherograph especially when the profiles have a low signal intensity. These artefacts can be removed by using a simple procedure based on BigDye® XTerminator™. This procedure requires limited amounts of PCR product, allows to do several loadings on a capillary sequencer starting from the same purified PCR product and also increases the sensitivity for detection of less amplified loci.

DNA typing from lipstick prints left on the skin

A. Barbaro, P. Cormaci, A. Barbaro — 2009-09-18

Abstract: New technologies permit DNA typing from very small and degraded samples, even those that are as latent as traces. In another previous study we demonstrated the possibility of obtaining reliable DNA profiles from prints left onto various surfaces: now we studied the ability to obtain a reliable genetic profile even from prints left by made up lips on the skin.

SNPs in paternity investigation: The simple future

Paulo Dario, Teresa Ribeiro, Rosa Espinheira, Helena Geada — 2009-10-12

Abstract: Based on the 52 SNP-plex developed by the SNPforID Consortium, we designed two 10-plex to study single nucleotide polymorphisms (SNPs) for human identification and to establish its usefulness in paternity casework. This 20 autosomal SNP set was studied in 56 paternity investigation cases from South Portuguese resident population, also analyzed with 17 Short Tandem Repeats (STRs). Results obtained with both methodologies were consistent with each other, except for one case where the alleged father could not be excluded by SNPs. No mutation was found in the SNP loci, whereas a mismatch in STRs was detected. The use of SNPs as a complement to the analysis of autosomal STRs in paternity casework can result in paternity index and paternity probability values equivalent or higher than those obtained with more STR loci, but with lower costs. This study shows that instead of using additional STR loci, the analysis of 20 autosomal SNPs, as a complement technique to standard methodologies, is an appealing alternative in paternity investigation cases.

Internal validation of 29 autosomal SNP multiplex using a ABI 310 genetic analyser

2009-10-15

Abstract: In the last few years genetic identification and paternity testing have begun to make increasing use of autosomal SNP (Single Nucleotide Polymorphism) typing as a supplement or alternative to STR analysis. With the improvement in detection technology SNP analysis is likely to be easier and more sensitive, with the generation of new methods and multiplex systems for a growing array of SNP markers. SNPforID consortium developed 52 SNP PCR multiplex for human identification purposes detected with 23 plex and 29 plex single base extension reactions (Auto1 and 2 respectively). In this study, internal validation for the 29 SNPs of Auto2 was carried out by performing a 29 plex PCR and single base extension reaction on control samples and previously analyzed forensic casework and subsequent detection with an AB 310 Genetic Analyzer. We tested the accuracy, precision, sensitivity and reproducibility of the Auto2 multiplex with this instrument in our laboratory. We used 9947A control DNA samples of the AmpFℓSTR Identifiler™ kit to test the validation parameters together with non-probative DNA samples from whole blood and buccal swab samples of 29 healthy donors from different parts of Istanbul. Good results were obtained but interpretation of the peak patterns obtained on the AB 310 requires care and thorough optimization before they can be readily compard to those obtained from multiple capillary AB 31xx Analyzers. We succesfully optimized and validated the SNPforID Auto2 multiplex system for identification analyses in our laboratory.

ABO genotyping by duplex amplification and oligonucleotide arrays assay

Li Li, Chengtao Li, Yan Liu, Yao Li — 2009-12-01

Abstract: Objective: Research on the application feasibility of ABO genotyping for forensic identification by oligonucleotide arrays assay.Methods: Oligonucleotide microarrays which detect three different SNPs in exon 6 and exon 7 for ABO genotyping were used. After hybridization wash, the arrays were scanned and fluorescence intensities were analyzed using microarray population studies on ABO was carried out in a sample of 115 unrelated Chinese Han individuals oligonucleotide arrays for genotype detection. The method was also applied to cases.Results: Technique could identify six genotypes of ABO system and the results of GeneChip analyses confirmed by PCR–RFLP. According to the results of population studies, no significant deviations Hardy–Weinberg equilibrium could be found. The observed heterozygosity (H-obs) was 0.591. Expected heterozygosity (H-exp) was 0.616. The polymorphic information content (PIC) was the average exclusion probability in paternity testing for duos (PE (1)) was 0.188. The average exclusion probability in paternity testing for trios (PE(2)) was 0.344. The discrimination power 0.777.Conclusion: The data and case application demonstrated that ABO typing by oligonucleotide probe arrays was a useful technique for paternity testing and individual identification.

Comparative analysis of ABO genotyping and serological typing in northern Chinese Han population

Xianhua Jiang — 2009-10-23

Abstract: In this paper, the comparative analysis of ABO genotyping and serological typing was conducted in 360 unrelated blood samples from northern Chinese Han population using genotyping method and serological typing method, respectively. The results of ABO genotyping were obtained by Goldeneye 16BT STR plus ABO kit. The ABO serological types were determined by the antigen–antibody agglutination test. The ABO types were confirmed by the two methods and no contradiction types were found; two more types were obtained using the ABO genotyping method and the discrimination power was further improved; the information of ABO genotyping and 15 STRs could be obtained at the same time using the Goldeneye 16BT STR plus ABO kit.

Trace DNA success rates relating to volume crime offences

2009-09-28

Abstract: In this study, 252 trace DNA samples (from handled surfaces) from 201 burglary, robbery and drugs cases were compiled to assess success rates and to interpret the value of trace DNA evidence in volume crime investigations. The average amount of DNA recovered from the trace DNA samples collected was 1.7ng. Full or major (12 or more alleles) profiles were recovered from 14% of samples. Samples from firearms and burglary points of entry were the least successful. Mixtures were recovered from 21% of samples, presenting a case for the collection of more elimination profiles to enable more samples to be used for database purposes. The research highlighted the difficulties in collecting data relating to the success rates of samples. Computerised automation of this process would be extremely beneficial in the assistance of policy development, method application, training, and investigative usefulness.

Biological and DNA evidence in 1000 sexual assault cases

2009-10-26

Abstract: Using a Filemaker-based database (DNA Pro-FILES, Synchrone Infosystème Inc.), we have conducted a large-scale study on 1000 sexual assault (SA) cases where a standardized kit was submitted to our laboratory alone or with other types of exhibits. We looked at the likelihood of obtaining good quality DNA evidence, allegedly from the assailant, according to a number of parameters.The overall proportion of SA cases with DNA evidence is nearly 50%. A little more than 30% of SA kits provided DNA evidence while for 16% of cases DNA evidence could be obtained only from other exhibits.The likelihood of obtaining DNA evidence is approximately 50% in teenager and adult SA cases, but much lower for children 10 years old or younger (15%). In children cases, profiles were found mostly on clothing or skin swabs.The likelihood of obtaining DNA evidence from vaginal swabs remains good for up to 3 days after the assault (from 35% on the first day to 23% on the third day). A DNA profile was obtained from approximately 22% of anal/rectal swabs and 41% of skin swabs taken less than 1 day after the assault. Less than 10% of oral washes provided DNA evidence, all having been collected within 24h of the assault.We found that in bodily samples, a negative result for acid phosphate (AP) is a poor predictor of the likelihood of obtaining good quality DNA evidence. Approximately 15% of vaginal swabs and 8% of anal swabs negative for AP nevertheless provided good quality DNA evidence.

DNA recovery from different evidences in 300 cases of sexual assault

W.R. Bozzo, A.G. Colussi, M.I. Ortiz, M.M. Lojo — 2009-10-26

Abstract: Almost 60% of the DNA evidences analyzed in our laboratory correspond to sexual assault cases. With the aim to assess the efficiency of the DNA IQ System (Promega) in recovering the perpetrator DNA profile, the statistical analysis of results obtained in 300 casework was performed. In such cases, 850 evidence samples were processed. In 71% of the cases the perpetrator DNA profile was detected in at least one of the submitted casework samples, with a minimum of 13 STRs markers typed using the AmpFlSTR Identifiler PCR amplification kit (Applied Biosystem). When the suspect DNA profile was available, 67% matched with the evidence.With regard to the type of evidence, the best performance corresponded to panties, with more than 70% of success in recovering male profile, whereas the efficiency of vaginal swabs was almost 60%, with a higher incidence of victim/perpetrator mixed profiles.

Sexual assault cases related to unknown perpetrator: Almost 50% of the analyzed cases corresponded to serial offenders

A. Colussi, M. Viegas, M.I. Ortiz, W.R. Bozzo, M. Lojo — 2009-10-23

Abstract: As a collaborative effort in solving sexual assault cases where there is no suspect, the DNA profiles associated with 273 cases were analyzed. Such cases were submitted from different Criminal Investigation Units belonging to eight judicial departments of Buenos Aires Province. A single NN male profile was recovered from the evidences by differential lysis with DNA IQ System (Promega) and typing with IdentiFiler kit. Comparative analysis of the compiled DNA profiles showed that in 45% of the cases the evidence DNA profile matched in at least two unrelated cases. Associations between groups of unsolved cases provided a valuable tool in aiding law enforcement investigations.

Female criminals—It's not always the offender!

2009-09-14

Abstract: Numerous crimes (including murder), all having a common denominator, occurred in Germany and Austria between 1993 and 2009. All of these cases presented with identical female DNA traces being found at the crime scene. The crimes committed differed markedly, as did the suspects involved, which were of varied origin. Many of these cases could be solved. However, none of the suspects could identify an involved female. Fourteen of these cases (including one murder) occurred in Upper Austria.A special task force of the Austrian police, together with the Institute of Legal Medicine in Salzburg, began systematically searching for errors in the investigative process after the cases became more and more incoherent and nebulous. In the end, the DNA trace evidence was shown to be contaminated. A woman involved in the manufacture of the cotton swabs turned out to be the source of the female DNA profile.Following this, several products of other manufacturers were tested for contamination with DNA. It was noted that cotton swabs which had been sterilised with radiation were often contaminated. As a result, it is recommended that the manufacturing process, as well as the products themselves used in collection of DNA trace evidence, should be re-evaluated with the emphasis on preventing contamination.

Analysis of forensic samples in Banco Nacional de Datos Genéticos

O. Santapa, S. Filippini, S. Valente, M.B. Rodriguez Cardozo — 2009-10-12

Abstract: Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye® Terminator v 1.1 Kit and analyzed in ABIPRISM® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler®, Minifiler® and YFiler® Kit (AB) and analyzed in ABI PRISM® 3100 Genetic Analyzer and ABI PRISM® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.

Paternity investigation experience with a 40 autosomal SNP panel

M.R. Whittle, E.C. Favaro, D.R. Sumita — 2009-09-23

Abstract: With both the SNPforID and the Kidd panels of autosomal SNPs available, we selected the 40 most informative and population-independent SNPs from both these sets for evaluation in paternity testing and as a prelude for forensic human identification.We used the published primer sequences and constructed PCR multiplexes for genotyping using the SNaPshot assay. Fifty trios and 50 duos previously analysed using conventional autosomal STR markers were re-analysed using the 40 SNPs. We report our findings regarding the practical use of these markers including unexpected mutations which impacted significantly on the use of this panel.

DNA profile evidence in complex disputed paternity cases: The analysis of 300 real cases

M.A. Pena, M.A. Gomez, P. Percow, M.M. Lojo — 2009-11-02

Abstract: In disputed paternity cases where the putative father is unavailable DNA from one or more of his relatives could be used. However, interpreting results is often difficult, because of the partial information regarding the parental genotype obtained from his relatives. We analyzed results obtained in 300 real paternity cases performed through close relatives of the real father (sib, half-sibs, one grandparent and/or uncle). DNA was typed with PowerPlex (Promega) and the LR estimated with the Software BDGen. As expected the higher LR values were achieved with sibs and half-sibs (in such cases where his/her mother was available for testing). The LR values were tight related to the number of uninformative loci, which varied between 0 and 13. In 10% of the reviewed cases, 10 or more non-informative loci were observed; all of them associated LR values below 0.01. Thus, providing evidence in favor of no relatedness.

Supplementary markers for deficient immigration cases: Additional STRs or SNPs?

2009-10-23

Abstract: Analysis with commonly available STR kits can sometimes fail to produce sufficient information in immigration cases containing only one parent. In these cases, not only does paternity/maternity need to be assured, but also other possible relationships dismissed (e.g. avuncular relationships).We have taken more than 50 of these cases and investigated which type of additional marker produces the greatest benefits: 48 SNPs or 6 additional informative STRs (including 5 additional markers from the new European extended set). The results of this analysis show the SNPs to be of greater value.

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

2009-10-08

Abstract: This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.

STR genotyping of DNA extracted from used Triage kits

M. Hara, A. Kido, A. Takada, T. Miyazaki, K. Saito — 2009-10-12

Abstract: This study examined whether short tandem repeat (STR) genotyping can be performed using DNA remaining in Triage kits used to screen for drugs of abuse in urine. STR genotyping was successful for 15 loci using 12 kits stored for 1–6 months at room temperature. These results suggest that STR genotyping for human identification can be performed using DNA extracted from used Triage kits.

Extraction of high quality DNA from biological materials and calcified tissues

2009-09-28

Abstract: Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and CT values for the internal PCR control of Quantifiler® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpFℓSTR® Identifiler® PCR Amplification Kit. The protocol is easily adapted for automation.

Effect of blood stained soils and time period on DNA and allele drop out using Promega 16 Powerplex® kit

M.S. Shahzad, Ozlem Bulbul, Gonul Filoglu, Mujgan Cengiz, Salih Cengiz — 2009-10-05

Abstract: Blood stained soils may be of great interest in forensic incidents. Amplification of DNA from soil is often inhibited by co-purified contaminants. Different soils types from Pakistan and Turkey were stained with blood and samples were collected systematically after specified intervals. Rapid, inexpensive, large-scale DNA extraction method involving minimal purification was developed. DNA was quantized using Spectrophotometer and Fluorometer and was confirmed by Agarose Gel Electrophoresis. DNA extracted from different soils in different periods showed a remarkable decrease in yield as well as degradation in every extraction. PCR amplification was performed using various DNA targets present in Promega 16 Powerplex® System kit. Amplification could not carry out in all loci especially in degraded samples taken after 20 days. Allele n locus drop out was noticed which shows that DNA was degraded. For some loci more than 2 alleles were also noticed showing contamination while working with the blood stained soils.

Prevalence of mixed DNA profiles in fingernail swabs from autoptic cases

N. Cerri, A. Verzeletti, V. Cortellini, A. Cincotta, F. De Ferrari — 2009-09-07

Abstract: Physical contact between two or more persons can give rise to the transfer of DNA from one person to another and biological material can accumulate under the fingernail hyponichium. The purpose of this study is to value the normal levels of foreign DNA profiles under the fingernail of individuals deceased of a non-violent death, in order to define the usefulness of this approach in the recovery of a suspect's DNA profile. No foreign DNA was found in the majority of the cases.

A standard procedure for accommodating forensic anthropological and genetic analysis of decomposing human remains from tropical climates

2009-09-22

Abstract: At the Medical Legal Center in Ribeirão Preto, Brazil (CEMEL/FMRP-USP), unidentified decomposing bodies routinely undergo soft tissue removal (by immersion in water at 80–90°C for 24h) prior to an anthropological analysis intended to yield a biological profile of age, sex, ancestry, height, pathology and so on. In the event that this analysis is unsuccessful, samples may be submitted for DNA profiling. The tropical climate and the defleshing process may confound preservation, recovery and analysis of DNA, however. In order to establish an optimal standardized protocol for identification of decomposing human remains from a tropical climatic region, the outcome of anthropological and genetic analyses was compared, along with the utility of bone (mainly femur and sternum) and teeth (mainly molar) specimens for DNA analysis. In a sample (n=39) of partially skeletonized remains, anthropological analysis was sufficient for identification in eight cases. In further six cases, DNA profiling was successfully attempted. As a consequence of our study, we recommend collection of 1–2 well preserved teeth prior to defleshing and anthropological analysis in these circumstances.

Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs

2009-10-05

Abstract: The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of FFTIP (spleen/lung) and hairs, with or without bulbs, were analyzed using three methods of extraction (QIAamp DNA mini, QIAamp DNA micro-kit and phenol–chloroform followed by microcon YM-30). The amount of DNA recovered was quantified by spectrophotometer. The β-actin, amelogenin gene and the profiles of STR were analyzed. Based on experimental results, a general guideline concerning the appropriate extraction method according to the tissue and the quantity of the starting material for the analysis of DNA from FFTIP and hairs could be suggested.

Genetic identification of degraded DNA samples buried in different types of soil

V. Bogas, M. Carvalho, M.J. Anjos, M.F. Pinheiro, F. Corte-Real — 2009-10-07

Abstract: Biological samples buried in different types of soil are often found in crime scenes. These samples are usually highly degraded which difficult their analysis. Several factors contribute to the degradation of biological material including temperature variation, humidity, UV light and especially the presence of microorganisms.Blood was collected from three non-related male donors and blood stains were made in fabrics such as jeans, cotton and lycra. Blood stains were dried at room temperature and buried in three different types of soil (sand, marsh and clay), to promote its natural degradation.The buried samples suffered a high degradation over time which difficult their genetic identification. The marshy soil proved to be the most destructive one, leading to rapid degradation of the different analyzed fabrics, which disabled the obtainment of the genetic profiles.

Utility validation of extraction of genomic DNA from hard tissues, bone and nail, using PrepFiler™ Forensic DNA Extraction Kit

Masaki Hashiyada, Nori Nakayashiki, Masato Funayama — 2009-09-24

Abstract: STR profiling using hard tissues obtained from a severely decomposed body is sometimes a laborious work. There is now on a market a new DNA extraction kit, PrepFiler™ Forensic DNA Extraction Kit (AppliedBiosystems), and we tested it for missing persons. Postmortem intervals ranged from weeks to several years. Fifteen bone fragments and eleven nails were used in this report. Genomic DNA was quantified by QuantiFiler® DUO Quantification Kit (AppliedBiosystems), and STRs were analyzed using AmpFlSTR® Identifiler® PCR Amplification Kit (AppliedBiosystems). The profiling of 16 STR loci was successful in all nail samples. However, STR profiling was successful in only 6 of 15 bone materials. Nine cases failed to analyze STR polymorphisms using another DNA extraction kit, the QIAamp DNA Mini Kit (QIAGEN). For bone samples, it seems that STR profiling depends on the quality of samples.

A new approach in the identification of degraded paternity samples

2009-10-12

Abstract: DNA extraction from bone is an important issue particularly in paternity cases when bones are the only remaining material to obtain and analyze DNA. The difficulties arising from bacterial damages, taphonomic factors and diagenesis might negatively affect the extraction and the amplification of DNA. This makes the laboratory procedure a hard and time-consuming process, and the analysis can fail. Analyzing mini-STRs in this type of degraded samples is highly recommended. In this study a new extraction technique was carried on bone samples which were then typed for mini-STRs. The aim was to differentiate two genetically related skeletons found in the same familial grave for a paternity test. The analysis revealed that this new extraction technique along with mini-STR analysis can properly be an effective way to obtain and analyze DNA in bones in the field of forensic sciences.

Validation of PrepFiler™ forensic DNA extraction kit (Applied Biosystems)

A. Barbaro, P. Cormaci, A. Agostino — 2009-09-17

Abstract: The PrepFiler™ is a new kit recently introduced by Applied Biosystems for DNA extraction from a wide range of forensic samples. In the present study we tested the performance of PrepFiler™ kit against other commonly used commercially available kits on a variety of real forensic casework samples: bloodstains on different substrates, washed bloodstains, semen stains, saliva stains, hairs, bones, tissues, nails, prints after chemical treatments, skin swabs.

STR and SNP analysis of human DNA from Lucilia sericata larvae's gut contents

2009-10-05

Abstract: Importance of forensic entomology becomes inevitable when come across some incident where corpse is unidentifiable and lot of maggots or other insects are present. The most common application of forensic entomology is to use insects for the identification of specimens or human remains. DNA analysis recovered from a larva's gut contents can be used to identify a missing body. The obtained human STR and SNP profile support the association of a maggot to a specific patients or corpse. Main aim of this research was the identification of human DNA from gut contents of third instar maggots (larvae of Lucilia sericata) placed on diabetic patient's wounds for treatment purpose. Maggots (8–15) were taken from each diabetic patients (no. of the patients 8) and DNA was extracted from the gut contents manually by using Qiagen tissue protocol. Agarose gel electrophoresis was performed and the total size of DNA was seen using UV transilluminator. PCR amplification, STRs and SNPs profiling was then performed using PCR 9700 and AmpFLSTR Identifiler and SnaPshot Multiplex Kit (Applied Biosystems) respectively. The results were analyzed on ABI 310. SNP profiles were good and identifiable compared to the STRs where amplification was poor and the peaks were low. This may be the fact of the enzymatic activity present in the gut of the larvae which cause tremendous reduction in DNA size and thus yield. The results of this study reveals that it is possible to obtain a complete human profile using STRs and SNPs even if DNA is recovered from gut contents of maggots.

The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Annalisa Durdle, Roland A.H. van Oorschot, Robert John Mitchell — 2009-11-04

Abstract: Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

2009-10-12

Abstract: It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpFℓSTR® SGM Plus® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.

The problem of DNA contamination in forensic case work—How to get rid of unwanted DNA?

2009-10-12

Abstract: The PCR technique has become a powerful and very sensitive tool in a broad field of research, that is, molecular biology, medical diagnostics, population genetics, ancient DNA analysis and forensic casework.However, the high sensitivity down to single molecules can easily cause false-positive PCR results due to different types of contamination. In this study, artificial DNA contaminations (saliva and pure DNA) were treated with UV irradiation and other decontamination procedures. A satisfactory DNA removal could not be achieved, emphasizing the necessity of contamination avoidance.

MtSNP typing before mtDNA sequencing: Why do it?

Paulo Dario, Joana Bom, Teresa Ribeiro, Helena Geada — 2009-10-12

Abstract: Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.

Trace DNA collection—Performance of minitape and three different swabs

2009-09-25

Abstract: In this study two types of synthetic swabs and one commercially available minitape were tested and compared with the currently used cotton swab. The results indicate that there is no major difference in performance between the swabs for recovery of trace samples, and that the minitape is better suitable for recovering from absorbent materials than swabs are. However, no statistical calculations were carried out due to the low number of samples in each category.

Optimisation of cellular DNA recovery from tape-lifts

R. May, J. Thomson — 2009-10-12

Abstract: Adhesive tape-lifts are a commonly used technique for the recovery of DNA from forensic exhibits. Examination of large forensic exhibits or tape-lifts from old “cold” cases can make the direct submission of the tapes for extraction difficult. By applying a swab loaded with an organic solvent to the tape-lift, any DNA bound to the tape can be transferred and concentrated on to the swab head. Whilst removing any DNA, the tape's glue adhesive is also transferred. This requires a modified extraction technique that can dissolve the adhesive whilst maintaining the integrity of any DNA. Of several solvents tested, xylene was shown to be the solvent of choice, efficiently removing the adhesive and any bound DNA. A modified chelex extraction method, again incorporating xylene, provided optimal conditions for dissolving the adhesive and releasing the DNA for lysis.

One method of collecting fallen off epithelial cell

Xianhua Jiang — 2009-11-13

Abstract: A person usually falls off epithelial cells as many as 400,000 every day, in which the majority adheres to the internal surface of clothes contacting the skin. So many cells disperse on the collar, sleeve cuff and so on. In order to extract DNA from this kind of forensic samples, the normal method is to cut down the cloth with the fallen cells, then the cloth is digested. Due to the restriction of the cut-down size, only a very small amount of cells are collected by this method. Moreover, also because the existence of the dye, the detergent residuum will inhibit the PCR reactions, the good STR profiles are hardly obtained from a considerable number of these samples. Now a vacuum-collection method was established to collect the fallen cells on the surface of forensic samples in this article. The device was made based on the vacuum cleaner, and a special filter membrane with 5mm diameter was used. When the device was used to collect cells, the filter membrane was sealed on the inhaling mouth. Then the large area of the forensic can be scanned repeatedly to collect cells. After scanning, the cells were deposited on the external surface of the membrane. The membrane was digested to extract DNA by chelex-100 method . The successful rate of STRs analysis was greatly improved by this method. This method was used in many samples, such as gloves, jacket underwear, shoes, hat, cup and headgear from forensic cases, and the fallen cells were collected from most of them. The satisfied STR profiles were obtained. This vacuum-collection method can collect cells without destroying carriers, and was suitable to the DNA analysis of forensic cases.

The analysis of biological samples from crime scene for a future human DNA profile confrontation. Effects of presumptive test reagents on the ability to obtain STR profiles for human identification

2009-10-05

Abstract: Because of the increase of evidence of blood stains, that have been washed or cleaned in an attempt to mask the analysis of DNA profiles, there is also an increase in the use of presumptive tests on samples sent to laboratories. Some of the presumptive tests, used to identify blood and semen stains, could potentially affect the recovery of high molecular weight DNA from the samples, or extinguish them, especially those already present in small quantities. After the presumptive tests, often these samples are discarded. This study aimed to examine the possibility of obtaining a DNA profile from samples submitted for presumptive testing and cleaned with bleaches with and without chlorine. Two different protocols were conducted: (a) A unique sample of human blood in natura (5μL), already typed through the DNA techniques with the genetic profile previously known (control), was distributed onto cotton fabrics and dried at room temperature. Four samples of fabric were macerated in saline solution and Coombs serum and then stored for three months (room temperature and freezer −20°C). (b) Another sample of human blood, type A, in natura, already typed through the techniques of DNA (control) was used. Aliquots of 200μL were distributed in: cotton, denim and synthetic fabric. The samples were dried at room temperature for 24h. The blood stains in those fabrics (cotton, denim and synthetic) were then divided into three groups: unwashed, cleaned with chlorine bleach and cleaned with chlorine bleach and soap powder. The samples were again dried at room temperature for 24h, before the use of luminol. The DNA were extracted with Chelex 100 and amplified with the Identifiler Kit (Applied Biosystems). The blood stains exposed to saline and Coombs serum had DNA profiles consistent with untreated samples (controls). This result shows that the experts should keep and store the samples treated with saline and Coombs serum for future DNA confrontation when necessary. Also discussed in this paper the pattern of blood stains after washing with bleaching solutions, as well as the quantity of DNA obtained from these samples.

Influence of the luminol chemiluminescence reaction on the confirmatory tests for the detection and characterization of bloodstains in forensic analysis

V.R.D. Santos, W.X. Paula, E. Kalapothakis — 2009-10-05

Abstract: Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40 years in forensic science. This reaction is based on the emission of light through the chemical reaction of luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron from the hemoglobin) (Laux [1]).This work evaluates the luminol interference and its effect on subsequent serological and DNA testing. Samples prepared with blood and different concentrations of luminol solution containing luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests with luminol do not establish the characterization and identification of stains at crime scenes, preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.

Grading a rape case followed by death from the study of autosomal STRs and STRs of the Y chromosome—Case study

2009-10-05

Abstract: Two women were found dead inside a residence. Choke causes death in one that had been naked in a bed and contusion injury in another that was found on a sofa. Were received samples of vaginal and anal swabs of the two victims of homicide with suspected of having suffered sexual violence. References also received samples of two victims and a suspect. We performed genetic analysis for identification of samples from the meeting of any possibility of overlap between patterns and profiles of sequences of deoxyribonucleic acid (DNA) based on genetic relationship between those involved. The reference samples were subjected to the procedure of extraction of nuclear DNA by Chelex method and the swabs samples by differential extraction. For all the samples were performed for amplification of STRs loci and autosomal STRs of chromosome Y. The profiles of DNA sequences were obtained by the Polymerase Chain Reaction (PCR), using sequences starting with marked substances emitting fluorescence detected by reading the optical laser in 3100 Avant automatic sequencer from Applied Biosystems. The information of consecutive loci of Short Repeats or STRs of autosomal chromosomes and the Y chromosome was obtained using the systems or products sold in multilocus, methodologies recommended by the supplier and valid for analysis of DNA. We used the multilocus Identifiler and YFiler system of Applied Biosystems to the amplification of samples. The validation of results has shown a genetic profile in male anal secretion of the victims with a complete coincidence with the suspect.

Case report of a homicide resolved 15 years later: The robustness of Chelex extraction

L. Caenazzo, E. Ponzano, N. Cerri — 2009-09-07

Abstract: DNA typing techniques is one of the most advanced tools for human identification. During the last 10 years, a great number of methods for DNA extraction and analysis have been introduced to forensic genetic, with considerable success but also with considerable controversy. The success and validation of a criminal investigation are very closely related to the process used for obtaining and preserving biological evidence.We report the strategy that we employed to analyze evidences belonging to a homicide happened in Brescia (Italy) in 1992, not resolved at that time, with the forensic genetic analysis. After 16 years the analysis were conducted on DNA samples extracted with Chelex maintained at −80°C, bloodstain, and biological specimens of perpetrators. Standard autosomal and Y-chromosome STR analysis identified the persons involved and victim's profiles. This case is of interest as a demonstration of a more successful application of DNA typing in well conserved DNA samples than in bloodstains kept in the Court Office.

Statistical analysis of DNA mixtures using peak area information and allelic drop out

2009-09-24

Abstract: A case of a woman killed in Perugia is reported. The woman was beaten to death and the body showed evidence of bites, kicks and punches. The request of the Court was to verify the presence of bites and if they belonged to humans. Morphological examination and genetic analysis with human Y-specific markers were performed in order to verify the origin of the bites. The DNA profile from the surrounded area of the traces was compared with the profile of the victim's husband (the suspect).The results showed a match between the profile of the suspect and that of the traces for all loci examined. Due to the fact that also other relatives of the husband's male lineage lived in the same house, it was not possible to identify the man who really contributed to the traces. Therefore, the analysis was implemented with autosomal STR markers, which showed a mixed genetic profile. In order to verify the number and the identity of the contributors, statistical analysis based upon peak area information was performed with Probabilistic Expert Systems.

Identification of gestational trophoblastic disease in a sexual assault case

2009-11-09

Abstract: In this study, gestational trophoblastic disease (GTD) was observed by short tandem repeat (STR) typing from the aborted tissues in a sexual assault case. By histological screening, the fetal tissue could not be distinguished from the maternal tissue in this case. Therefore, five specimens were collected randomly from the aborted tissues for DNA analysis. STR typing was performed by the commercial ABI Identifiler kit. The results showed that three specimens were of the maternal origin, one was a mixture of the mother and male fetus, and the other one was of male fetal origin with partial triploid. Three alleles were identified in each locus of D8S1179, D7S820 and VWA for the fetal specimen. For these three alleles, one matched the maternal origin and the others matched the putative paternal origin (suspect). Analysis of the Y-STR by using the commercial ABI Y-Filer kit, the fetal types matched the types of the suspect. We reported the case of partial mole on forensic evidence and gave the valuable information from its identification.

Forensic application of Y chromosome SNPs in inconclusive cases

2009-10-05

Abstract: Biallelic markers, Single Nucleotide Polymorphisms (SNPs), are nowadays a powerful tool in the analysis of degraded samples. Namely, Y chromosome SNPs allow to determine the gender of the analyzed sample and to establish its haplogroup, making possible to attribute the ethnicity of male individuals. The aim of this study is to obtain Y-SNPs in forensic samples without STRs results, checking methodologies previously used.

Traffic accident vs homicide: The contribution of DNA analysis to clarify this mystery. A case report

2009-11-13

Abstract: A marriage procession was going through the road when the vehicle met with a fatal accident and the wife of the driver died. The autopsy revealed lesions according with fatal traffic accident. But, a second autopsy revealed that there were injuries, but it was not reported in the first autopsy protocol. We analyze several autosomal STRs to typify some evidences collected inside the vehicle of traffic accident which were stained by the blood of the woman mortal victim. The results of the analysis of DNA suggested that the victim bled inside the vehicle and died and then, she was placed on the pavement and her husband simulated an accident.

Human being eaten by his own dogs: Genetic confirmation through analysis of bones recovered in a dog's stomach content

2009-09-02

Abstract: A part of a decomposed human body from an individual was found at his home, together with the decomposed bodies of his 3 dogs. The disappearance of half of the human body was hardly explained. Hypothetically the dogs, starving, could have eaten their owner after his death. All the bodies were recovered for autopsy.Small bone fragments were recovered in one dog's stomach and identified as human by anthropological analysis. DNA was extracted and cytochrome b gene analysis was made in order to determine their origin, confirmed as human.Genetic identification allowed achieving an eight mini-STR profile with MiniFiler (Applied Biosystems) identical to the bone material collected at the victim's autopsy, confirmed that the dogs had effectively eaten their owner.The results showed that it is possible to obtain nuclear DNA in samples subjected to gastric acids and the combination of different techniques allowed us to determine, in each step, the most convenient workflow for the remains identification.

DNA analysis of biological material on perforating bullets and crime scene reconstruction

2009-10-12

Abstract: DNA analysis of biological material present on a bullet surface may be an important tool for the reconstruction of a crime scene. In shooting incidents when there is more than one gunshot or victim or when the crime scene has been changed, the presence of DNA on the implicated bullet may determine that it hit the victim. The comparison of this bullet and the firearm(s) involved may identify the gun from which the bullet was fired and help to define the shooter. We report two cases of homicide by firearms in which biological material was recovered by swabbing the bullets. DNA was extracted and PCR typing of STR was carried out using the AmpFLSTR® Identifiler (Applied Biosystems). In both cases we obtained genetic profiles from different regions on the surfaces of the two projectiles. In the two situations, DNA typing played a decisive role in the reconstruction of the crime scenes.

Forensic application of mitochondrial DNA SNPs

2009-09-22

Abstract: Mitochondrial DNA (mtDNA) has enormous potential in forensic genetics, allowing identification of genetic material from degraded samples. Genetic testing can also be performed using mtDNA coding region SNPs. SNPs have a number of characteristics that make them unique for forensic analysis, allowing the successful analysis of degraded samples. The aim of the present study was to evaluate the effectiveness of a mtSNP typing assay on skeletal remains that were buried directly in soil for more than thirty years in adverse climate conditions.

Crime investigation set on mitochondrial DNA analysis

2009-09-17

Abstract: Mitochondrial DNA analysis is a useful tool for typing evidences with small amounts or no nuclear DNA. A homicide case with several hairs is reported. Hair samples were washed in ethanol (70%) for 30min followed by a further wash step in sterile distilled water for 30min. The sequencing strategy BigDye/BetterBuffer/XTerminator was applied. In the presented case a mixture of both victim/aggressor haplotype was detected in hair samples previously washed.

An homoplasmic large deletion in mtDNA Control Region: Case report

2009-10-08

Abstract: We report a new case of a large, homoplasmic Control Region deletion in human mitochondrial DNA. A missing 154bp fragment spanning positions 16154–16307 was found in an apparently healthy blood donor from Salta (NW Argentina) whose maternal lineage was attributable to Native American haplogroup D1. The same mutation, to the best of our knowledge, has been independently reported before only twice, in both homoplasmic and heteroplasmic states.

A paternal mutation in the Penta D STR locus

C.R. Thacker, E. Musgrave-Brown, D. Ballard, Y.D. Syndercombe-Court — 2009-12-01

Abstract: In the field of paternity testing, inconsistencies between tested individuals can point to non-paternity or the presence of a mutation or mutations. We report on a case where one inconsistency was observed between a putative father and his daughter following routine STR typing using Powerplex-16 (Promega) and SGM Plus (Applied Biosystems). A three-step mutation was detected in the Penta D STR locus. A paternity index of 99.9999% was obtained. Following the additional analysis of over 50 different independent tests and the testing of other family members, no further inconsistencies were detected.

Non-exclusion maternity case with two genetic incompatibilities, a mutation and a null allele

2009-09-01

Abstract: We present an immigration case with an apparent double Mendelian inconsistency between mother and child with different underlying mechanisms. The first inconsistency was caused by a null allele in the vWA locus. The second inconsistency was due to a one step maternal mutation in the D2S1338 system. The 17 autosomal STRs of the Identifiler and Powerplex 16 multiplexes, with additional HLA A-B-DR haplotypes yielded a maternity index of 78747, providing sufficient evidence for maternity of the alleged mother. This case shows that paternity or maternity cannot be excluded on the basis of only two parent/child mismatches.

Chimerism detected in fraternal twins using ABI AmpFlSTR® Identifiler

2009-10-05

Abstract: A female proband (one of fraternal twins) presented for molecular analysis of Fragile X syndrome. Southern Blot analysis indicated a normal female methylation profile at the FMR-1 gene, but atypical intensities of the non-methylated and methylated alleles, suggesting reduced dosage of the methylated locus. Cytogenetic analysis of the patient's lymphoblasts showed a mosaic chromosome profile in which half the cells exhibited a 46,XX karyotype and the remaining cells were 46,XY. Subsequent analysis of buccal cells of the patient showed predominantly a 46,XX karyotype. These data were taken to suggest anastomosis and haematopoietic stem cell exchange (“haematopoietic chimerism”) with the proband's male twin early in development. To confirm this suggestion lymphoblast and buccal DNA from each twin were subjected to analysis using the Applied Biosystems Identifiler kit. The profile of each twin exhibited multiple alleles at most autosomal loci using DNA extracted from lymphoblasts, but one–two alleles dominate at loci using buccal cell DNA, thereby confirming haematopoietic chimerism.

A case of chimerism in a paternity study

2009-10-30

Abstract: The STR profile of a phenotipically normal woman typed from blood tissue in a paternity case, revealed more than two alleles in some autosomal loci, result that was interpreted as a chimera due to the coexistence of two genetically distinct cell populations. The aim of this work was to solve the paternity study, elucidate the origin of the chimera, and evaluate its consequence in forensic genetics. Buccal epithelium and hair root tissues showed a single autosomal STR profile. Instead, blood tissue exhibited chimerism in seven out of the twenty autosomal loci tested. The paternity analysis was resolved comparing the unique STR pattern typed from buccal and hair root cells. The detection in blood tissue of the Y allele in Amelogenin locus and typing of a Y-chromosome haplotype confirmed the presence of male cells in the chimeric woman. The deduced male profile cannot be excluded as a son of the alleged father. Based on the results we interpret that the woman subject of the study is a partial or a whole-body chimera. Chimerism may be a pitfall in forensic investigations like paternity testing and crime cases.

Paternity testing involving human remains identification and putative half sister: Usefulness of an X-hexaplex STR markers

2009-10-12

Abstract: Paternity testing is being increasingly requested with the aim of challenging presumptive fatherhood. The ability to establish the biological father or mother is usually based on the genotyping of autosomal short tandem repeats (STR). However, in the last few years the use of X chromosomal STR (chr-XSTRs) markers gained strong interest in paternity involving females and specially in kinship analysis where the alleged father is absent since true daughters share the single X-chromosome from their biological father. In this work, we present an approach that combines data from fifteen autosomal and six chr-X STRs in order to know the DNA profiles of presumed relatives by genotyping human remains and/or investigate female as putative half sister or unrelated. As a result, the LR over all available loci for two cases were 1,432,331,424 and 9,357 corresponding to probabilities of 99.9999999% and 99.989%, respectively. The LR values associated to the high probabilities confirm the usefulness of the X-hexaplex STR markers in resolving relationships even thought genotyping human remains.

Prenatal testing in paternity testing: A positive perspective

C.R. Thacker, D. Ballard, Y.D. Syndercombe Court — 2009-11-23

Abstract: Prenatal paternity testing is an area of human identification that raises many ethical questions and is often portrayed in a negative light. In most cases, fetal samples are obtained using invasive techniques and the associated risks to the pregnancy necessitate careful consideration before embarking on such a course of action. Although the risk of miscarriage or other damage to the fetus can be avoided through the use of maternal blood sampling for prenatal diagnosis of paternity, until this form of testing has been shown to be reliable there remains the possibility that it will lead to erroneous termination of pregnancy.We report on two independent cases where doubts had been raised over the quality of results offered after non-invasive tests were performed. Subsequent tests gave different results and, in each case, a planned termination of pregnancy was avoided.

X-STRs analysis in paternity testing when the alleged father is related to the biological father

Ulises Toscanini, Gabriela Berardi, Andrea Gómez, Eduardo Raimondi — 2009-10-12

Abstract: We report a case where a woman (W) claimed the paternity of a man (M). This man could be her biological father, but he could also be her uncle (i.e. a brother of her actual biological father). Routinely used autosomal markers were first typed yielding one incompatibility among the 17 short tandem repeats (STRs) analyzed. Next a set of 10 X-chromosome STRs (X-STRs) additionally typed allowed to achieve a conclusive result. Thus, the use of this set of X-STRs could be even more informative than the autosomal STRs markers in situations similar to the one presented here.

Utility of Y- and X-STRs in the research of complex biological relationship

J.J. Builes, A. Manrique, D. Aguirre, Y. Puerto, M.L. Bravo, L. Gusmão — 2009-10-15

Abstract: The social situation in the past years has led to increase in the paternity test with absent parents in Colombia. We present here three cases of biological relationship investigation of an extramarital son without parents; the first case is the biological relationship research between granddaughter and grandparents with dead parents, the second one is between niece and three paternal uncles without father and mother and the third one is between grandson and grandparents with dead parents. In the three cases the research started with the typification of 15 autosomal STRs markers. Cases 1 and 2 were complemented with X-STR markers and Case 3 was complemented with Y-STR haplotype, all cases were solved with a conclusive diagnosis. This report shows the efficiency of STRs markers linked to sexual chromosomes, like complement, when autosomal STRs are not allowed to get the necessary probability.

DNA recovery from a 44-year old umbilical cord

M.A. Pena, Laborde Lisandro, M.M. Lojo — 2009-10-26

Abstract: A 44-year-old umbilical cord was used to perform a post-mortem paternity test. DNA was extracted with Quiamp DNA Minikit (Qiagen) and typing with IdentiFiler and YFiler Kits (Applied Biosystems). As results obtained excluded the alleged father, a further identification of the recovered DNA profile was done by DNA from the father of the alleged father as reference sample.

Evaluation of deleted region from Yp11.2 of two amelogenin negative related males

Stefania Turrina, Giulia Filippini, Domenico De Leo — 2009-10-05

Abstract: The amelogenin locus is encoded by two single copy genes located on the short arm of X (AMELX, on Xp22.1–22.3) and Y (AMELY, on Yp11.2) chromosomes. Few cases of AMELY deletion have been reported and some studies have described that AMELY dropout is due to a large deletion encompassing AMELY on Yp11.2. In this study, we describe a large deleted region on the short arm of the Y chromosome discovered during routine paternity testing in two related males.The complete absence of AMELY and DYS458 marker and the presence of SRY gene in both samples, induced to conclude that a deletion was occurred in a portion of the short arm of Y chromosome. Twenty Y-specific Short Tagged Sequences (STSs) were selected to delineate the breakpoints of deletion.Seven STS between 4.91Mb and 7.97Mb were completely absent. The deletion of 3.06Mb occurred 1.88Mb upstream and 1.18Mb downstream of the amelogenin locus.Considering the consequences of a misidentification of a male sample, the use of Y chromosome markers and SRY gene are always crucial in sex determination in criminal investigation.

Investigation of illegal graves in Argentina by using STR, mini-STR, Y-STR and mitochondrial DNA analysis

2009-10-05

Abstract: During the military governments in Argentina in the 1970s, the bodies of people who had been detained and later killed were deposited in individual graves as well as common graves. The identity of the majority of the bodies was not provided.During 2008, the Argentine Forensic Anthropology Team (EAAF) processed nine bone fragments from bodies found in a group of graves in the Province of Buenos Aires.Nuclear DNA (nDNA) profiles were obtained by amplifying autosomal STRs (using the Identifiler®, Profiler® and COfiler® kits), Y-STR (Y-Filer®) and mini-STR using kits developed “in-house”. The mitochondrial DNA (mtDNA) HV1 and HV2 regions were also sequenced on the same bone samples.Blood samples were analyzed from 21 individuals who reported to the EAAF that they had a family member missing from the aforementioned time period.This study presents the results of the comparison made between the bone sample genetic profiles and those of the family members, based on the genetic marker used (STR, Y-STR and/or mtDNA) and on the proposed kinships for each hypothesis. From the 9 bone samples analyzed, a total of 8 individual skeletons were observed. Of those 8 individuals, 4 were identified through kinship analysis.

DNA profiling of skeletal samples from the disappeared in Latin America

2009-11-09

Abstract: The identification of missing persons can be quite challenging; particularly in instances where a significant number of individuals went missing during conflicts from decades past. In these situations, DNA profiling and kinship analysis are necessary tools to aid with the identification of compromised skeletal remains such as those analyzed for the Latin American Initiative for the Identification of the “Disappeared.” In 2008, Bode was awarded contracts for this initiative to perform DNA analysis on hundreds of skeletal remains and family references. Bode's success in obtaining profiles utilizing advanced extraction and purification techniques along with mini-STR technology has, to date, resulted in the identification of greater than 80 individuals.

Disaster carbonized victims identification in State of Rondonia, Brazil

2009-10-23

Abstract: This work relates the identification of the 13 carbonized corpses in a car crash in which DNA analysis was necessary. The corpses and relatives samples were tested using polymerase chain reaction (PCR) amplification and short tandem repeat (STR and Y-STR) typing. Amplification products were analyzed by an automated sequencer and statistical analyses were performed using Familias 1.7 software. Full profiles were obtained for all samples. All victim samples had relationship to the reference samples, with agreement for all autosomal markers and the Y chromosome haplotype. Human identification through DNA analysis is an extremely efficient tool in forensic casework when other methods are not successful, even in cases of carbonized bodies.

Analysis of complex kinship cases for human identification of civil war victims in Guatemala using M-FISys software

Marco García, Luis Martinez, Mishel Stephenson, John Crews, Fredy Peccerelli — 2009-10-05

Abstract: Twelve years after the end of Guatemala's 36-year internal conflict, the depth and breadth of loss of human life during this time is now being calculated, and the number is staggering. FAFG has been working for several years trying to reconstruct the historic memory. Now with DNA technology running at FAFG laboratory, our ability to identify victims is much greater, but the data behind these identifications has become much more complicated. To help manage the data FAFG has employed M-FISys software, which contains tools and algorithms for use in elucidating very complex kinship relationships that cross familial as well as generational lines, giving alternative approaches to complex kinship relationships and management of population genetic information. In FAFG's first comparison of 67 victims to 451 family references numerous genetic leads were made and here we describe a family tree compound of 3 family groups that are biologically related to one another and in total consist of 42 individuals spread over 5 generations, including: 14 missing persons; 12 living family members who donated biological samples for DNA analysis and 16 individuals who were either deceased or unavailable for sample collection. This case exemplifies the depth and breadth of the losses as well as the nature of complex kinship between victims and relatives. Through the use of integrated software to combine ante-mortem and post-mortem data into one system, large-scale human identification can be made easier and simpler for those tasked with large-scale or complex human identifications.

Genetic identification of fire deaths

Anke Heinrich, Thorsten Schwark, Eva Simeoni, Nicole von Wurmb-Schwark — 2009-10-12

Abstract: We investigated whether authentic DNA patterns can be obtained from human bones showing different stages of burning (well-preserved, semi-burned, black burned, blue-grey burned, blue-grey-white burned) by comparing STR-analysis, mitochondrial SNP-analysis and mitochondrial sequencing. Altogether, 71 bone fragments from 13 fire victims showing different grades of burning were obtained at autopsy and genetic profiles were compared to the original genetic pattern from undamaged tissues such as internal organs or unburned bones.The results show that an identification with all three methods is reliably and reproducibly possible from well-preserved and semi-burned bones. In black burned bones the DNA is usually highly degraded, and in some cases no nuclear DNA is left, leaving mitochondrial DNA (mtDNA) sequencing and SNP-analysis of mtDNA as an option. Blue-grey burned bones however only lead to sporadic authentic profiles, and blue-grey-white burned bones were almost impossible to reliably investigate with any DNA method.

Missing and unidentified persons database

2009-10-12

Abstract: Missing persons and unidentified human remains constitute a global problem. Brazil, like other countries, has a large number of cadavers and human remains that need to be identified.In order to help to solve this problem, we developed a bioinformatics tool named Missing Person Database-gen (MPD-gen) to register and compare genetic and non-genetic information from missing persons, relatives of missing persons, unidentified cadavers and human remains. The MPD-gen was developed in PHP5 with the MYSQL database. This tool stores and compares genetic information from the relatives of missing persons, unidentified cadavers and unidentified human remains. The genetic comparison is made with autosomal and Y-chromosomal STR markers. The system makes a pairwise comparison, calculates the likelihood ratio and probabilities for a relationship, including that of parent, child, sibling and second degree relative, using an algorithm based on the heterozygosity of the markers. The system performance was tested and accurately identified the genetic profiles of parents, children and siblings. This software program can be used as a national database as well as for identification in the event of a mass disaster.

Use of alternative samples in the restitution of missing persons descendants

2009-10-12

Abstract: The Banco Nacional de Datos Genéticos (BNDG) in Argentina was created by National Law No. 23.511. This law establishes our laboratory as the official expert in causes related to civil state suppression, during the military government between 1976 and 1983.Aim: To present the effort of the BNDG for the identification of missing persons’ descendants through the obtention of the genetic profile in alternative samples different from blood. The genetic data obtained was compared with the families’ genetic data stored in our institution.Thirty-seven alternative samples were remitted by Argentine Justice to the laboratory. DNA was isolated by phenol–chloroform method.STR: AmpFlSTR® Identifiler and AmpFLSTR® Yfiler kit (Applied Biosystems) were used. Electrophoresis of the amplification products was performed on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Data Collection, GeneScan® Analysis v3.1 and Genotyper® Analysis v2.5.2 softwares were used.Mitochondrial DNA: HV1 and HV2 fragments were amplified with: L-15997/H-16255; L-16209/H-16401 and L-00030/H-00412 primers. Sequencing was performed with BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems). PCR products were purified and then analyzed in an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems).Statistical analysis: Familias software was used.Toothbrushes resulted a good source for DNA recovery, as described in the international literature. So, we considered the toothbrushes as the alternative sample of preference to be analyzed for genetic identification. We recovered DNA from all the samples processed obtaining 100% efficiency. Nine persons were restituted to their biological families by using alternative samples.

Missing people: Problems of identification of unknown bodies using DNA database

2009-10-12

Abstract: Databases of unknown bodies and relatives of missing persons were created in the Republic of Belarus as a part of national DNA database. More than 350 unknown bodies which have been found in the republic since 1997 were exhumed in the last three years in order to study DNA and put results into the database.Several cases of erroneous matching were registered after direct bone's genotypes comparison to dataset of missing people relatives. Firstly, the database is organized with one mismatch considered as unexclusion. Secondly, sometimes only one relative (parent or child) is available. Thirdly, less than 15 loci genotype either from relative or bone sample was put into the database. Fourthly, 15 loci match between unrelated people is possible if you are operating with a big dataset. Additional examinations of Y-chromosome, mitochondrial DNA or samples of other relatives were performed that allowed to prove or exclude relationship and identify the unknown body.

Mitochondrial DNA analysis of human skeletal remains unearthed from Northern area of Kanagawa Prefecture, Japan

2009-10-12

Abstract: The mitochondrial DNA (mtDNA) hypervariable regions (HVRs) I and II have been analyzed to obtain the nucleotide variation data in eight skeletal remains unearthed from Northern area of Kanagawa Prefecture, followed by capillary electrophoresis using the ABI PRISM 310 genetic analyzer. The remains DNA were analyzed successfully, and several nucleotide substitutions and insertions were detected. The polymorphic profiles were essentially the same as those obtained from a modern Japanese living in Kanagawa.

A mini-primer set covering the mtDNA hypervariable regions for the genetic typing of old skeletal remains

P. Grignani, G. Peloso, P. Fattorini, C. Previderè — 2009-10-05

Abstract: A mini-primer set to amplify the mtDNA control region was developed and tested on fourteen long compact bone samples (femur and humerus samples) recovered from a World War II mass grave. This approach gave successful PCR amplifications for all but one of the bone samples. The following sequencing analysis identified seven different mtDNA haplotypes, three of which shared by more than one bone sample. These haplotypes were then compared to living relatives of missing persons disappeared in that area at the end of the WWII (1945).

Assessment of the effectiveness of human remains DNA typing: Analysis of 134 cases

A. Colussi, M. Viegas, J. Beltramo, M. Lojo — 2009-10-26

Abstract: Almost 10% of our casework involved DNA typing of human remains. In order to assess the efficiency of our protocol, results obtained in 134 cases were analyzed with regard to the success in DNA typing. In such cases an overall of 331 samples were processed, most of them corresponding to bones (68%), teeth (28%), and cartilage recovered from the bone joints (6%). After decalcification samples were subjected to DNA extraction with DNA Qiamp Minikit (Qiagen). DNA samples were quantified by real-time PCR and typed with AmpFl STR IdentiFiler PCR Amplification Kit (Applied Biosystems). AmpFl STR MiniFiler PCR Amplification Kit (Applied Biosystems) was used, to complete information in degraded samples.A complete DNA profile was achieved in 79% of the cases. The best performance was obtained with cartilage, from where 68% of the processed samples rendered a complete profile, whereas from the bone and teeth samples just almost 45% lead to a successful DNA typing.

Sampling of the cranium for mitochondrial DNA analysis of human skeletal remains

2009-10-12

Abstract: Sampling of cranial fragments for mitochondrial DNA (mtDNA) analysis is a common practice for identification of skeletonized human remains. The Armed Forces DNA Identification Laboratory (AFDIL) and Joint POW/MIA Accounting Command – Central Identification Laboratory (JPAC-CIL) work in concert to identify the remains of US service members killed in past military conflicts. When dealing with samples taken from remains that are commingled or lack a secure archaeological context, multiple elements must be sampled from the same burial or case. In such cases, the cranium is often fragmentary. Previous work examined the success rate of approximately 4000 skeletal elements for sequencing of mtDNA. The approximately 50% success of the crania seemed anomalous considering the frequency with which they are sampled. Subsequent studies of the 558 cranial fragments tested from 1992 to August 2009 were done to examine the independent rates of success of the portions of the skull. It was found that each yielded reportable mtDNA sequence at rates that exhibited statistically significant differences.

First identification of human remains using mtDNA sequence analysis in Genetic Laboratory of Royal Gendarmerie in Morocco

2009-10-06

Abstract: Mitochondrial DNA analyses are increasingly recognized as a viable option in the pursuit of DNA evidence in forensic cases for which nuclear analyses are unsuccessful or cannot be performed on the available evidence. The standard forensic mtDNA analysis at Genetic laboratory of Royal Gendarmerie (LGGR) in Morocco examines two hypervariable regions HVI and HVII in the mitochondrial DNA D-loop region, each of these regions is amplified in two pieces of approximately 250base pairs (bp). The paper presents the results of forensic mitochondrial DNA analyses which were aimed at typing bone samples up to 52 years old.

Population data for 12 STR loci in Northern European brown bear (Ursus arctos) and application of DNA profiles for forensic casework

2009-09-07

Abstract: The development of wildlife genetics combined with non-invasive sampling might be both an economic benefit for the society and a benefit for the survival of the threatened species. The aims of this study are to develop a quality assured approach for DNA profiling of brown bears (Ursus arctos) in Northern Europe using material from non-invasive sampling and to generate a population database that can be used for conservation management as well as a reference database for forensic purposes. Non-invasive sampling was performed by the collection of scats in the field and by using sets of hair traps in a grid pattern in specified geographical areas. Genotypes from 12 STR loci were determined for 232 Norwegian bears. Initial analysis of the entire sample indicated a high level of substructure. Thus, the sample was divided into four geographically different populations consisting of 206 individuals for further validation of the markers. Ten STRs (G1D, G10L, Mu05, Mu09, Mu10, Mu15, Mu23, Mu50, Mu51, and Mu59) conformed to Hardy–Weinberg equilibrium (HWE) expectations with only minor deviations, while the remaining two STR loci (Mu26 and G10B) were excluded from our set of putative forensic profiling system markers after revealing significant deviations from HWE in all four sub-populations. The average estimate of population substructure for Norwegian bears using 10 STRs (FST) was determined to be 0.1, while the estimate for inbreeding (FIS) was −0.02. Accounting for the FST-value, the average probability of identity (PIave) was 5.67×10−10 and the average probability of sibling identity (PIsib) was 1.68×10−4. In Norway, this brown bear DNA profiling system has been applied to forensic casework.

The use of mitochondrial DNA genes to identify closely related avian species

Sansook Boonseub, Shanan S. Tobe, Adrian M.T. Linacre — 2009-12-01

Abstract: Species identification using mitochondrial DNA (mtDNA) loci is a standard method for mammalian species testing. Less is understood about the conservation and variability in the avian mitochondrial genome, yet many exotic bird species are threatened with extinction and are traded illegally. In this study 80 different avian species were chosen from 22 different Orders and their gene sequences for the cytochrome b, cytochrome oxidase I and the ND2 genes were obtained from the NCBI web site. Alignments of the sequence determined the areas of greatest variation and conservation. The alignment result of DNA sequence showed that the cytochrome b gene placed the most number of avian species into their appropriate Orders, ND2 was next closest and COI the poorest of the three loci. These data support the use of cytochrome b over the other two mitochondrial loci for avian species identification.

Identifying NUMT contamination in mtDNA analyses

Ana Goios, Ana Carvalho, António Amorim — 2009-12-01

Abstract: NUMTs are insertions of mitochondrial DNA (mtDNA) sequences into the nuclear genome. They present different degrees of homology and may be present in various copies throughout the genome. Their presence has been identified firstly in the cat genome, and since then in many other species, namely in humans. When highly homologous to mtDNA and/or present in a high number of copies, they may be co-amplified with or instead of the desired mtDNA sequence, thus becoming a source of contamination in mtDNA analyses. This problem varies from species to species, and has been much discussed for human sequence analyses. Since pets have been more and more targeted in forensics, it is important to understand the extent to which NUMT sequences may interfere with the results of mtDNA analysis in these species.The domestic cat represents a particular case due to the high prevalence of NUMTs. We have performed amplification and sequencing of the cat mtDNA control region (n≈30) and of ND5 and ND6 genes (n≈70) using newly designed and previously described primers, respectively. We analysed the sequences with different softwares and constructed phylogenetic networks.By analysing and comparing phylogenies resultant from both sequenced regions we concluded that amplification with control region primers resulted in NUMT contamination. We here discuss how it is possible to distinguish the two kinds of sequences through a careful observation of electropherograms, alignments and phylogenetic networks and recommend critical analyses to be performed after obtaining the sequences in order to safely assign sequence origin.

Forensic DNA profiling of Cervus elaphus species in the United Kingdom

Eleni Socratous, Eleanor A.M. Graham, Guy N. Rutty — 2009-10-12

Abstract: Following the immense impact that human DNA profiling has had upon forensic investigations, many researchers and commercial organisations are now expanding the technology to allow for interrogation of additional species. In the United Kingdom illegal poaching of Cervus elaphus (red deer) species is threatening the welfare of this indigenous protected animal. This project has been designed to identify and test STR markers for the individualisation of red deer species residing in Grizedale Forest, Cumbria, UK, to aid the investigation of this crime. Muscle tissue samples have been collected from 156 red deer to provide a source of DNA for this project. Of a potential 57 STR markers identified during this project, 16 markers, displaying forensically important characteristics have been selected for further investigation. Eight of these loci have been characterised by direct sequencing multiplex PCR systems have been designed for their co-amplification.

Genetic typing of dogs’ traces in biological samples

2009-09-07

Abstract: Dog-bite related injuries and fatalities are increasing in incidence and represents an important public health concern, as dogs are intensely integrated in human social life. Forensic investigations in dog attack usually involve the examination of bite marks and toothprints. Generally, it was possible to obtain a canine-specific STR profile from the dog's saliva left on the wound area, even when high background of human DNA was present (blood). But dogs can also be victims of injuries. The authors describe two cases: a case of a Labrador Retriever dog, found quite lifeless in a dust-bin, and a case of a child slaughtered by dogs in the garden adjacent to his house.

Belgian dog mitochondrial DNA database for forensics

Stijn Desmyter, Sylvie Comblez — 2009-11-02

Abstract: A Belgian population sample of 117 unrelated breed dogs and mongrels, was mitochondrial DNA profiled by sequencing the two hypervariable regions HV1 and HV2. The complete control region of each dog was amplified in a single PCR. The combination of eight sequencing reactions compiles the mtDNA profile, of which each position was covered at least twice. 48 haplotypes were identified, of which 32 were unique in the studied population sample. The most frequent profile was observed in 18% of the analysed dogs. The occurrence of the profiles in the Belgian population sample is similar to those observed in other at random sampled populations of unrelated dogs. The exclusion capacity is 0.93, which is lower than for the human mtDNA control region, but is still informative in forensics.

Genetic diversity analysis of 10 STR's loci used for forensic identification in canine hair samples

2009-09-22

Abstract: Pets live with people and place biological samples everywhere, which may be useful in a forensic context linking suspects and victims, to an occurrence.The genetic diversity of canine microsatellite loci PEZ1, FHC2054, FHC2010, PEZ5, PEZ20, PEZ12, PEZ3, PEZ6, PEZ8 and FHC2079 was investigated in a population of 63 Portuguese dogs.Preliminary results show that the studied markers appeared to be polymorphic, thus contributing to increase the potential of forensic samples of animal origin. The number of alleles per locus, varied between five (FHC2010) to 20 (PEZ3) and allelic frequencies were estimated.

Advances in the DNA analysis of canine trace evidence for serious crime investigation in the UK

Rob Ogden, Elizabeth Heap, Ross McEwing — 2009-10-19

Abstract: Non-human evidence is becoming widespread in serious crime investigations. Canine hair samples have proved especially useful due to the high rate of transfer that naturally occurs between dogs, people and property. A murder investigation in the UK during 2008 provided the opportunity to undertake further research into the application and limitations of non-human hairs for linking victim to suspect.The overall aim of the work was to maximize the genetic data that could be obtained from shed hairs. This was attempted by conducting four short experiments to examine: (i) the recovery of exogenous human DNA from the surface of hairs, (ii) the potential for canine DNA originating from saliva to be obtained from hair shafts, (iii) the presence of residual DNA on tape lifts following hair removal and (iv) the potential for enhanced profile recovery following whole genome amplification of canine DNA.Results showed that DNA could be recovered from the surface of hairs following simulated licking and skin contact. When applied to casework samples, additional human DNA evidence was recovered from dog hairs in this manner. Attempted DNA recovery from tape lifts was unsuccessful. Whole genome amplification improved the recovery of full and partial profiles under reducing DNA concentration.The work demonstrates several novel approaches for recovering trace genetic evidence from animal hairs. Although such detailed analysis is unlikely to be applied to routine casework investigations, it increases the range of forensic tools available to investigate serious crimes.

Gene expression analysis as a tool for age estimation of blowfly pupae

Richard Zehner, Jens Amendt, Petra Boehme — 2009-09-14

Abstract: Forensic entomology, the use of insects in medicolegal investigations, mainly focus on the estimation of the postmortem interval (PMI) calculating the age of necrophagous specimens like blowflies, typically examined using maggot stages.While staging the age of the larvae is possible at a quite detailed scale, the age of the pupae is not easy to specify without rearing up to the adult stage so far, which is time-consuming or might be difficult. However, the pupal stage represents about 50% of the immature development time and therefore pupal age may serve as an important tool in entomological PMI estimation.Using a specific ddRT-PCR protocol we were able to monitor a differential gene expression in Calliphora vicina pupae at different ages by development dependent banding patterns on an agarose gel. Transcripts of interest were isolated from the gel and their nucleotide sequence determined. Primer/probe sets for quantitative RT-PCR using an ABI 7300 real-time instrument were designed and applied on cDNA from pupae of different development stages.Quantitation of these transcripts leads to age specific expression patterns. So far they have been worked out for pupae at the beginning, the middle and the end of their metamorphosis.

FishPopTrace—Developing SNP-based population genetic assignment methods to investigate illegal fishing

Jann Th. Martinsohn, Rob Ogden, FishPopTrace Consortium — 2009-10-12

Abstract: The FAO estimates that 80% of marine fish stocks are fully or overexploited worldwide. Illegal unreported and unregulated (IUU) fishing contributes vastly to this condition, and poses a severe threat to marine ecosystems. Controlling for compliance and enforcing fishing regulations is hampered by difficulties in identifying the geographic origin of fish and fish products, at point of landing and further down the food supply chain. While forensic genetic species identification methods are routinely employed to investigate commercial fraud, there are at present no validated methods for identifying the geographic origin of marine fish.FishPopTrace is an international project, funded by the EU framework programme (FP7), aiming to generate forensically validated panels of SNP markers for geographic origin assignment in four commercially important fish species, cod (Gadus morhua), hake (Merluccius merluccius), herring (Clupea harengus) and common sole (Solea solea). 454-sequencing with sample tagging is employed to generate large numbers of population informative candidate SNP loci in each species. Selected SNPs are subsequently genotyped using Illumina 1536-arrays across populations to provide high resolution maps of genetic variation. Panels comprising subsets of these markers will ultimately be validated for traceability and enforcement applications.

Microbial forensics: Do Aspergillus fumigatus strains present local or regional differentiation?

Ricardo Araujo, António Amorim, Leonor Gusmão — 2009-10-05

Abstract: Microbial community profiling is an important issue for microbial forensics. Some works support a large-scale dispersion of microbes and weak or absent biogeography, while others report the existence of endemic strains. Fungi have recently been used for soil discriminatory and definition of specific ecosystems. The advent of large-scale genotyping studies on fungal populations may provide a unique opportunity to compare genetic diversity within and among populations.In this work, we studied the pathogenic mould Aspergillus fumigatus that is frequently associated to several human disorders. A set of clinical and environmental isolates of A. fumigatus from Hospital S. João (Porto, Portugal) was tested, in addition to another group from other Portuguese and American Hospitals. A. fumigatus isolates were genotyped using a microsatellite-based single-multiplex PCR with eight short tandem repeat markers and a single nucleotide polymorphism (SNP).This SNP could split A. fumigatus population in two groups: 357 isolates with an additional nucleotide A and 55 isolates without this base insertion. The smallest group, comprising 27 genotypes, contained exclusively strains from Hospital S. João. The occurrence of microvariation events (strains differing in a single marker) was very common among environmental isolates. A larger study including more strains from diverse locations may improve the categorisation of local/regional strains. Additionally, the inclusion of more SNPs for A. fumigatus genotyping will improve the characterisation of A. fumigatus population.

Whole-genome typing of Bacillus anthracis isolates by next-generation sequencing accurately and rapidly identifies strain-specific diagnostic polymorphisms

2009-12-01

Abstract: In the event of bioterrorism, infectious disease outbreaks in the food supply chain, or hospital acquired infections, rapid, high-resolution genetic characterization is critical for determining the identity of the agent and attributing it to a specific source. Four Bacillus anthracis strains were sequenced to demonstrate the utility of next-generation sequencing technology, specifically the SOLiD™ system (Applied Biosystems™, Foster City, CA), for microbial forensics investigations. Reads from the B. anthracis Ames ancestor strain detected only a single false positive SNP. Of the 148 SNPs that distinguish the Ames and Sterne strains, 126 (85%) were identified with 19 of the 22 uncalled SNPs in two dense clusters that precluded mapping of reads. Three previously unsequenced, geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames ancestor reference genome. The high throughput, multiplexing capability, and accuracy of the SOLiD™ system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation.

Simultaneous identification of multiple mammalian species from mixed forensic samples based on mtDNA control region length polymorphism

Luca Fumagalli, Catherine Jan Cabrita, Vincent Castella — 2009-09-02

Abstract: Molecular species identification in mixed or contaminated biological material has always been problematic. We developed a simple and accurate method for mammal DNA identification in mixtures, based on interspecific mitochondrial DNA control region length polymorphism. Contrary to other published methods dealing with species mixtures, our protocol requires a single universal primer pair and amplification step, and is not based on a pre-defined panel of species. This protocol has been routinely employed by our laboratory for species identification in dozens of human and animal forensic caseworks. Six representative forensic caseworks involving the specific identification of mixed animal samples are reported in this paper, in order to demonstrate the applicability and usefulness of the method.

Identifying endangered species from degraded mixtures at low levels

Shanan S. Tobe, Adrian Linacre — 2009-09-23

Abstract: Although endangered species are afforded protection under national and international laws, trade in products containing endangered species is still one of the most lucrative criminal enterprises in the world. Forensic science is employed to determine if endangered species are present in commercial products but runs into problems due to the extremely low levels of DNA and degraded nature of the majority of samples. Therefore a section of the mitochondrial genome is generally amplified and sequenced to determine if endangered species are present. This technique is sensitive and accurate, but cannot identify components of a DNA mixture due to the use of universal primers and can fail with subcellular levels of DNA. A multiplex PCR was therefore developed, based on the cytochrome b and 12S rRNA genes, to identify the presence of endangered mammalian species, if present, in commercial products. Four species [tiger (Panthera tigris ssp.), leopard (Panthera pardus), musk deer (Moschus sp.) and Asiatic black bear (Ursus thibetanus ssp.)] in addition to several common non-protected mammalian species can be identified using species-specific primers. A specific PCR product is produced depending on which species is/are present. The test is accurate and sensitive to low levels of DNA. It has been tested using traditional East Asian Medicine claiming to contain one or more endangered mammalian species. The test can be performed using standard DNA analysis techniques and implemented by any laboratory with these facilities.

Cytochrome b or cytochrome c oxidase subunit I for mammalian species identification—An answer to the debate

Shanan S. Tobe, Andrew Kitchener, Adrian Linacre — 2009-09-23

Abstract: Species identification for forensic purposes is being increasingly used, as the value of non-human evidence is realized. This requires the identification of the species before individual analysis can take place. Traditionally the cytochrome b (cyt b) gene was used for species identification, but in 2003 the cytochrome c oxidase subunit 1 (CO1) gene was introduced under the terminology ‘barcoding’. This started an ongoing debate as to which gene offers the best template for species identification (high inter-species variability and low intra-species variation). Sequence data from 236 mammals were compared with multiple sequence alignments for a large number of human, cow and dog samples. Comparisons were made based on the number of inter-species variations between the different species and the intra-species variation between members of the same species.

Genus identification of toxic plant by DNA

S. Matsuyama, M. Taniguchi, J. Tsukioka, K. Goto, K. Nishi — 2009-10-12

Abstract: In this study we designed specific primer pairs for identification of toxic plants such as subgenus Aconitum, genus Ricinus and genus Illicium in internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA. Allied species of target plants and foods were not detected, but each primer pairs give a PCR product in the presence of target plant.This PCR method is useful for identification of toxic plants and can detect subgenus Aconitum, genus Illicium and genus Ricinus with template DNA of 1ng, 100pg and 10pg, respectively.

Tiger species identification based on molecular approach

Thitika Kitpipit, Adrian Linacre, Shanan S. Tobe — 2009-12-01

Abstract: The trade in samples of tiger (Panthera tigris), or parts derived from tiger, is controlled through the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which lists all subspecies as protected at the highest level. Tiger has been used as a component in traditional medicines for centuries, often as powder thus making its presence difficult to identify. It is therefore necessary to use a molecular approach for the unambiguous identification of the species. Some countries require knowledge of the exact subspecies present in order to prosecute anyone alleged to trade in tiger products. To this end, mitochondrial DNA from two individuals of four of the five subspecies of tiger were sequenced to determine if subspecies-specific variation could be identified that could be the basis for a molecular test. We report on the determination of a total of 7891bp of the tiger mitochondrial genome spanning 16S rRNA through ND4.

Mitochondrial analysis revealed high homogeneity in the Waorani population—The last nomadic group of hunter-gatherers from Ecuador

2009-09-18

Abstract: It reports the preliminary analysis of the mitochondrial DNA of the last nomadic ethnic group of hunter-gatherers from Ecuador: the Waorani. The control region (HVI and HVII) of 111 Waorani individuals from two Amazonian communities was studied. It founds only two different haplotypes, which can be included in two (A and D) of the four major Native Americans mtDNA haplogroups (A–D). The haplogroup A2 was the most prevalent in both communities. The high homogeneity observed in the Waorani population might mirror the geographical and social isolation that have distinguished this population. Further studies will be necessary to deepen in the anthropological meaning of these results.

The impact of jumping alignments on mtDNA population analysis and database searching

Bobi K. Den Hartog, John W. Elling, Bruce Budowle — 2009-10-19

Abstract: Describing human mitochondrial DNA sequences by listing only those sites that differ from an aligned reference sequence is the standard practice for nomenclature. However this different-from-reference description can produce artificial alignments when comparing two non-reference sequences which in some situations may exaggerate the difference between the non-reference sequences—a problem called “jumping alignments.” The impact of jumping alignments in database searching and population studies is evaluated. Alternative phylogenetic approaches for sequence alignment are also compared. The data show that a small percentage of jumping alignments occur with a standardized nomenclature system and with a phylogenetic approach, with a nominal impact on false exclusion database searching errors.

Clustering for forensic mitotype quality analysis

Bobi K. Den Hartog, John W. Elling — 2009-11-02

Abstract: Errors in mitochondrial DNA (mtDNA) sequencing can result in unusual patterns of polymorphisms which can be detected by the lack of similarity to the sequences in a database. In this work, an approach to routine data quality review is described that employs cluster analysis to identify conserved sequence similarities. Comparison of new sequences to the clusters identifies unusual polymorphisms and sequence regions that may warrant further attention by the analyst. This approach is accessible, easily automatable and can be tailored specifically to targeted population groups in individual investigations.

HVIII discrimination power to distinguish HVI and HVII common sequences

C. Fridman, R.S. Gonzalez — 2009-09-07

Abstract: The mtDNA sequence analysis has been found important applications in the human identification field, and important properties as the exclusively maternal inheritance, the absence of recombination and high rate of mutation provide a high index of variability, mainly in hypervariable regions HVI and HVII. Although highly polymorphic, the presence of many highly common polymorphisms, resulting in sequences (haplotypes) common to more than one individual, is a limitation to the use of these regions in some cases. The use of additional mtDNA markers to the classic sequencing of HVI/HVII can increase the discrimination power of common haplotypes and, therefore, of individuals. Herein, we analyzed polymorphisms in mtDNA HVIII region in 19 pairs of Brazilian mother/child who could not previously be individualized and matched through the analysis of HVI and HVII, in order to try to make these matches, evaluating the discrimination power of HVIII. After HVIII analysis we were able to differentiate 4 pairs out of 19 (21%). Two mothers and two children presented the haplotype 16182C 16183C 16189 16217 16249 16312 16344 73 152 263 271 309.1C 315.1C, and could be distinguished in two pairs with HVIII sequences 460C 499A and 499A, respectively. Other 2 pairs showed rCRS 263 309.C 315.1C in HVI/HVII and could be matched later since one pair presented HVIII 477C polymorphism and the other showed 523D 524D sequence. In conclusion, about one-fifth of the cases could be distinguished by HVIII polymorphisms, being a useful additional analysis in some cases.

MtDNA SNP analysis in a Central Portuguese population

2009-12-01

Abstract: Mitochondrial DNA (mtDNA) has a great potential in forensic and population genetics, allowing the identification of population origin of individuals and related trace samples. In order to apply this methodology as inclusion evidence in forensic practice it is necessary to determine the population genetic structure. Here, we present haplogroup discrimination of a Central Portuguese population using 16 mtSNPs. Haplogroups in the Central Portuguese population were determined with the following distribution: Hg H: 40.19%, Hg V: 4.90%; Hg HV: 4.90%, Hg U: 13.72%, Hg K: 5.88%; Hg T: 10.78%; Hg J: 9.80%, Hg I: 1.96%, Hg X: 2.94% and others: 4.90%, being this distribution in accordance with other European populations.

Mitochondrial DNA control region database in Banco Nacional de Datos Genéticos, Argentina

2009-10-05

Abstract: To analyze the distribution of different haplotypes of mitochondrial control region DNA in a large sample of argentine population who assisted to the Banco Nacional de Datos Genéticos (BNDG) looking for their identity in cases of civil state suppression during the dictatorial government (1976–1983).A total of 1168 unrelated argentine individuals who assisted to the BNDG were sampled for the analysis.Mitochondrial DNA Control Region sequences were determined for both hypervariable regions 1 (HV1) and 2 (HV2).Blood samples were used as the DNA source.The amplification for both hypervariable regions was performed in a Perkin Elmer 9700 thermal cycler.The cycle sequencing was performed using the BigDye® Terminator v 1.1 Cycle Sequencing Kit (Applied Biosystems) and automated sequencing was performed in an ABIPRISM® 3100 Genetic Analyzer (Applied Biosystems).Analysis of mitochondrial DNA sequencing data was performed by manual method.The hypervariable region 1 (HV1) was analyzed between positions 16023 and 16428 and the hypervariable region 2 (HV2) between 50 and 480 according to the Anderson Sequence.To analyze these results a database was created and statistical parameters were calculated.From 1168 haplotypes analyzed, 794 were unique. Haplotype frequencies were estimated by haplotype counting. Values of Genetic Identity (P) and Genetic Diversity (h) were calculated, being 3.3939×10−3 and 0.9974 respectively.This Mitochondrial Control Region DNA database provides useful information for genetic profiles comparison and maternal lineage determination in our lab.

Preliminary results of mitochondrial DNA sequence variation in Spanish Pyrenean populations

2009-09-22

Abstract: Mitochondrial DNA sequences of the two hypervariable regions HVS-I and HVS-II were determined for 233 unrelated autochthonous individuals from East, Central and West Pyrenees. Resulting haplogroups were confirmed with RFLPs. Although the distribution of mitochondrial DNA haplogroups in the Pyrenees presents differences between populations, the results obtained may well support the hypothesis that the mountain chain did not act as a barrier to gene flow among Pyrenean populations. These preliminary data could be helpful for the understanding of the present distribution of mitochondrial DNA haplogroups in Iberian Peninsula and its phylogeny.

Common mitochondrial DNA haplogroups observed in an argentine population database sample

2009-10-08

Abstract: Mitochondrial DNA hypervariable regions I and II were sequenced from 403 unrelated Argentine individuals. The aim of this study was to create a population database as well as to identify the population diversity for this genetic marker by classifying it into haplogroups.The sequence polymorphisms of the HVI and HVII regions were determined by PCR and direct sequencing. The haplotypes found were checked by phylogenetic haplogroup analysis to decrease haplotype assignation errors and to avoid artificial recombination.We found 78 different haplogroups in this set of samples. A high percentage of haplotypes (53%) belong to European haplogroups due to the large flow of European immigrants from colonial times. However, we also observed a high percentage of haplotypes that belong to Amerindian haplogroups (39%), which were conserved through the female Amerindian population contribution. Furthermore, we found a small group of haplotypes with Sub-Saharan African origin (3.5%) due to the slave trade at the beginning of Argentina's colonization.The sequences found showed that this set of samples has an abundant haplogroup diversity because of the European and Amerindian ethnic group contribution.

Mitochondrial DNA control region sequence analysis of Mataco–Guaicurú speaking tribes from Argentina

2009-09-27

Abstract: In this work we analyze three Mataco–Guaicurú speaking Amerindian tribes that inhabit the Northern Argentinean portion of Gran Chaco region. One hundred and sixty-eight samples of unrelated males belonging to two linguistic families: Guaycurú (Toba from Chaco, n=27; Toba from Formosa, n=37 and Pilaga from Formosa, n=56) and Mataco (Wichi from Formosa, n=48) were investigated. The entire Control Region of the mtDNA from position 16024 to 576 was sequenced. EMPOP sequencing strategy was employed including the use of 10 primers for each sample. Specific haplotypes were found in these populations with very high frequency. These findings could provide clues to address the ethnicity of a sample in routine forensic casework.

Haplotype diversity in mitochondrial DNA hypervariable regions I and II in Maracaibo population (Venezuela)

2009-10-12

Abstract: The control region of the human mitochondrial DNA (mtDNA) is highly polymorphic due to a rapid rate of evolution. The hypervariable segments HVI and HVII of the mitochondrial control region (D-loop) are the most variable part of human mitochondrial DNA. Blood samples from 50 unrelated individuals born from Maracaibo City (Venezuela) were obtained from routine paternity. Polymerase chain reaction amplification products were purified and fluorescent-based capillary electrophoresis sequencing method, the sequences were analysed and it is compared DNA sequences with the rCRS. The dominant haplogroups corresponded to Amerindians followed by African. The nucleotide positions most common for HVII was 263G (100%), 315.1C (100%), 73G (94%), and 309.1C (66%) and for HVI 16223T (74%), 16362C (56%), 16319A (40%) and 16111T (34%).

Variability of mitochondrial DNA HVS-III segment in a human isolate from the Pas Valley (northern Spain)

2009-10-05

Abstract: In this study, we analyzed the HVS-III region of the mitochondrial DNA in 61 maternally unrelated individuals from the Pas Valley (Cantabria), a human isolate from northern Spain whose HVS-I and HVS-II segments were also analyzed. Our results demonstrated that even the analysis of the three hypervariable segments of the mtDNA control region might constitute a limited approach for forensic purposes in human isolates. Nevertheless, polymorphic positions of HVS-III may be useful to confirm and refine the haplogroup assignment.

Segments HVS-I and HVS-II of mitochondrial DNA in a population from Santa Catarina (Brazil): Predominance of European lineages

2009-10-05

Abstract: The study of the hypervariable segments HVS-I and HVS-II of the mitochondrial DNA (mtDNA) control region of 80 healthy and maternally unrelated individuals revealed that the population from Santa Catarina is extremely heterogeneous, mainly due to the impact of relatively recent migratory waves from Europe. In spite of this, Native American lineages as well as African lineages incorporated much earlier are also present at noticeable proportions. This strikingly high variability generated by the intense gene flow is mirrored in a high power of discrimination (97.69%), which makes the analysis of mitochondrial HVS-I and HVS-II segments very useful for forensic genetic purposes in this Brazilian population.

Mitochondrial DNA control region in native population from the province of Jujuy (northwestern Argentina)

2009-10-05

Abstract: The province of Jujuy is located in northwestern Argentina. We analyzed mitochondrial DNA (mtDNA) haplogroup composition in 100 autochthonous individuals living at altitudes between 1200 and 3500m above sea level, in San Salvador de Jujuy, Quebrada de Humahuaca and Puna. The purpose was to explore the influence of non-Native American maternal lineages. Additionally, we evaluated the efficacy of analyzing the entire mtDNA control region to increase its power of discrimination with forensic purposes. Results showed that the population sample was entirely composed by Native American haplogroups, with haplotypes belonging to haplogroup B as the most common lineages. Analysis of the entire mtDNA D-loop region proved to be useful to increase the power of discrimination provided by the analysis of HVS-I and HVS-II segments and to refine haplogroup assignment.

The genetic composition of Argentina prior to the massive immigration era: Insights from matrilineages of extant criollos in central-western Argentina

2009-10-12

Abstract: Massive transatlantic immigration starting in 1860 significantly modified the human genetic landscape of Argentina. In an attempt to analyze the genetic composition of the country previous to this radical change, biological samples and genealogical info were obtained from individuals in La Rioja and San Juan cities in central-western Argentina. MtDNA control region sequences were obtained from individuals of self-reported criollo maternal ancestry, assigned to the (sub)haplogroups they belong to, and assigned a major continental origin. A high proportion of maternal lineages of Native American ancestry (>86%) was found in both populations, as well as similar inputs stemming from West Eurasia and sub-Saharan Africa. In sharp contrast, significant differences in the contribution of Native American (sub)haplogroups were observed. We propose that our results reflect both the differential distribution of Native American populations that contributed to the present-day criollo mtDNA gene pool and a preferential input of immigrants of Chilean origin to San Juan.

Swedish population data on the SNPforID consortium autosomal SNP-multiplex

Kerstin Montelius, Andreas O. Tillmar, Jonny Kumlin, Bertil Lindblom — 2009-10-16

Abstract: To meet the demands to solve more complex cases, we are in need of more markers than the short tandem repeats (STR-markers) at present available in the forensic community. As a complement, the single nucleotide polymorphisms (SNP's) may play an important role. A basis for genetic calculations is a solid frequency database. The aim of this work has been to build a database with Swedish frequencies appropriate for 49 of the 52 SNP-multiplex developed by the SNPforID consortium.A set of 78 samples from unrelated individuals with Swedish names was amplified with SNP-primers and run in a capillary instrument as described by the consortium. All data were checked for Hardy–Weinberg equilibrium and linkage disequilibrium. Significant p-values could not be found. An additional aim was to get an idea of the mutation frequency of the SNP's. 236 meiosis were tested, which correspond to 11,564 possible mutation spots. Mutations were not found in the material.

The polymorphisms of 9 SNP loci on mitochondrial DNA in the Chinese Han population

Xiaoming Sun, Yi Ye, Yingbi Li, Jin Wu, Yiping Hou — 2009-09-24

Abstract: The single nucleotide polymorphisms on mitochondrial DNA (mtDNA) were potential markers for analysis of difficult samples in casework and played a role on forensic science. In order to increase the useful loci which could be used in forensic casework, we investigated the frequencies of 9 SNP loci on mitochondrial DNA in the Chinese Han population in Chengdu through the allele-specific primer extension, and got the haplogroups with these loci. Our results showed there were 32 haplogroups totally among 205 non-relational persons in our population sample. The results implied that the analysis of the mtDNA-SNP loci was proved to be suitable for forensic application and provided new genetic markers for the forensic purpose.

The Africa male lineages of Bahia's people—Northeast Brazil: A preliminary SNPs study

2009-09-07

Abstract: The non-recombining portion of the human Y (NRY) chromosome has various types of variation, including single nucleotide polymorphism (SNP) variation. The study of this specific region can be helpful in forensics, since Y haplogroups show regional specificity, providing useful information about geographic origin of an individual or evidence under investigation. In this study, single nucleotide polymorphisms (SNPs) located on the Y chromosome specific region (M96, M2, M35, M78, M81, M123, M34, M201, M170, M304, M26 and M172) were used to characterize a population sample of 100 males from Bahia, in order to investigate the frequency distribution of the male lineages. These SNPs were selected from the set of 24 SNPs that determine the 11 frequent haplogroups present in Brazilian population. Genotyping was made using SNaPshot kit (Applied Biosystems) in two multiplexes: one with five Y-SNPs (M304, M201, M170, M26 and M172) and another with seven Y-SNPs (M34, M35, M2, M78, M96, M123 and M81). The results validation was done using the software GeneMapper v3.2 (Applied Biosystems). The Y chromosomes not characterized by the SNP set tested (48.0%) were assigned ABCDF(xG,I,J) and will be analyzed with additional markers set. The most frequent haplogroup identified was E1b1a*-M2 (19.0%), followed by J2*-M172 (7.0%), E1b1b1a*-M78 (5.00%), G*-M201 (5.00%), I*(xI2a2)-M170 (4.0%), J*(XJ2)-M304 (4.00%), E1b1b1c1-M34 (3.0%), I2a2*-M26 (3.00%), E*(xE1b1a-b)-M96 (1.0%) and E1b1b1b*-M81 (1.0%). The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.

Population data of 52 autosomal SNPs in Italian population

A. Barbaro, C. Phillips, M. Fondevila, Á. Carracedo, M.V. Lareu — 2009-10-05

Abstract: SNPs show a range of characteristics that make them well suited to forensic analysis including a low mutation rate, much reduced amplicon sizes and relatively simple multiplex assays that use established capillary analyzers. In the present study we characterized variation within Italy, studying the geographically separated Italian populations: Sicily, Calabria (South), Lazio, Toscana (Centre), and Veneto (North) using a validated 52plex SNaPshot. Statistical analysis of SNP genotypes indicated no significant differences in allele frequencies distributions between the populations studied.

Database of the polymorphic genetic markers D19S433 and D2S1338 from the population of Buenos Aires Province, Argentina

Celia Iudica, Stella Maris Jaureguiberry, María Laura Parolin, Lorena Sambuco — 2009-10-12

Abstract: Short tandem repeat (STR) loci are the most informative DNA genetic markers for attempting to individualize biological material for application at paternity and forensic cases. Before a new marker system can be introduced into forensic casework, a database for the relevant population must be established for statistical evaluation of the evidence. Therefore, this report presents allele frequency data and parameters of biological or legal interest, such as heterozygosity value and power of exclusion in Buenos Aires Province (Argentina) population sample for the loci D19S433 and D2S1338. Blood samples (N=473) were collected form individuals unrelated throughout Buenos Aires Province on FTA cards. Following DNA extractions, multiplex PCR amplifications were carried out using AmpFlSTR Identifiler PCR Amplification kit (Applied Biosystems) and amplified products analyzed on an Applied Biosystems DNA sequencer (Model 3130). Heterozygosity value is estimated as 0.81184 for D19S433, and 0.85201 for D2S1338. Both STR loci are in Hardy–Weinberg equilibrium. For D19S433, power of exclusion and power of discrimination are estimated as 0.621 and 0.937, respectively. For D2S1338 the same parameters are 0.699 and 0.968. The allele frequency data can be used for deriving estimates of multiple locus profile frequencies for identity testing purposes.

A highly polymorphic STR-locus within the MHC-region close to HLA-DR/DQ: Austrian population data of DQIV (alias M2_4_32)

E.M. Dauber, E.M. Schwartz-Jungl, S. Wenda, G. Dorner, B. Glock, W.R. Mayr — 2009-09-18

Abstract: The tetranucleotide repeat locus DQIV (UniSTS:239151) is located within the HLA region (6p21.3), close to HLA-DRB1 and HLA-DRQ1. STR-loci within the MHC region can add useful information in stem cell transplantation and are used for a better characterization of HLA-haplotypes. The study showed in an Austrian population sample (n=292), that DQIV is approximately as informative as other commonly applied loci in the forensic field.

Population genetic data for F13A01, FES/FPS, F13B and LPL in Colombia (Department of Santander)

2009-09-18

Abstract: Allele frequencies for 4 STR autosomal loci (F13A01, FES/FPS, F13B and LPL) were obtained from a sample of 395 unrelated individuals from the Department of Santander (Northeast of Colombia).

Genetic analysis of 9 non-CODIS miniSTR loci in the Brazilian population of Parana

Marcelo Malaghini, Vicente Schneider, Fabio Leite — 2009-10-12

Abstract: DNA typing was performed on 155 unrelated volunteers from Parana (South Brazil) using 9 non-CODIS mini-short tandem repeat (miniSTR) markers (D10S1248, D14S1434, D22S1045, D1S1677, D2S441, D4S2364, D20S482, D3S3053 and D6S474), comprising three different multiplex systems. The allele frequencies, heterozygosity, power of discrimination, mean exclusion chance, polymorphism information content and typical paternity index of all loci were calculated by statistical analysis. The combined matching probability and the combined mean exclusion chance was 1 in 9,09×107 and 0.995856, respectively. The typical paternity index varied from 1077 (D4S2364) to 2588 (D3S3053). No evidence of deviation from Hardy–Weinberg equilibrium was observed for any loci, except for D10S1248 (p value=0.00101). There was also no evidence for correlation of alleles between loci. Five of the six loci showed good levels of polymorphisms, with heterozygosities greater than 0.65. This study demonstrates that these multiplex systems are useful and convenient tools for forensic identification and parentage testing in the Brazilian population of Parana.

pop.STR—An online population frequency browser for established and new forensic STRs

2009-10-12

Abstract: We recently produced allele frequency data for 20 forensic STRs in more than 50 worldwide populations. The STRs characterized include 5 new European Standard Set (ESS) STRs where novel low frequency and intermediate-repeat genotypes found were confirmed by sequence analysis. Data for the 20 STRs has been collated into an open-access online frequency browser at: http://spsmart.cesga.es/popstr.php that allows users to combine populations into groups to generate re-calculated allele frequency estimates from the merged genotype data. The flexibility to combine populations in this way and the graphical summaries provided for each marker's allele frequencies offers the forensic analyst an informative system to consult STR variability in a global range of populations.

Population data for 15 STRs loci in an immigrant population sample living in Northern Italy

N. Cerri, A. Verzeletti, V. Cortellini, F. De Ferrari — 2009-09-14

Abstract: Allelic frequencies and statistical parameters of forensic interest for the 15 STRs loci of the PowerPlex® 16 System were estimated in a population sample of 292 unrelated individuals from non-EU countries: 62 from Pakistan, 90 from Albania and 140 from Maghreb (Morocco, Tunisia and Egypt).

Allele frequencies of six miniSTR loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441, D1S1677) in two Italian populations

2009-09-23

Abstract: MiniSTR loci have demonstrated to be an effective tool to recover genetic information from degraded sample due to the small PCR products. The use of these non-CODIS miniSTRs can increase the probability that a degraded sample can be typed and in addition these systems can be used in complex paternity cases where more markers are needed.Six autosomal loci (D10S1248, D14S1434, D22S1045, D4S2364, D2S441, D1S1677) were investigated in 200 individuals from Italian regions, Umbria and Sardinia.Statistical analysis and comparison between two groups were performed.

Allele frequencies of 15 STRs loci in an Argentine population sample

2009-12-01

Abstract: It is well known that the use of STR (Short Tandem Repeats) for forensic purposes requires the existence of databases that reflect the allele distribution in the population they will be applied. During the last years we have received a greater affluence of samples from different regions of Argentina, instead of receiving almost only from Buenos Aires city, as we did six years ago.Because of this change in the population we are working with, our aim is to study the distribution of allele frequencies for 15 STR loci and calculate the statistical parameters of forensic interest, in a sample provenient from different regions of Argentina.A total of 375 unrelated individuals from different provinces in Argentina, were included for the analysis. DNA was obtained from blood samples with salting out Miller's method, and AmpFlSTR® Identifiler was applied. Capillary electrophoresis of the amplification products were performed on an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). The 3100 Data Collection Software–v1.0.1 (Applied Biosystems) and GeneScan–v3.7 (Applied Biosystems) and Genotyper Software-v3.7 (Applied Biosystems) were used. Statistical analysis for allele frequencies, power of discrimination and power of exclusion were calculated using PowerStats software. Deviation from Hardy–Weinberg equilibrium, observed and expected heterozygosity was calculated with Arlequin V3.1 software.All the loci studied were in Hardy–Weinberg equilibrium, and statistical parameters calculated were consistent with the bibliography. This database is a useful tool for routine forensic applications in our population.

The presence of tri-allelic TPOX genotypes in Dominican Population

Vilma Díaz, Patria Rivas, Angel Carracedo — 2009-10-16

Abstract: The presence of the tri-allelic TPOX genotypes represents a new finding about the Dominican Population origins. The STRs in our population have been characterized in our previous investigation, now we are beginning the process to confirm the true components of our ethnical structure. About 90% of the contemporary Dominican Population has African ancestry or has African roots, for this reason we strongly consider the nature of the tri-allelic TPOX genotypes as African origins. In this work, we report tri-allelic TPOX genotypes by routine relationship testing using PowerPlex 16 (Promega Corporation) kits.

Genetic profile of Federal District of Brazil based on 18 STR autosomal loci

2009-10-12

Abstract: Allelic frequencies for 18 DNA STR autosomal markers (D16S539, D7S820, D13S317, D5S818, CSF1PO, TPOX, TH01, vWA, F13A01, FESFPS, F13B, LPL, Penta E, D18S51, D21S11, D3S1358, FGA and D8S1179) were obtained from a sample of unrelated individuals from the Federal District, in the Center-West of Brazil.

Usefulness of a hundred of autosomal tetranucleotide STR markers for genetic analysis among geographically close human regional populations in East Asia

2009-10-21

Abstract: DNA samples collected from totally 16 regional populations from 6 countries in East/Southeast Asia were analyzed for 105 autosomal tetranucleotide STR markers. Both the genetic structural and 3D-plots analytical results were not inconsistent with their historical backgrounds and geographical distributions. Analysis for more than one hundreds STR loci would be potentially useful to genetically differ even between geographically very close populations such as Korean and Japanese.

Allele frequencies of three mini-STR loci (D22S1045, D14S1434 and D10S1248) in North-East Italy

Solange Sorçaburu Cigliero, Fabio Gerin, Carlo Previderè, Paolo Fattorini — 2009-09-27

Abstract: The analysis of mini-STR loci represents a powerful tool for genetic typing of degraded DNA samples. Since the forensic application of genetic markers requires reference databases, a population sample from North-East Italy was tested at the mini-STR loci D10S1248, D14S1434 and D22S1045. DNA was extracted from fresh blood samples collected from 100 healthy unrelated subjects living in that area. The genetic typing was performed amplifying each STR marker in a single PCR. The amplicons were separated through acrylamide gels. The alleles were scored by comparison with an allelic ladder created by mixing up sequenced alleles. Allele nomenclature was performed according to Coble and Butler . Allele frequencies and other forensic parameters (HE, PD, PIC, PE and PPI) are provided. No Hardy–Weinberg disequilibrium was found. Our results are in agreement with those previously found in other Italian population samples.

Use of non-CODIS miniSTR markers: Creation of a database in Argentina

2009-10-05

Abstract: The use of miniSTR markers is a valuable tool in the forensic laboratory since it allows high-quality genetic profiles to be obtained on low copy number (LCN) or degraded DNA taken from different samples, such as cadaveric material, paraffin-embedded tissue, chewing gum, traces of saliva, pieces of cloth, cigarette butts, bone samples, etc.In addition, it is useful to have additional STR loci to solve paternity cases with one or more inconsistencies or to increase the amount of genetic information for incomplete family studies, where the genetic profile of the alleged father has to be reconstructed.Genetic frequencies and forensic parameters were calculated for 12 non-CODIS miniSTR loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, D17S1290, D10S1248, D14S1434, D22S1045, D4S2364, D2S441 and D1S1677) from a total of 506 unrelated individuals from various provinces in the central region of Argentina (Cordoba, Buenos Aires, Salta, Entre Rios and Santa Fe).The application of these miniSTR markers allowed LR>1000 values to be obtained in kinship cases with incomplete families whose results had not been significant when using commercial kits.In conclusion, a database with Argentine population frequencies from these 12 miniSTR markers may be very useful in adding supplementary information for the forensic genetic laboratory.

Update of an on-line autosomal STR and Y-STR reference database of Argentina

2009-10-05

Abstract: Allele frequency data of genetic markers employed in paternity testing and forensic casework is essential for the statistical analysis of the results obtained by these molecular markers.In 2004, our group developed and launched a free on-line reference database including autosomal STRs allele and Y-STR haplotype frequencies for 10 Argentinean provinces (http://www.ffyb.uba.ar/marcadores/antecedentes.asp). The dataset included 2710 individuals typed by means of 13 autosomal STRs and 239 individuals with the minimal Y-STRs haplotype (YHRD).In order to further improve the available information on-line, the database was increased for both autosomal and Y-STR markers. A total of 6501 genotypes from unrelated donors were added to the previous database. These samples were obtained from Buenos Aires, Chubut, Rio Negro, Santa Fe, Mendoza, Misiones, Corrientes, Chaco, Formosa and Salta, and typed at least with 13 autosomal STRs and 1336 unrelated males were also typed for Y-STRs. In parallel, protocols for DNA analysis and reference bibliography were updated.The present update attained a total of 9211 genotypes and 1575 Y-chromosome haplotypes from the Argentinean population. This contribution offers on-line statistical information about highly standardized genetic markers to the forensic and molecular anthropology research communities.

Population data about the distribution of 15 autosomal STRs and 17 Y-STRs in South of Italy (Calabria)

A. Barbaro, P. Cormaci, A. La Marca, S. Votano, A. Barbaro — 2009-09-23

Abstract: In the present study we investigated the distribution of 15 autosomal STRs loci and 17 Y-STRs loci in a population from Southern Italy (Calabria). Samples for the study were obtained form more than 300 unrelated healthy individuals belonging to the analysed population since at least three generations.Different bio-statistical values of forensic interest were calculated for the loci examined in the present study. The test for Hardy–Weinberg equilibrium showed that the genotype distribution was correspondent with the expected.

Population data of 5 next generation STRs in Southern Italy

A. Barbaro, C. Phillips, L. Fernandez Formoso, Á. Carracedo, M.V. Lareu — 2009-10-08

Abstract: MiniSTRs analysis has been demonstrated useful to increase the success rate of degraded samples typing. In the present study we investigated the distribution of D10S1248, D12S391, D1S1656, D22S1045, and D2S441 in a population from Southern Italy (Calabria).Saliva/blood samples were obtained from around 150 unrelated healthy individuals belonging to tested population since at least 3 generations.Statistical analysis was performed and results obtained showed that all loci met Hardy–Weinberg expectations.

Forensic evaluation of 15 STR loci in Venezuelan Military Aircrew

2009-10-09

Abstract: Allele frequencies of fifteen autosomal short tandem repeat (STR) loci (CSF1P0, D7S820, D8S1179, D21S11, D2S1338, D3S1358, D13S317, D16S539, TH01, D18S51, D19S433, TPOX, vWA, D5S818 and FGA, included in the AmpFLSTR Identifiler, Applied Biosystems) were analyzed in a sample of 245 unrelated individuals from the military aircrew component from different geographic regions of Venezuela. Gene frequencies, the adjustment to Hardy–Weinberg equilibrium and some statistical parameters of forensic interest were estimated. The main aim of the present analysis is to establish a Venezuelan aircrew DNA database for forensic testing purpose.

Association between STRs from the X chromosome in a sample of Portuguese Gypsies

2009-10-05

Abstract: In the present work 10 X-STRs were analyzed in a sample of 123 unrelated males belonging to the Portuguese Gypsy community. Average diversity level among the Gypsy group (0.721) was lower compared to the Portuguese non-Gypsy (0.745). DXS6809 showed to be the most polymorphic marker (0.832) and DXS7133 the less informative one (0.599). The genetic distance between Portuguese Gypsies and non-Gypsies was found to be statistically significant (FST=0.019; P≤0.000).In the Gypsy sample, the evaluation of pairwise linkage disequilibrium (LD) through gametic determinant (D′) allowed to detect significant associations between DXS6809 and DXS6789 and between DXS6789 and DXS9898 (after Bonferroni's correction). To our knowledge, this is the first study where a significant association between the latter pair of markers is reported. In recently admixed populations, as the Gypsies are, high level of LD across loci are not unexpected and might have important forensic implications. The finding indicates, for instance, that among the Portuguese Gypsies, associations between X-STRs should be considered in forensic applications, instead of dealing with locus frequencies independently. Evaluation of the forensic efficiency of this decaplex system in the Portuguese Gypsies revealed not only high levels of power of discrimination among males (0.999997) and females (0.9999999993), but also strong combined mean exclusion chance values in duos and trios (0.9995 and 0.99998, respectively).

Genetic data of 10 X-chromosomal loci in Vitória population (Espírito Santo State, Brazil)

2009-10-12

Abstract: Genetic population data for 10 X-STR (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 and DXS6789) were obtained from Vitória population (Espírito Santo State, Brazil). No deviations from the Hardy–Weinberg equilibrium and linkage disequilibrium were observed. The combined powers of discrimination in males and females were 0.9999995 and 0.99999999996, respectively. These high values show the potential of this system in human identification in Vitória population, Brazil.

Genetic studies of eight X-STRs in a Northeast Italian population

Stefania Turrina, Giulia Filippini, Domenico De Leo — 2009-10-05

Abstract: Population genetic parameters of eight X-STR (DXS7132, DXS7423, DXS8378, DXS10074, DXS10101, DXS10134, DXS10135, HPRTB) markers located on the X-chromosome in four closely linkage groups were analysed in 176 unrelated Italian individuals (147 females and 29 males) using Mentype® Argus X-8 PCR Amplification Kit (Biotype). The Chi-square test for genotype distribution showed no significant deviation from Hardy–Weinberg equilibrium (HWE). Several microvariant and rare alleles have been observed in some of the X-STR markers studied. PIC of the eight X-STRs ranged from 0.617 to 0.925.

Chromosome X centromere region—Haplotype frequencies for different populations

2009-10-15

Abstract: Searching for suitable and closely linked STRs on the X-chromosome (ChrX) we evaluated several polymorphic markers located within the human ChrX centromere region. Stable haplotypes can be expected in this region because of low recombination rates. The five markers investigated here show a tetranucleotide or pentanucleotide structure and exhibit high or medium polymorphic information content. We validated a pentaplex PCR for these STRs with a special 3 primer system for the amplification of the combined STR-INDEL polymorphism DXS10163. Additionally we present allele and haplotype frequency data for populations from Germany, Ethiopia, Egypt and Somalia.

Analysis of 12 X-chromosomal STRs in an Algerian population sample

2009-09-22

Abstract: Twelve X-chromosomal short tandem repeat (STR) loci (DXS6789, DXS6809, GATA172D05, DXS101, DXS8378, DXS8377, DXS7132, DXS6800, DXS6801, DXS7424, HPRTB, DXS10011), including two clusters of closely linked markers (DXS6801–DXS6809–DXS6789 mapping in Xq21 and DXS7424–DXS101 mapping in Xq22) were typed in a northwestern Algerian population sample (n=210; 104 men and 106 women). The calculated allele and haplotype frequencies were compared with those previously obtained for the same set of markers in the Italian population. No evidence of linkage disequilibrium was observed between pairs of loci within clusters of strictly linked markers.

Genetic patterns of 10 X chromosome short tandem repeats in an Asian population from Macau

Iva Gomes, António Amorim, Vânia Pereira, Angel Carracedo, Leonor Gusmão — 2009-10-12

Abstract: Genetic data was obtained in a sample from Macau using an X-STR decaplex system. Allele frequencies were calculated for all loci. Observed genotype distributions did not show deviations from Hardy–Weinberg expectations. DXS6809 was the most polymorphic marker in Macau while DXS7133 was the least discriminating. Single locus comparisons with other Asian populations showed significant genetic distances with Pakistan, Japan, Mongolia and Korea. With samples from Taiwan and US Asians no significant genetic differences were obtained. No significant association was found between any of the pairs studied after applying Bonferroni's correction (p<0.001). However, low p values were found between DXS6809 and DXS6789 (p=0.0030) and DXS8378–DXS9902 (p=0.0035) that belong to two described STR clusters on the X chromosome. The decaplex system used in this work was evaluated for potential application in human identification and kinship testing in the present population: high overall values of power of female (99.9999995%) and male (99.998%) discrimination were obtained, as well as high overall values of power of exclusions in father/mother/daughter trios (99.996%), in father/daughter duos (99.92%) and in half-sisters with same father (98%).

Genetic data of 10 X-STR in two Native American populations of Argentina

Ulises Toscanini, Leonor Gusmão, Gabriela Berardi, Eduardo Raimondi — 2009-10-12

Abstract: We present genetic data for ten X-STRs (DXS8378, DXS9898, DXS7133, GATA31E08, GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902 y DXS6789) in two Native American populations from the North and Northwest regions of Argentina, namely Toba and Colla.The present study results support the usefulness of these markers in kinship investigation and also in population genetics studies.

Y-chromosome haplotype database in Venezuelan central region and its comparison with other Venezuelan populations

2009-10-05

Abstract: One hundred and fifteen unrelated male individuals were sampled and Y-STR genotypes were determined using the PowerPlexY-System (Promega Corp.) and the ABIPRISM 3130 Genetic Analyzer.Allele frequencies and gene diversity for each of the 11 markers analyzed are shown. One hundred and seven different haplotypes were found. Five of them were present in more than one individual, and 101 were not previously found in Caracas and Maracaibo populations .When genetic distance between Central Region and Caracas was estimated, non-significant difference was found. Likewise, differences were not found in markers either. However, when Central Region and Maracaibo were compared, significant differences were found in both genetic distance and DYS385 and DYS438 allele distributions.

Y-chromosomal STR haplotypes in an Arab population from Somalia

U.D. Immel, M. Kleiber — 2009-09-10

Abstract: We analyzed Y-chromosomal STRs in an Arabic population sample of 33 males from Somalia and found 29 different haplotypes. Most of these haplotypes were never observed in any population study so far.

Knowing your DNA database: Issues with determining ancestral Y haplotypes in a Y-Filer database

2009-10-16

Abstract: Y-chromosome STR profiles are being increasingly used in forensic investigations. Differences between populations in male-specific DNA markers can be large (relative to autosomal ones) and this knowledge has implications for appropriate database construction. Also, in cosmopolitan populations, such as Australia, asymmetrical admixture between groups can have important effects. This paper reports on these issues as they relate to the South Australian Y chromosome Aboriginal database.

Population data of 12 Y-STR loci from a Somali population

Andreas O. Tillmar, Kerstin Montelius — 2009-10-05

Abstract: 12 Y-chromosome STR loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS385ab) were typed for 147 males from Somalia. A total of 66 haplotypes were identified of which 44 were unique. The most common haplotype was found 26 times corresponding to a population frequency of 17.7%. The haplotype diversity was computed to be 0.956. No significant differences were found when the Somali samples were compared with two other Somali sample sets.

New single nucleotide polymorphisms on Y chromosome in the Chinese Han population

Wei Wei, Jing Yan, Haibo Luo, Yiping Hou — 2009-09-25

Abstract: The single nucleotide polymorphisms on Y chromosome (Y-SNP) were potential markers not only for typing of degradation DNA but also for analysis of mixed biological stains in sexual assault cases. Y-SNP genotyping is becoming a key technology for forensic DNA studies. However, the difficulty of Y-SNP genotyping for forensic purpose was that there was a lack of Y-SNP loci in Chinese population so far. Exploring the genetic information in whole genome on Y chromosome and finding out new Y-SNP loci play an important role on DNA typing for forensic purpose. Our aim was to find out new Y-SNP loci in Chinese population. A total of eight loci on Y chromosome were investigated by the pyrosequencing method with pooling samples. The samples were collected from non-relational individuals in Chinese Han population in Chengdu. Our results showed at least there were two new Y-SNP loci suitable for forensic purposes in Chinese population.

Evaluating Y-chromosome STRs mutation rates: A collaborative study of the Ge.F.I.-ISFG Italian Group

Valerio Onofri, Loredana Buscemi, Adriano Tagliabracci, the Ge.F.I. Group — 2009-10-12

Abstract: A collaborative study was carried out by the Italian ISFG Working Group in order to improve the data on Y-STR mutations at the loci mostly used in forensic analysis, following recommendations of the ISFG DNA Commission.The knowledge on Y-STR mutation rates needs to be considered in the paternity probabilities, especially in deficiency cases of disputed paternity involving male offspring where the alleged father is not available for DNA analysis. Furthermore, the mutation rate represents a precious tool to estimate the local and temporal origin of a given Y-SNP based haplogroup.The sample consisted of 433 father/son pairs from paternity cases in 15 different laboratories from Italy. The biological relationship of all father/son pairs was previously confirmed by using autosomal microsatellites. The laboratories used AmpFlSTR YFiler kit (AB) and PowerPlex Y System (Promega); DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, GATA C4, and GATA H4.1 data were collected. The participants were also asked to provide the age of the biological father and, if possible, male descendants beyond the first generation.20 mutations were observed among all of the allele transfers in the sample (19 single step and 1 double step), and mutations in the same father/son pair were found in three cases. Locus-specific mutation rates were calculated. Forensic implication of the average age of the father as well as the number of locus deletions and amplifications were discussed.

Analysis of Y chromosome SNPs in Alagoas, Northeastern Brazil

2009-10-12

Abstract: Alagoas State is located in Northeast Brazil. Like many other South American populations, the genetic composition of Alagoas is the result of admixture between Amerindians, Europeans and Africans. In this work, 24 Y chromosome SNPs were analysed (SRY10831, M168, M213, M9, M22, M45, M3, M173, M17, P25, M269, M70, M201, M170, M26, M304, M172, M96, M2, M35, M78, M81, M123 and M34) in a sample of 247 unrelated men belonging to Alagoas population, aiming to determine the different Amerindian, European and African male contributions to the current population. SNP typing was done by a single base extension method, using the SNaPshot minisequencing kit. A total of 16 haplogroups were found in our sample. The main contribution to the present day Y chromosome genepool of Alagoas was found to be European (94.74%), and a much lower percentage of African (4.45%) and Amerindian (0.81%) ancestry lineages have been detected. When our sample was compared with others from Portugal and Rio de Janeiro, no significant genetic differences could be observed.

Investigation of population structure in the Victorian Italian and Greek population using Y chromosome STR haplotype analysis

Anjali A. Goundar, Roland A.H. van Oorschot, Runa Daniel, R. John Mitchell — 2009-11-09

Abstract: Investigation of Y chromosome STR haplotypes, using AmpFlSTR® Yfiler™ PCR Amplification Kit (Applied Biosystems), in the Greek and Italian populations of Victoria, Australia, reveal that Y haplotype variation is largely structured according to Y SNP haplogroup rather than by nationality or population of origin.

Mutations at 17 Y-STR loci in father–son pairs from Southern Spain

M.J. Farfán, V. Prieto — 2009-09-01

Abstract: We have analyzed 113 confirmed and unrelated father–son pairs from Andalucía and Extremadura (Southern and Southwestern Spain) for the 17 Y-chromosome microsatellite loci included in the AmpFlSTR® Yfiler® PCR amplification kit (Applied Biosystems). Among the 113 sons, 111 different haplotypes were observed of which 109 were unique. Out of the 1920 allele transfers studied, three single-step mutations between father and son were detected (overall mutation rate: 1.563×10−3; 95% CI, 0.322×10−3 to 4.559×10−3) which is in agreement with previous reported data. These results will be part of a collaborative work carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG).

Y-chromosome SNP analysis in the Brazilian population of São Paulo state (Ribeirão Preto)

2009-10-12

Abstract: In recent years, an interest has grown with Y-chromosome single nucleotide polymorphisms (Y-SNPs) and their potential applications, especially in evolutionary biology, forensics and medical genetics. A total of 81 samples from unrelated males of the Brazilian population of São Paulo State (Ribeirão Preto) have been analyzed for 14 Y-SNPs (SRY1532, M213, M9, M70, M22, TAT, 92R7, M173, P25—multiplex 1; M170, M62, M172, M26 and M201—multiplex 2) using the SNaPshot™ methodology, and haplogroups frequencies were determined. European influence (mainly due to the male Portuguese settlers, but also the later arrival of Spanish and Italian) is therefore confirmed, given the fact that the most predominant European haplogroup R1b1 is also the most representative haplogroup (53.09%), followed by haplogroup J2 (with not more than 12.35%). The remaining haplogroups have made a minor contribution, between 9.88% and 1.23%, yet very significant.

Haplotyping of Y-chromosomal short tandem repeats DYS481, DYS570, DYS576 and DYS643 in three Baltic populations

R. Lessig, J. Edelmann, J. Dressler, M. Krawczak — 2009-09-23

Abstract: The aim of the present study was to characterize four less intensively investigated Y-STRs, namely DYS481, DYS570, DYS576, and DYS643, that were introduced by Lim et al. as being more diverse than commonly used markers. The haplotype distribution of these Y-STRs in a German sample from West Saxony has been published earlier this year . Here, we investigated three additional populations from the Baltics and compared the results to those obtained in the Germans. The Y-STRs were found to be highly polymorphic in the Baltic populations with a discrimination capacity similar to that seen in other studies. Our results highlight shows the usefulness of the investigated markers for forensic purposes as an addition to the minimal or extended Y-STR haplotypes. Their inclusion in routine genotyping would allow the definition of unique haplotypes in most practical cases.

Comparison of Y-chromosome haplogroup frequencies in eight Provinces of Argentina

2009-09-25

Abstract: Y-chromosome haplogroups constructed from SNP markers of Y-specific regions have been largely used in population genetic trials. These data might also be applied to forensic studies on human populations showing multiethnic admixture. Haplogroups have evidenced differential ethnic and geographic distribution. The objective of the present report was to analyze 775 male samples for 16 Y-SNP by PCR-RFLP and to describe haplogroup frequencies in three Argentine geographic regions: Central-West (Cuyo), Center, and North-West.

Genetic structure of Mendoza province population inferred from autosomal and Y-chromosome STRs analysis

Miguel Marino, Sandra Furfuro, Daniel Corach — 2009-11-02

Abstract: The population of Mendoza, like the entire population of Argentina, has a heterogeneous genetic constitution. It is the result of two major contributors; Native Americans and Europeans and, in a lesser degree, Western Africans, brought to the territory as slaves during colonial times. In order to investigate the proportion of genetic contributions of the parental populations to nowadays population of Mendoza, a set of 13 autosomal and 9 Y-STRs routinely used in forensic typing were selected. Although these markers are not the best suited for tracing ancestry, they might provide clues about the genetic structure of a population. A total of 242 male unrelated donors inhabiting Mendoza province were analyzed and compared with Argentinean Amerindian, Caucasian and African-American, reference samples.Genetic distance, based on Rst values, and STRUCTURE analysis were performed for both polymorphic systems.Our results demonstrate a tri-parental genetic contribution whose proportions differs between autosomal or Y-STRs markers. Autosomal STRs denoted 46.8% European, 31.6% Native American and 21.5% African contributions. Instead, Y-STRs showed 67% European, 21% Amerindian, and 12% African ancestral lineages.These results are consistent with the history of submission undergone by the native populations during the conquest of the Americas and underscore the impact of the European male genetic contribution to extant population of Argentina. Probably our results underestimate the Caucasian and overestimate the Native American and African contributions since the polymorphisms employed are not as sensitive as the ancestry informative markers (AIMs).

Distribution of Y-chromosomal SNP-haplogroups between males from Ethiopia

M. Kohl, R. Lessig, J. Edelmann, J. Dressler, K. Thiele — 2009-11-13

Abstract: Y-chromosomal SNPs are used in phylogenetical studies because in a paternal lineage the Y-SNP “set” is only changed by mutational events. Lower migration rates of males in the past in local scale cause different haplogroup distributions in closed populations. In the actual haplogroup tree (ISOGG 2008) 20 main haplogroups (A-T) are listed.The Y-chromosomes of 173 unrelated males from Ethiopia (most of them contain to the Amharics) were analysed. For typing, a set of 40 SNPs, which include most of the main haplogroups of the actual phylogenetical tree, were used in combination with the Multiplex PCR Kit (Qiagen) and the SNaPshot™ Multiplex Kit (Applied Biosystems). The ABI PRISM™ 310 Genetic Sequenzer was used for allele calling.The distribution of the five detected main haplogroups was calculated against the total number of the 173 analysed samples. 40 males (23.1% of analysed samples) belong to the haplogroup A*. 88 samples (50.9%) were related to haplogroup E*, 43 (24.9%) to haplogroup J* and one sample (0.6%) to each haplogroup F* and G*.

Y-STR haplotype variation in a sample from Buenos Aires (Argentina)

C.I. Catanesi, L.A. Glesmann, M. Silbestro, L. Vidal-Rioja — 2009-10-05

Abstract: We present the haplotype analysis of 9 Y-STRs in 93 males from Buenos Aires province, sampled for paternity testing in our laboratory. We found a high haplotype diversity, 0.985, and only 6 haplotypes out of 76, were present in more than one male. About 25% of the haplotypes found do not appear in the YHRD Haplotype Reference database, while more than a half of them are not present in the SHDG database. Among the 76 haplotypes, 30% and 20% of them are described in the database as present in Italy and Spain respectively. These values are in accordance to the migration history of the country, since European immigrants mainly came from Spain and Italy. On the other hand, it is relevant to find and describe haplotypes which are not currently included in the databases.

Phylogeography of French male lineages

2009-10-23

Abstract: The genetic landscape of European Y-chromosomes has been widely surveyed. However, French male lineages are still poorly characterized, being scarce the current genetic contribution to the demographic history of France.We have studied, at Y-chromosome level, French population from 7 different regions throughout France: Nord-Pas-de-Calais (Lille), Bretagne (Rennes), Alsace (Strasbourg), Île-de-France (Paris), Auvergne (Clermont-Ferrand), Provence-Alpes-Côte d’Azur (Marseille) and Midi-Pyrénées (Toulouse).More than five hundred male samples were genotyped for 27 Y-chromosome biallelic markers and 17 Y-STRs, in order to assess the degree of population sub-structuring of male lineages on this population.The results obtained show a significant level of population sub-structuring when Bretagne was compared with the remaining regions grouping together as a single cluster, obtaining a value of 6.83% (p=0.0000) for the proportion of the total variance explained by differences between populations, when using Y-SNPs data.Our data will contribute to elucidate the genetic composition of France as well as to reconstruct the demographic history of European populations. Likewise, the population sub-structuring detected entails that special care must be taken when Y-chromosome databases of France are used for forensic casework.

Analysis of Y chromosome lineages in a sample from Sub-Saharan Africa descendents in Rio de Janeiro

Andréa M. Oliveira, Leonor Gusmão, Patrícia Domingues, Elizeu F. de Carvalho — 2009-10-05

Abstract: Genetic differentiation can exist not only between populations but also between different groups within a population, which must have to be taken into account in forensic databasing. The analysis of Y chromosome SNPs has shown to be a powerful tool to detect population substructure, critical in admixed populations as it is the case of Brazilian as well as most American populations. With this work, we aimed to study the origin of paternal lineages (Y-SNPs) in a sample of Sub-Saharan Africa descendents living in Rio de Janeiro. A low proportion of YAP+ chromosomes were found (≈42%), a mutation usually represented in almost 80% of the male lineages in Bantu populations. Concerning the YAP− chromosomes, all of them carried the M89 mutation, and the most common lineages were those belonging to the European R1b1-P25 haplogroup, with a frequency of 37.12%. As expected, a much higher proportion of YAP+ chromosomes were found in this sample than in Rio de Janeiro general population (19%). Nevertheless, in accordance with other studies on African ancestry groups in America, our results revealed a high proportion of European Y lineages in Afro-Brazilians from Rio de Janeiro.

Development of Y-SNP typing assay for forensic application in Venezuelan population

2009-09-22

Abstract: Single-nucleotide polymorphisms of Y-chromosome (Y-SNPs) are genetic markers that can display regional specificity, thus providing useful information about the geographic origin of a subject or evidence under investigation. In this study, we developed 2 multiplex PCRs to analyze 12 SNPs (M2, M3, M9, M35, M45, M96, M170, M174, M201, M207, M304, P25), which occur in basal branches or specific subclades of the Y-chromosome phylogenetic tree and allows the assignation of a subject haplotype to the most frequent Venezuelan haplogroups. We used SNaPshot minisequencing methodology for SNP typing. Our assay proved to be appropriated for assessing the most frequent haplogroups in Venezuela, and it could be useful to solve problems in the forensic casework.

Evaluation of 21 Y-STRs for population and forensic studies

M. Eugenia D’Amato, Mongi Benjeddou, Sean Davison — 2009-10-05

Abstract: In the present study we evaluated the applicability for forensic and evolutionary studies of 21 Y-STR loci (DYS437, DYS447, DYS448, DYS449, DYS456, DYS481, DYS504, DYS510, DYS518, DYS532, DYS536, DYS542, DYS552, DYS562, DYS576, DYS587, DYS612, DYS626, DYS644, DYS710, and Y-GATA-H4). Allele sequence analysis, allele diversity, gene diversity, allele frequency spectrum, discrimination capacity and informativeness for assignment were studied in European English, Asian Indian and Xhosa population groups sampled in South Africa.Seven loci showed size homoplasy. Individuals with mixed ancestry were identified using a statistical method for population assignment and a phylogenetic network. In comparison to our previous minimal haplotype data for the same population groups these loci showed significant increase in discrimination capacity (overall, from 0.773 to 0.958).

“South to North increasing gradient of paternal European ancestry throughout the Mexican territory: Evidence of Y-linked short tandem repeats”

2009-09-02

Abstract: We genotyped at least 12 Y-STRs in DNA samples of 986 Mestizos from five states: Aguascalientes (n=293), Jalisco (n=185), Guanajuato (n=168), Chiapas (n=170), and Yucatán (n=170). Powerplex-Y and AmpFℓSTR® Y-filer® kits were employed, offering a minimum haplotype diversity of 99.69% and 99.88%, respectively. The inclusion of additional Y-STR databases to the analyses allowed obtaining a Y-STR variability landscape from Mexico. Genetic relatedness and preliminary admixture estimates confirmed a population differentiation gradient throughout the Mexican territory: the European ancestry increments to the Northwest and, correspondingly, the Amerindian ancestry increments to the Southeast. These results must be considered for human identification purposes given that most states in Mexico do not have a proper Y-STR database.

Origin of paternal lineages in an admixed population of Northern Argentina (La Esperanza, Jujuy)

2009-10-12

Abstract: The development of the molecular biology techniques has made possible the study of the genetic background of American populations where a substantial contribution of the aboriginal component exists, but its individuals had lost the ethnic identity because of dramatic historical, cultural and social changes. The aim of the present work was to investigate the origin of the paternal lineages present in La Esperanza (Jujuy, Argentina), a population originated at the end of XVIII century as a consequence of the installation of a sugar plantation.DNA of 37 non-related males was purified from buccal swabs and subjected to STR PCR amplification of 11 Y chromosome loci using a commercial kit. In addition, 4 binary markers commonly used in diagnosis of ethnic origin (DYS199, M89, M207, and M173) were characterized by RFLP-PCR.From the 37 individuals analyzed, 34 different STRs based haplotypes were observed, 31 of them being unique, which shows the high heterogeneity of the genetic composition of the population. A search in the Y-Haplotype Chromosome Reference Database allowed to identify 11 Amerindian, 14 European, 2 Arabs and 2 Africans haplotypes. The 8 remaining haplotypes were not possible to assign unequivocally. On the other hand, the analysis of binary markers permits to include all the individuals analyzed (29) into a known haplogroup: 17 individuals carry the haplogroup Q1a3a (Amerindian), 9 the R1 (European), 1 the R (European) and 2 F (Eurasian). We conclude that the STRs and binary markers should be used jointly to get more accurate information about the ethnic origin and genetic diversity of human populations.

Distinguishing kinship from genealogical likelihoods

Nádia Pinto, Leonor Gusmão, António Amorim — 2009-09-18

Abstract: Kinship analyses, besides paternity, are increasingly common in forensic genetics routine. The evaluation of the probability of a disputed kinship between individuals using unlinked autosomal markers employs a likelihood ratio format: the probability of the observations assuming the kinship is true against the probability of the same observations under the alternative hypothesis of a different degree of relationship (usually its absence). However, with very few exceptions (identity, first degree relatedness—paternity or maternity, full-brotherhood or unrelated), the algebraic expressions are not unique for a given kinship, the same probability value being obtained for various genealogical constellations. Then, in a likelihood ratio, what is compared are sets of possible kinship configurations, which are defined by identity-by-descent probabilities known as Jacquard's coefficients. Pedigrees with the same identity-by-descent probabilities are thus undistinguishable by unlinked autosomal markers. In the literature the most common reported undistinguishable kinships, by autosomal transmission, are avuncular, half-sibs and grandparent–grandchild. Therefore, when reporting the results of genetic information upon a specific kinship case a particular caution must be taken in order to avoid misconceptions and fallacious inferences.

Overdispersion in allelic counts and -correction in forensic genetics

Torben Tvedebrink — 2009-10-05

Abstract: A statistical model for incorporating the extra variability in allelic counts due to subpopulation structures is presented. In forensic genetics, this effect is modelled by the identical-by-decent-parameter, . It is shown, that may be defined as an overdispersion parameter capturing the extra variability from subpopulation structures. This allows derivation of maximum likelihood estimates of the allele probabilities and together with the computation of profile log-likelihood, confidence intervals and hypothesis testing.

More for the same? Enhancing the investigative potential of forensic DNA databases (REF 0415)

Susan Pope, Timothy Clayton, Jonathan Whitaker, John Lowe, Roberto Puch-Solis — 2009-09-24

Abstract: The UK has had a National DNA Database [NDNAD] since 1995. It now contains more than 4.5 million records representing 7.5% of the UK population. The Forensic Science Service has introduced two additional services designed to supplement and enhance the ability of law enforcement agencies to investigate offences. Both employ novel and bespoke software that exploit data held on the NDNAD to greater effect than using direct matching algorithms.The first service, ‘familial DNA searching’ [fDNA] was introduced in 2003 and has now been applied in over one hundred serious offences. In these cases, although a crime DNA profile was in existence, the offender's profile was not present on the NDNAD; instead the focus is to search for a potential relative of the offender. A number of successful detections have been obtained and the data generated from those operations have been collated and studied. We present an overview of the data and discuss a number of refinements which have been implemented from the experience gained.The second and most recent service to be introduced employs proprietary software called DNAboost(r) and applies it to complex DNA mixtures which cannot be resolved to allow for a standard search. A mixed DNA profile is ‘de-convoluted’ to generate all feasible DNA profiles in order to interrogate a DNA database of named individuals. The paper will discuss how this has been achieved and the initial results of the operational testing of the software.

Assigning weight of DNA evidence using a continuous model that takes into account stutter and dropout

R. Puch-Solis, L. Rodgers, S. Pope, I. Evett — 2009-10-05

Abstract: Currently practitioners use a binary approach to DNA profile interpretation. However it is recognised that methods considering peak heights/areas are preferable. In this paper we give an example that shows how peaks in stutter position and preferential amplification can be included in a calculation of likelihood ratios using a model that takes into account peak heights.

RMNE probability of forensic DNA profiles with allelic drop-out

F. Van Nieuwerburgh, E. Goetghebeur, M. Vandewoestyne, D. Deforce — 2009-10-05

Abstract: Two methods are commonly used to report on evidence carried by forensic DNA profiles: the “Random Man Not Excluded” (RMNE) approach and the likelihood ratio (LR) approach. Each approach has its advantages and disadvantages. The RMNE approach is much more straightforward to implement, requires less interpretation (which is subject to non-objectivity) and is easier to explain in court. The RNME approach returns a value which is valid, independent of the knowledge of possible contributors.It is often claimed a major advantage of the LR method that drop-out can be assessed probabilistically. We propose new RMNE calculations that likewise account for allelic drop-out in an observed forensic DNA profile. The reported calculations present a non suspect-driven alternative to the poor practice of omitting an inconvenient locus from the standard RMNE calculation when there are loci that require dropped out alleles to allow for a match with the suspect sample. In contrast with the LR approach, the presented RMNE approach does not need error-prone assumptions on the probability P(D) that an allelic drop-out has occurred in the evidence profile. An Excel file with pre-programmed calculations of RMNE probabilities for DNA profiles up to 16 loci and with a maximum of 2 drop-outs is available at: http://www.labfbt.UGent.be/RMNE.php.

Searching a DNA databank with complex mixtures from two individuals

Josée Noël, Léo Lavergne, France Mailly, Dominique Roberge, Christine Jolicoeur — 2009-10-23

Abstract: In 2000, Canada established a CODIS-based National DNA Databank (NDDB) composed of two indices: a Convicted Offender Index (COI) and a Crime Scene Index (CSI). Our laboratory populates the CSI index with single-source and mixed profiles on the 13 P+/COfiler® loci. These mixtures are coded based on the number of mixed loci (loci with more than 2 alleles).We first compared the behaviour of several hundreds low complexity mixtures (≤3 mixed loci), moderate complexity mixtures (4–6 mixed loci) and single-source profiles uploaded over the past 9 years, when run against 180000 COI profiles. We demonstrated the value of searching a databank with mixtures as hundreds of matches were reported to investigators, while the proportion of rejected matches was similar across the 3 types of samples.Based on these results, we designed a new study to evaluate 130 complex (7–13 mixed loci), two-contributor mixtures which were run against the COI. Results show that such mixtures return a manageable number of candidate matches in spite of their complexity: 70% returned no candidates while only a few generated more than one candidate.Databanking strategies coupled with mixture interpretation guidelines and review of original electropherograms maximise the use of a databank while minimising the risk of adventitious hits.

Bayesian networks for victim identification on the basis of DNA profiles

C.J. Bruijning-van Dongen, K. Slooten, W. Burgers, W. Wiegerinck — 2009-12-01

Abstract: We have developed software to improve screening and matching routine for victim identification based on DNA profiles. The software, called Napoleon/Bonaparte, uses Bayesian networks for the analysis. It is designed for effective handling of the identification process in case of a large disaster with many victims and can be applied in the missing person program. In this paper we will describe the Bayesian network approach and we will discuss some of the additional features to handle events with many victims.

Use of animal/plant freeware for calculating likelihood ratio for paternity and kinship in complicated human pedigrees

Jiří Drábek — 2009-10-09

Abstract: According to survey of International Paternity Testing Workshop (IPTW) 2009, none of the laboratories referred the use of plant and/or animal software for parentage and kinship calculation though laws of genetics are universal.The aim of this project is to test whether non-commercial software primarily written for plant and/or animal geneticists is suitable for kinship analysis of human autosomal microsatellite data in complex cases.Software familias 1.81 applied to DNA profiles of Romanov royal family executed by communists in 1918 was used as golden standard for software applicability assessment.Out of many freeware programs available from Internet, only Cervus 3, FaMoz, and Kingroup v2 were chosen for evaluation. Similarities and differences from familias 1.81 and results derived from using distinct non-human genetics versus forensic genetics approaches are presented.

Analysis of complex family cases with probabilistic expert systems

2009-12-01

Abstract: Forensic identification with genetic markers is currently experiencing various scenarios of inference, spanning from direct to indirect comparisons in matter of family relationship and criminal analysis. The discrimination power of current genetic polymorphisms is such that the inferential process underlying this field of human identification can efficiently be deployed even in cases where there is a lack of direct knowledge on the genetic asset on one of the terms in comparison. The most promising of these software facilities are the probabilistic expert systems (PES). To verify the power of these systems to confirm relationships among individuals in defective cases, we analyzed four different groups of complex families with known relationships. To test the goodness of the software employed (FINEX), we created different pedigrees by deleting one or more individuals from each family and calculating the different probability values obtained.

STRs and AIMs informativeness for forensic purposes in an admixed Brazilian population

2009-11-13

Abstract: Forensic parameters estimated for genetic markers signalize their potential in forensic genetics. Microsatellite markers had shown to be more adequate for identification than SNPs. Among the SNPs are the ancestry informative markers (AIMs), which are makers with significant frequency differential between populations, particularly useful in ancestry studies. In this work, we analyze whether they are adequate for individual identification in comparison to STRs. Data on 15 AIMs and 17 STRs of an admixed Brazilian population were employed to evaluate MP, PD, PE, PIC, and TPI. Such forensic parameters estimates suggest that AIM markers can be useful for individual identification in admixed populations, although a higher number of markers is necessary to accomplish the informativeness of STR markers.

Partial forensic validation of a 16plex SNP assay for the inference of biogeographical ancestry

Runa Daniel, Juan J. Sanchez, Najah T. Nassif, Alexis Hernandez, Simon J. Walsh — 2009-09-23

Abstract: A partial forensic validation study demonstrated the suitability of an ancestry informative 16 locus SNP assay to forensic casework.

Forensic application of an individual ancestry index in Brazilian populations

2009-10-12

Abstract: We aimed to evaluate whether it is possible to identify the original population of an individual in Brazil using data on nine AIMs. Therefore, we analyzed the genotypic distribution and the structure of five populations (n=578) divided into two ancestry groups: euro- and afro-derived. Although it was possible to cluster most of the individuals into their correct ancestry group, only 25% could be clustered into their actual population. An individual ancestry index was generated, which could distinguish one group from the other and, therefore, the ancestry of the samples, but not the populations to which they belong. Despite the limitation of the methodology employed, it was possible to identify the individual ancestry, which makes it a potential forensic tool.

DRD2TaqI haplotypes in three urban Brazilian populations

2009-10-12

Abstract: Brazil is a continental-sized country divided into five geographic regions, each one with different history of colonization and human settlement. Manaus, located at the North region, is the capital of Amazonas. It suffered large populational influx mainly during the Rubber Age (1870–1920), and due to the Zona Franca de Manaus (established in 1967). Goiás is a Middle-Western state and its population increased with the discovery of gold, on the 17th century. The Federal District was relocated from Rio de Janeiro to the Middle-West in 1960, and since then has received people from all other regions of Brazil. Therefore, this population has been being considered as a reflex of Brazil's population. Here we describe the distribution of the DRD2TaqI A, B and D haplotypes in Federal District, Manaus and Goiás. For this purpose, a total of 353 samples from Manaus (42), Federal District (134) and Goiás (177) were analyzed by using PCR and RFLP techniques. Data were analyzed employing Arlequin 3.11 and GENEPOP 3.4. Manaus was the only population that showed absence of a haplotype (haplotype 7) and also the lowest frequency of haplotype 6, indicating lower African parental contribution than the Middle-Western populations. Contrary to our expectations, all populations showed low frequencies of haplotype 8, a characteristically European marker. The DRD2TaqI haplotype is an AIM with great power of discrimination, therefore being recommended in the analysis of genetic mixture, but it would be necessary to expand the set of AIM markers to obtain a more concrete admixture pattern in those populations.

Analysis of CYP2D6 gene variation in Venezuelan population: Implications for forensic toxicology

2009-09-22

Abstract: Slow drug degradation by CYP enzymes can lead to its accumulation in the body with subsequent toxic effects. CYP2D6 is involved in the metabolism of many toxicologically important drugs such as tricyclic antidepressants, commonly implicated in fatal drug poisonings. Since in human populations the gene coding for this enzyme exhibits high genetic polymorphism, they are of major pharmacogenetic importance. The aim of this work is to describe the distribution of genetic polymorphisms of CYP2D6 responsible for poor and intermediate metabolizers phenotypes in Venezuelan population and provide guidance on the importance of conducting postmortem forensic examinations to elucidate the role of genetic variation in drug intoxications. We carried out the analysis of CYP2D6*3, *4, *5, *6 and 10* alleles of 110 unrelated healthy individuals by allele-specific PCR tetra-primer. The allelic frequencies obtained for *4 (15.9%) and *10 (16.4%) in the Venezuelan population highlights the importance of CYP2D6 genotyping in cases where a high concentration of a drug metabolized by this route has been used, without suspicion of acute overdose.

CYP2D6 polymorphism studies: How forensic genetics helps clinical medicine

2009-09-07

Abstract: CYP2D6 polymorphism, recently studied by minisequencing analysis, lets physicians know the phenotype of a patient or a worker, allowing the correct therapy or the development of a preventive occupational medicine, since recently evidence in favour of CYP2D6 phenotype and pesticide exposure interaction in parkinsonism has been described. In the forensic field the usefulness of CYP2D6 polymorphism is known, in the future joining the classical autopsy with the forensic genetic laboratory. With the aim to genotype a rural area where the pesticides are widely employed, we studied a population sample from Ravenna in the North of Italy. We also analyzed an African population sample, in order to characterize a geographic area where the CYP2D6 ultra metabolizer phenotype is more frequent, to compare the genetic makeup and to enlarge the CYP2D6 population data.A total of 122 individuals resident in the Ravenna area and 65 individuals from North and East Africa were tested by minisequencing method for 11 SNPs positions together with gene deletions and duplications. The Ravenna population, even if in agreement with population data available up to date, showed a slightly higher rate of non-functional alleles defining the poor metabolizer phenotype. African population genotyping confirms the higher rate of gene duplication already reported.The obtained data highlight the need to know the genetic background of a geographical area related to the economical and social setting to address the healthy policies in the occupational medicine nevertheless considering the today admixed population structure.

Genetic susceptibility for addiction: Searching of risk loci for the widespread drugs of abuse

2009-09-25

Abstract: Addiction to drugs of abuse is the major health and social issue, for its morbidity and mortality, individual and society cost, violence and legal problems involvement. The susceptibility to addiction has a complex genetic basis characterized by phenotypic and genetic heterogeneity as well as polygenicity. Both linkage mapping and association mapping have identified susceptibility loci for addiction-related phenotypes.In this study we limit our focus to gamma-amino butyric acid A receptor alpha 2 (GABRA2), one of the candidate gene in addiction, in order to investigate the genetic susceptibility of alcoholism. 10 SNPs across the 3′-GABRA2 were analyzed in an Italian sample of 149 cases and 278 healthy controls. No SNP and haplotype association of 3′-GABRA2 with AUDs was found, in contrast to previous reports in other Caucasian populations.

The relevance between dopamine D3 receptor gene variations and drug addiction

2009-10-08

Abstract: Drug addiction is characterized by impairment of reward, compulsive behavior and inhibitory control deficits. Addiction genetic studies are focused on identifying the candidate genes responsible for the disease. However studies performed in different populations reveal different and sometimes conflicting results. In this study we aimed to determine the association of dopamine DRD3 receptor gene polymorphism and drug addiction in Turkish population. The patient group included 126 addicts diagnosed according to DSM-IV criteria. The results were compared to 100 individuals with no history of addiction. The gene frequencies were tested with Hardy–Weinberg equilibrium. Socio-demographic data including educational level, marital status and occupation were also evaluated. The frequencies of A1 homozygote individuals were 46%, A1 and A2 heterozygotes were 41.6% and A2 homozygotes were 12.7%, while they were respectively 46%, 43% and 11% in controls. According to the overall results, no statistically significant difference in DRD3 polymorphism was observed between the patient and control groups. There was a slight but significant association between DRD3 receptor gene, heroin and cocaine addiction, which was found to be consistent with some of the literature.

Genetics of addiction in legal medicine and forensic investigation: SNPs variations associated with nicotine and cannabis dependence

2009-10-05

Abstract: Substance addiction is a complex chronic brain disorder, characterized by neurobiological changes leading to compulsive drug seeking and taking. Although environmental factors contribute to drug addiction, evidence showed that genetic factors with multiple genes also play a significant role. Cannabis and tobacco result as the most common widely abused substances. Epidemiological studies have strongly implicated genetics in nicotine and marijuana consuming and vulnerability to subsequent dependence, estimating the range of heritability from 34% to 78% for cannabis and approximately from 50% to 70% for nicotine. Furthermore, varying aspects of impulsive personality and principal psychiatric disorders co-occur with tobacco and cannabis dependence status. We evaluate the possibility of identifying an individual's risk probability to become an addict, based on a genotype analysis and the different possible applications in legal medicine and forensic genetics.

The role of CAV3 gene in channelopathies

F. Alessandrini, M. Pesaresi, V. Cappelli, A. Tagliabracci — 2009-12-01

Abstract: Congenital long-QT syndrome (LQTS) is a hereditary cardiac disease characterized by a high risk of life-threatening arrhythmias.Until recently, LQTS was exclusively a cardiac channelopathy. It was observed that the LQT3 associated, SCN5A-encoded cardiac sodium channel localizes in caveolae and it was hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS (LQT9).Caveolae are characterized by the presence of caveolins, scaffolding proteins that interact with cholesterol and provide the structural framework for macromolecular signaling complexes.Using polymerase chain reaction and direct DNA sequencing, mutational analysis on CAV3 gene was performed on DNA extracted from 50 patients with LQTS diagnosis, but with no mutations on the entire coding regions of the major LQTS associated genes – KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and KCNJ2 – and targeted analysis of ANK2 and RyR2.Mutational analysis of CAV3 in 50 unrelated LQTS subjects identified 9 mutations; two of them were found in the coding region. They are missense mutations and are located in a very conserved region.Mutations in the intronic sequences were analyzed by SpliceAid (www.introni.it), a web resource to predict the effect of the DNA mutations at the level of the target sequences of the RNA-binding proteins that determine the pattern of mRNA splicing. Some of them may alter the correct mRNA splicing.Further studies are needed to characterize the impact of these mutations in vivo.

Involvement of hypertrophic cardiomyopathy genes in sudden infant death syndrome (SIDS)

2009-11-02

Abstract: Despite declines in prevalence during the last years, sudden infant death syndrome (SIDS) continues to be the leading cause of death for infants aged between 1 month and 1 year in developed countries. Epidemiological studies have described a number of environmental factors related, as placing infants prone for sleep, maternal smoking during pregnancy or postnatal exposure to tobacco smoke; however, the main cause of SIDS remains obscure.Since hypertrophic cardiomyopathy (HCM) is one of the most prevalent causes of sudden cardiac death in younger adults, although in many cases the disease is not clearly evident at younger ages, we hypothesize about the possibility of founding HCM genes implicated in the development of sudden death in infants. In order to ascertain whether our hypothesis is correct, we have analysed more than 600 HCM mutations in 140 cases of SIDS. Mutation detection was performed with the MassArray genotyping platform of Sequenom and positive mutations were consequently confirmed by resequencing.

Sequenom MassArray™ application in the long QT syndrome mutation detection

2009-11-19

Abstract: The cause of death cannot be established in a large percentage of the deaths after traditional autopsy. This is then sudden cardiac death with an incidence of 30–200/100,000 people each year. The development of a fatal cardiac event because of an inherited channelopathy may explain these deaths. Long QT syndrome is one of these channelopathies.We present the results of 433 mutations described in the three most prevalent genes associated with long QT and Brugada syndromes studied in 502 samples of European origin with different clinical conditions and family history. The semi-automated MALDI–TOF system by using the Sequenom MassArray™mass spectromic system with iPLEX GOLD Chemistry allow us to get an efficient mutation and genetic polymorphisms detection in our samples.

Analysis of three major sarcomeric genes (MYH7, TNNT2, MYBPC3) in cardiomyopathy

2009-10-12

Abstract: Sarcomeric gene mutations are said to be important factor of cardiomyopathy. In those genes, β-myosin heavy chain gene (MYH7), troponin T gene (TNNT2) and myosin binding protein C gene (MYBPC3) account for most known mutations. In this study, we examined some cases of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) and arrhythmogenic right ventricular cardiomyopathy (ARVCM). There were various mutations in each gene and also detected new mutations. MYBPC3 was the most high frequency gene of the mutations and it was indicated that MYBPC3 was the general genetic cause of cardiomyopathy. Moreover, more than half of the cases of HCM and DCM had some mutations. From these results, genetic analysis of MYH7, TNNT2 and MYBPC3 were useful to diagnose cardiomyopathy for forensic autopsy cases.

Forensic mitochondrial DNA analysis in HIV-infected patients treated with nucleoside reverse transcriptase inhibitors

C. Robino, C. Colla, A. Biglino, M. Degioanni, S. Gino, C. Torre — 2009-09-07

Abstract: Hypervariable regions HV1 and HV2 of the mitochondrial DNA control region were sequenced in pre-therapy (serum) and post-therapy (saliva, hair) biological samples collected from HIV-infected patients (n=13) treated with nucleoside reverse transcriptase inhibitors (NRTIs). Occurrence of length/point heteroplasmic mutations as a consequence of treatment with NRTIs was evaluated. The observed rate of length heteroplasmy in the analyzed samples was consistent with values previously reported in untreated subjects, whereas no evidence of point heteroplasmy in post-treatment tissue samples was seen.

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs

E. Hanson, H. Lubenow, J. Ballantyne — 2009-10-08

Abstract: Numerous studies have demonstrated the ability to identify the body fluid origin of forensic biological stains using messenger RNA (mRNA) profiling. However, the size of the amplimer used in these assays may not be ideal for use with degraded or compromised samples frequently encountered in forensic casework. Therefore, a novel approach to body fluid identification through the use of microRNA (miRNA) profiling was developed. MicroRNAs are only 20–25 bases in length and may be more suitable for use with degraded or environmentally compromised biological samples. We have identified a set of nine differentially expressed miRNAs that permit the identification of the body fluid origin of forensic biological stains using as little as 50pg of total RNA.

Degraded DNA sample analysis using DNA repair enzymes, mini-STRs and (tri-allelic) SNPs

Antoinette A. Westen, Titia Sijen — 2009-09-01

Abstract: DNA degradation may cause the loss of the longer short tandem repeat (STR) markers, resulting in DNA profiles with lower discrimination power. We compared standard STR profiling with DNA repair enzyme incubation, and genotyping with mini-STRs or (tri-allelic) single nucleotide polymorphisms (SNPs) in progressively degraded, UV-irradiated DNA samples. In highly degraded DNA samples, most of the standard STR markers fail to amplify, while mini-STRs and especially (tri-allelic) SNPs still provide valuable information.

SNP typing of forensic samples with the GenPlex™ HID system: A collaborative study

2009-10-05

Abstract: We report the results of an inter-laboratory exercise on typing autosomal single nucleotide polymorphisms (SNPs) with the GenPlex™ HID system (Applied Biosystems—AB). A total of 12 European and US forensic genetic laboratories typed 10 reference DNA samples and 3 samples with partly degraded DNA that only gave strong reactions with amplicons shorter than approximately 135bp and weak reactions up to 185bp. The samples and the critical reagents were sent to the participating laboratories from Copenhagen. In addition, the laboratories typed the three degraded DNA samples with one or more STR kits. When 500pg degraded DNA was used, the success rates of SNP typing in the laboratories varied from 15.6% to 98.6% (median: 94.6%). Only 1.6% of the unsuccessful SNP types were due to wrong SNP calling, while the remaining 98.4% were due to lack of amplification and uncertain results. Two laboratories counted for more than two-thirds of the non-successful results while seven laboratories reported no wrong SNP types. Two laboratories typed the degraded DNA samples successfully with the MiniFiler kit, while other commonly used kits gave incomplete STR profiles in all laboratories. Titration of the degraded DNA showed that the SNP typing results became less reliable with 100pg DNA and less. The median success rate of SNP typing of 10 reference samples with 50pg reference DNA was 86% (range: 44.5–99.2% among laboratories). The success rate of SNP typing of the reference samples decreased with smaller amounts of DNA. SNP typing with the GenPlex™ HID system provided more information from degraded DNA samples than any of the commercially available kits used by the participating laboratories.

Indel polymorphisms—An additional set of markers on the X-chromosome

Jeanett Edelmann, Sandra Hering, Christa Augustin, Reinhard Szibor — 2009-10-16

Abstract: Insertion/deletion polymorphisms (indels) are diallelic markers which are underutilised in the forensic science. The simple structure of diallelic markers enables them to generate very short amplicons which facilitate successful typing of degraded DNA samples. Introducing indels into the ensemble of kinship testing tools we here present a panel of 26 X-chromosome indels, which can be amplified and analysed using 5 multiplex PCR setups.

Insertion/deletion polymorphisms: A multiplex assay and forensic applications

2009-10-19

Abstract: Insertion/deletion polymorphisms (indels) have considerable potential in the field of identification, since they combine desirable characteristics of both SNPs and STRs: (i) the use of reduced amplicon sizes comparable to those of forensic SNP assays thus improving amplification success of degraded samples and (ii) the simplicity of analysis through PCR and capillary electrophoresis, in the same way as STRs. We recently developed a simple assay capable of characterizing 38 non-coding bi-allelic indels distributed across all autosomes in a single PCR followed by capillary electrophoresis. As practical applications, the indel-plex was used with clear improvement in genotyping success in highly degraded DNA extracted from skeletal materials or paraffin-embedded tissues, and as a complementary tool in paternity investigations.

The combination of single cell micromanipulation with LV-PCR system and its application in forensic science

Caixia Li, Bing Qi, Anquan Ji, Xiulan Xu, Lan Hu — 2009-09-07

Abstract: Micromanipulation method was combined with the AmpliGrid LV-PCR system to select and detect single cells, in order to provide a possible solution for biological mixtures and trace samples. Three fresh buccal cells could be completely genotyped by two STR kits. Sixty parallel single cell LV-PCRs were performed using Identifiler®, 13 complete profiles (21.7%) and 13 acceptable profiles (13–15 loci) were obtained. Seventy single cells were typed by MiniFiler®, showing 48.6% full profiles and 18.6% acceptable profiles (6–8 loci). Two mock casework samples, cup and chewing gum, were assayed by single cell LV-PCR using MiniFiler®. Three out of four repeat experiments for the cup sample were fully genotyped. Twice of four chewing gum experiments yielded complete profiles. We re-analyzed one casework sample which had been shown to be a mixed profile tested by routine method, while single-person profile was obtained by the new method. These results showed great promise for mixtures and trace DNA analysis. Successful capture of intact cell is the key to the experiment. Replicate experiments are also necessitated to get reliable profiles.

Application of full mitochondrial genome sequencing using 454 GS FLX pyrosequencing

2009-10-15

Abstract: The GS FLX pyrosequencing platform using parallel tagged sequencing was tested on 10 Somali individuals for sequencing of the complete mitochondrial genome. The amplicons were sequenced twice with increasing coverage to establish the minimum of coverage needed to produce reliable sequence reads. The genome sequences were compared to previously obtained control regions sequences with Sanger sequencing and 49 SNPs in coding regions of the mitochondrial genome. No discrepancy was found with the three methods except in a poly-C stretch that was estimated to be 16193.1C by sequencing with terminator chemistry and no insert was observed with pyrosequencing. The sequence lengths of both pyrosequencing runs were between 225 and 235 nucleotides. The coverage for the first pyrosequencing run was between 0 and 159 and 10–577 for the second one.

Selective enrichment of human DNA from non-human DNAs for DNA typing of decomposed skeletal remains

Madhusudan R. Nandineni, Jeffrey P. Vedanayagam — 2009-10-12

Abstract: The degraded or decomposed body remains pose serious challenges for successful short tandem repeat (STR)-based DNA typing for human identification because of contamination with non-human DNAs and environment-induced changes. Owing to their tropical environment, which results in robust microbial infestation, obtaining good quality and quantity of human DNA for body identification by DNA profiling has not been very successful in those regions. To address this issue, we attempted to selectively recover the human DNA from the mixture of non-human DNAs by employing the biotinylated oligonucleotides, designed complementary to the regions flanking the STR loci to selectively capture the respective STR loci that are used for DNA profiling. Initially, the fragmented human DNA was hybridized with the biotinylated oligos, followed by conjugation of biotin with streptavidin-coated paramagnetic beads. After extensive washing to remove non-specific binding, repeat-containing DNA fragments were eluted employing a magnetic particle concentrator. Such repeat-enriched DNA fragments were subjected to PCR amplification to analyze STRs employing commercial human DNA typing kits. Our preliminary analysis has shown that there was selective recovery of STR-containing human DNAs when biotinylated oligos were used.

Reparation of degraded DNA improves PCR amplification of larger STR loci

2009-11-09

Abstract: Postmortem tissues frequently produce deficient STR profiles, which is a problem in forensic identification and paternity testing. This is because traditional STR typing samples can be affected by inhibitors that slow or inactivate the PCR reaction, and by DNA degradation that often leads to allele dropout and poor PCR amplification of the larger sized STR loci. The formaldehyde commonly used to fix soft tissues generates DNA degradation, due to single and double strand breaks. Likewise skeletal human remains are usually inadequate for PCR amplification since the DNA is often degraded due to exposure to heat, humidity, light and microorganisms.Therefore in order to improve the genotyping of traditional STR from degraded DNA samples using a simple approach, the objective of this work was to partially restore the DNA length by filling the single strand breaks before the PCR amplification.Formalin fixed soft tissues and bone remains were subjected to traditional DNA purification with solvent extraction and Amicon ultra filtration. The purified DNA was submitted to a restoration treatment involving a pre-polymerase chain reaction assay involving a incubation with dNTPs and Taq DNA polymerase at 72°C for 20min, followed by a denaturing step before the PCR amplification using the AmpFlSTR Identifiler kit. The pre-PCR reparation treatment of the DNA allowed to amplify the larger STRs loci that were missing in the untreated samples. Therefore, in situations where standard STR kit fails, the pre-PCR DNA repair increases the probability of obtaining a full DNA profile.

Analysis of DNA forensic markers using high throughput mass spectrometry

2009-10-12

Abstract: A novel DNA forensics platform has been developed that is based on automated high throughput electrospray ionization mass spectrometry (ESI-MS). The approach uses ESI-MS to “weigh” DNA markers with enough accuracy to yield an unambiguous base composition (Aw, Gx, Cy, Tz) which are then used to derive a DNA profile for an individual.A number of blinded studies have been performed to evaluate the platform for both STR and mtDNA typing. The same platform is used for both analyses and offers advantages over the conventional typing approaches.This approach facilitates the analysis of mtDNA samples containing length heteroplasmy in the HV1/HV2 regions in situations that are not amenable to sequencing. Based on the base compositions profiles from a 24-primer pair assay which covers CRS 15924–16428 and 31–576, this approach is more resolving than traditional sequencing approaches which cover 16024–16365 and 73–340 and facilitates mixture analysis.STR markers can be analyzed in a similar fashion. The high mass accuracy of the measurements affords the ability to detect SNPs within the STR markers. A survey of samples from different population groups suggests that the frequency of SNPs in several of the CODIS loci is in excess of 30%; SNPs were identified in 10 of the 13 core loci.

Validation of mass spectrometry analysis of mitochondrial DNA

2009-10-05

Abstract: The analysis of mitochondrial DNA (mtDNA) is used routinely to assist in determining the source of old bones, teeth, hair shafts, and other challenged biological samples. Sequencing is the method of choice for typing mtDNA. However, the method is laborious, time consuming, costly and currently not a practical approach for typing single nucleotide polymorphisms (SNPs) contained within the coding region of the mtDNA genome. Electrospray ionization mass spectrometry (ESI-MS) has several advantages for mtDNA analysis including speed, sensitivity and the ability to interrogate mixtures. As with sequencing and unlike many other SNP-based approaches, individual polymorphic positions do not need to be specifically targeted, as all variation within amplified regions is assayed simultaneously. Since each amplicon is assayed as an individual component, the technique easily resolves length heteroplasmy and is capable of quantitatively analyzing heteroplasmic and mixed samples. The ESI-MS approach originally described for mtDNA analysis has been improved using the Ibis T5000™ and a 24 amplicon tiling format and validation studies are underway. Results from these studies support that the use of mass spectrometry is a robust and reliable method for mtDNA analysis that offers resolution approaching that of sequencing, the ability to deconvolve mixtures and analyze heteroplasmy effectively, and a sensitivity of detection equivalent to current methods, with at least an order of magnitude increased throughput and a reduction in cost.

Enhancing resolution and statistical power by utilizing mass spectrometry for detection of SNPs within the short tandem repeats

2009-10-12

Abstract: Short tandem repeats (STRs) are used routinely for the analysis of DNA samples from evidentiary items, convicted offenders, relationship testing and other identity testing disciplines. The discriminatory power of the STRs is sufficient in most human identity testing comparisons to render an identification. However, STRs have some limitations in evaluations, such as parentage testing, identification of human remains, or pairwise evaluations of putative relatives by familial searching. A major assumption is that shared alleles in these associations stem from common ancestry, i.e. are Identical by Descent (IBD). However, STR alleles by definition are Identical by State (IBS). Using an electrospray ionization mass spectrometry (ESI-MS) system developed by Ibis Biosciences Inc., population databases were generated for the 13 core CODIS STRs from African Americans, Caucasians and Hispanics capturing both the length of the allele, as well as SNP variation contained within repeat motifs. SNPs were identified in 10 of the loci and some common alleles were subdivided with SNP typing. Inclusion of SNPs increases discrimination power significantly, whereby the seven most polymorphic SNP-containing STR loci have the discriminatory power of 10 traditionally typed loci. A system of nomenclature has been developed that facilitates the databasing, searching and analyses of these combined data forms.

Autosomal STR allele frequencies and Y-STR and mtDNA haplotypes in Chilean sample populations

2009-10-23

Abstract: DNA from 1020 unrelated male individuals sampled from five locations of Chile (Iquique, Santiago, Concepción, Temuco, and Punta Arenas) were typed for autosomal STRs, Y-STRs, and the mtDNA Control Region. The populations were selected to develop reference databases to support forensic casework and relationship testing. Allele frequencies for 15 autosomal STR loci across the five sampled sites were compiled. As there was considerable overlapping of birthplaces of subjects sampled from these five sites, the pooled dataset was re-grouped based on birthplaces of the subjects into eight geo-political birthplace regions of the country. Each of these populations was evaluated for conformance to Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between loci and within the populations was assessed. Descriptive statistics, i.e., power of discrimination (PD), power of exclusion (PE), and mean power of exclusion were determined. No deviations from HWE expectations (p<0.05) and LD were detected. Combined PD and PE for each population exceeded 0.99999. Y-STR and mtDNA haplotype frequencies were developed and haplotype sharing within and between populations was evaluated. The PD for the Y-STR database is 0.99841 and for the mtDNA database it is 0.99356. Population substructure on the haplotype data evaluated by AMOVA indicated approximately 0.03% of the variation detected originated from differences among the eight birthplace regions. Independence between Y-STR haplotypes, mtDNA haplotypes, and autosomal loci was assessed using a mismatch distribution approach.

mRNA profiling for the identification of sperm and seminal plasma

C. Haas, C. Muheim, A. Kratzer, W. Bär, C. Maake — 2009-10-05

Abstract: mRNA profiling is a promising new method for the identification of body fluids from biological stains. In this study we aimed to establish a multiplex RT-PCR protocol for the detection and differentiation of sperm and seminal plasma. The Agilent Bioanalyzer and Nanodrop spectrophotometer were shown not to be suitable for assessing RNA quality and quantity of forensic stains. Semen specificity of the mRNA markers was successfully confirmed with singleplex PCR. Our data indicated that semen samples down to 0.1μl and up to 20-year-old could be identified with mRNA profiling. With the semen multiplex, including 2 sperm markers (PRM1, PRM2) and 2 novel seminal plasma markers (SEMG1, PSA), samples from azoospermic men (absence of sperm in semen) are clearly distinguishable from those of normozoospermic men (having a normal sperm production). We think that our multiplex RT-PCR protocol is a reliable and sensitive method for the identification of semen in forensic samples.

How specific are the vaginal secretion mRNA-markers HBD1 and MUC4?

C. Cossu, U. Germann, A. Kratzer, W. Bär, C. Haas — 2009-10-05

Abstract: mRNA profiling is a new method for the identification of body fluids. We evaluated the specificity of the two supposedly vaginal secretion specific mRNA-markers HBD1 and MUC4 and the cross-reactivity with other mucous membranes. Both mRNA-markers reliably detect vaginal secretions from women of all ages (14–82 years), but they cross-react with other mucous membranes like buccal cells, or diverse mucous membranes taken from corpses.

Multiplexed SNP detection panels for human identification

2009-10-12

Abstract: SNP profiling is a very powerful tool for human identification. Two panels of 49 and 41 SNPs were selected based on high average heterozygosity (>0.4) and low global Fst values (<0.06) as a set of highly discriminative SNPs suitable for human identification in all geographic populations. SNPs were selected based on testing samples from 44 populations across the globe using TaqMan® allelic discrimination formats. Coding SNPs, and SNPs with known functional or phenotypic manifestations were excluded. To test the performance characteristics of the two panels of SNPs and to compare their utility to STR analysis for human identification and paternity testing, we genotyped a panel of 41 individuals from three different CEPH families spanning three generations. The test samples were genotyped using the two SNP panels and a 15-loci STR kit. Further, utility of the SNP panels in human ID testing and related applications was demonstrated using degraded DNA and DNA from blood, semen and saliva samples. The development of highly multiplexed SNP detection systems enabling lower cost and higher throughput via automation will result in increased use of SNP profiling in applications like human identification, cell line authentication, species identification and bacterial strain typing.

Investigation of SNP haplotypes in the H19 imprinted gene

2009-10-05

Abstract: Differentially methylated SNPs upstream of the human H19 imprinted gene were investigated in DNA samples from Japanese, German and African (Nigerian and Ghananian) individuals. We examined a roughly 1.2kb region of DNA and successfully detected a parental allele (haplotype of SNPs) after treatment of the genomic DNA by a methylation-sensitive enzyme. Our investigation revealed nine new SNPs that are largely specific to a single ethnic group, and the new haplotype system composed of over 20 SNPs, designated H19HP, revealed a high diversity of genotypes in each ethnic group. This haplotype system may therefore be a useful tool for ethnicity determination and personal identification.

Interpretation of low-copy-number DNA profile after post-PCR purification

Sabine Michel, Anne De Bast, Isabelle Vandenbroere, Olivier Froment — 2009-09-17

Abstract: DNA analysis using STR amplification become increasingly sensitive permitting the use of minutes quantities of DNA transferred through skin contact. However, in such case, the interpretation of mixed profiles is very difficult due to low intensity peak, allelic drop-out or stutter increase. We use a multiple PCR strategy with post-PCR purification to help interpret low-copy-number DNA profile. To ensure that this approach can be used with minimal risk, we tested post-PCR purification of limited amount of mixed known samples. Results were analysed for correct alleles, allelic drop-out, allelic drop-in and stutter increase. No allelic drop-in was observed and most of the lowest peaks were confirmed by post-PCR purification. However, careful interpretation of peaks present at a stutter position is necessary.

Trace DNA and street robbery: A criminalistic approach to DNA evidence

2009-10-16

Abstract: It is now routine to detect trace DNA from handled objects, and with such low quantities of DNA the principles of criminalistics are now more relevant to biological evidence. This study aimed to provide data into the abundance, transfer and persistence of trace DNA, in a particular crime scenario—street robbery.Items commonly stolen during a robbery (handbags and wallets) were swabbed to determine the background levels of DNA present. The likelihood of DNA transferring onto wallets during and after a robbery was investigated, as was the amount of handling time needed for the offender's DNA to become a major component in the recovered profile. A significant amount of DNA was recovered from wallets and bags in regular use, including small amounts of non-owner DNA. This indicates that background DNA may interfere with the recovery of offenders’ DNA. Profiles recovered from wallets stolen in a simulated robbery were in the majority mixtures, however the robber was a major component of the mixture or a single source profile in 40% of the profiles.The findings demonstrate that background data on the trace evidence characteristics of DNA will aid its interpretation and presentation in criminal trials.

Impact of relevant variables on the transfer of biological substances

Roland A.H. van Oorschot, Mariya Goray, Ece Eken, Robert J. Mitchell — 2009-10-12

Abstract: There is a paucity of data on the relative amounts of DNA containing material that is likely to be transferred given specific casework scenarios that incorporate multiple transfer steps. Availability and application of such data could be helpful in estimating the probability of a given set of circumstances actually occurring.Here we utilise data on transfer percentages, given knowledge of specific variables (type of biological substance, level of moisture of the biological substance, the substrate on which the sample is located, the substrate with which it comes into contact, and the manner of contact), as determined by Goray et al. [1,2] to extrapolate the amount of DNA expected to be retrieved from a targeted sample area, under particular multi-transfer step scenarios. We demonstrate that, in many scenarios incorporating multiple transfer steps, unrealistically large amounts of biological material would need to be present at source to generate a detectable level of DNA from the targeted crime scene surface.These findings will assist in comparing the likelihood of postulated alternative crime scenarios involving DNA transfer, at the investigation stage and/or during court proceedings.

Review of low template DNA typing

Adrian Linacre — 2009-09-01

Abstract: Low copy number (LCN) DNA profiling has played a prominent part in the investigation of many high profile crimes. It has been accepted by a limited number of criminal justice systems throughout the world but due to issues with interpretation and reporting of results there have been questions raised about the technique. In the UK the method was suspended when its use in a trial was questioned, after DNA was found on explosive devices used in the detonation of a bomb that killed 29 people in a town centre. In particular the validation procedures leading to the introduction of LCN were at issue. LCN had also come under question in a second high profile case leading to a review being commissioned by the UK Home Office. The review had unprecedented access to the validation process that the three UK suppliers of low template DNA typing had performed prior to introducing the technique. The review published its findings and made 21 recommendations while recommending that the technique be reintroduced into the UK criminal justice system. This presentation will comment upon the background to the review, its findings and the future of low template DNA typing.

Low copy number typing has yet to achieve “general acceptance”

Bruce Budowle, Arthur J. Eisenberg, Angela van Daal — 2009-09-22

Abstract: When processing a small number of starting DNA templates during the PCR, exaggerated stochastic sampling effects will occur, and because of increased sensitivity of detection there is a concomitant increased risk of observing contamination. Caution should be taken with using LCN typing and there should be awareness of the pitfalls that have been encountered by some users of LCN typing. The methodology does not yield reliable results; therefore interpretation strategies have been developed in an attempt to overcome the non-reproducibility of LCN typing. It is troubling that the LCN protocols that are available are scant and leave most of the interpretation to the discretion of the analyst. There is substantial evidence that the interpretation by practitioners often is not based on the results of validation studies and is steeped in practices of bias. In addition, no generally accepted approach(es) to statistical analyses and the values to account for the uncertainty associated with the stochastic phenomena, such as allele drop out, are described.

Low copy number typing—Where next?

Peter Gill, John Buckleton — 2009-10-12

Abstract: There has been much discussion of characterising DNA profiles according to whether they are deemed to be low-copy-number (LCN) or ‘conventional’. Previous authors have attempted to provide definitions relative to modified techniques (e.g. elevated cycle number). We do not believe that it is helpful to attempt delineation between two theoretical categories. This is because there is no possible natural delineator that can be used. The transition of the two ‘states’ is gradual rather than sudden and is independent of the methodology utilised. We prefer to work towards a single integrated approach that can be applied universally across all techniques that is independent of PCR cycle number, etc.