Volume 3, Issue 1 , Pages e1-e2, December 2011
DIP–STR: A new marker for resolving unbalanced DNA mixtures
Article Outline
- Abstract
- 1. Introduction
- 2. Materials and methods
- 3. Results and discussion
- Role of funding
- Conflict of interest
- Acknowledgement
- References
- Copyright
Abstract
The genetic characterization of unbalanced mixed stains remains an important area where improvement is imperative. Most cases of aggression, homicide and sexual assault produce biological traces with relatively large amount of the victim's DNA and small amount of the aggressor's DNA. If this ratio is smaller than 1:10 it is currently not possible to obtain a conventional autosomal DNA profile of the minor contributor, with potential loss of crucial DNA evidence. Y-STR analysis represents a solution for some cases but has several limitations. We propose here a method based on a new compound genetic marker formed by a Deletion/Insertion Polymorphism (DIP) linked to a Short Tandem Repeat polymorphism (STR), that we name DIP–STR. By means of allele-specific amplifications of DIP–STR haplotypes, we can produce a high resolution autosomal DNA profile of a donor that contributes less than 0.1% to a DNA mixture. Based on these features DIP–STR markers may outperform conventional Y-STR markers in mixed stain analysis.
Keywords: DNA mixture analysis, Insertion/Deletion Polymorphism, Sexual assault, Allele-specific PCR
1. Introduction
In forensic science, molecular biology tools have improved sensitivity to the point where the genomic DNA from a few dozen of cells can be retrieved. Hence, the number of cases with mixed DNA profiles has significantly increased. However, because of competition for PCR primers during amplification, the DNA that contributes less than 10% to a DNA mixture cannot be detected with conventional STR, SNP, Sanger sequencing, etc. [1], [2]. Below this threshold, the DNA profile of the minor contributor, who is potentially the perpetrator of the crime, is masked by the major DNA. Such situation happens for instance when analyzing touched objects or stains collected on the body of a victim. Y-STRs analysis is of great help for the detection of a trace male component over a female background [3]. However, depending on the frequency of a Y-haplotype in the population the result can be of limited statistical value [4] and patrilineal relatives of a suspect cannot be excluded as being the donor of the stain. Yet, there is a paramount need for the development of new methods that allow complete DNA analysis of unbalance mixed stains irrespective of the gender of the two donors.
Here, we propose an original method based on a compound genetic marker formed by a Deletion/Insertion Polymorphism [5], [6], [7], [8] linked to a Short Tandem Repeat polymorphism (DIP–STR). This marker offers two main advantages: first, the DIP sequence variation allows to design two allele-specific PCR primers, one for the insertion (L-DIP primer) and one for the deletion (S-DIP primer), that target the amplification of the minor DNA contributor for the alleles absent in the major DNA (Fig. 1A); second, the linked STR increases the discrimination power of this marker. Moreover, DIP–STRs are widespread in the genome and genotyping techniques are the same as for STR profiling. Here we report an example of casework resolution by DIP–STR analysis of a woman homicide where circumstantial evidences indicated three suspects, a man and his two sons.
Fig. 1.
(A) A DIP–STR marker is composed of a DIP polymorphism (S/L alleles) linked to an STR polymorphism (e.g., 3/4 repeats). Arrows indicate PCR primers. (B) DIP–STR results of the mixed stain A, 1
μl diluted 1/20 DNA was amplified with S-DIP and STR primers (left panel) and 2μl of DNA with L-DIP and STR primers (right panel). This last shows the positive amplification of the minor DNA component of the mixture which matches one of the suspects.
2. Materials and methods
DIP–STR markers located about 500bp apart were selected from 2 main databases: the Marshfield clinic (http://www.marshfiledclinic.org/) and the UCSCgenome browser (http://www.genome.ucsc.edu/). DNA was extracted with the QIAamp DNA mini kit following the manufacturer's (Qiagen) instructions. DNA was amplified by using 9700 thermocyclers and analyzed on an ABI Prism 3100 genetic analyzer with Genemapper software (Applera Europe) following standard procedures. DIP–STR markers were: MID1950–D20S473, MID1013–D5S490, MID1107–D5S1980. Preliminary data indicate specificity greater than 0.1% and sensitivity of 4ρg of DNA template (complete data will be published in other formats). Casework investigative material included: two stains collected on the body of the victim and the reference DNA of the victim and the suspects.
3. Results and discussion
DNA profiling of the two stains resulted in a complete autosomal STR profile, matching the victim and a complete Y STR profile, matching the three related male suspects. To discriminate between the 3 suspects we genotyped three DIP–STR markers. We report here the results of one informative marker: MID1950–D20S473. The victim showed the genotype S12–S13. Being the victim homozygous for the S-DIP allele, we specifically primed the minor DNA's amplification from the mixed stains by using the opposite L-DIP primer and same STR primer (Fig. 1B, right panel). DNA from both stains amplified the allele L12. Among the suspects only one matched the allele L12 of the stains (complete genotype L12, S11), the others turned out to be S11–S11 and S11–S12. With our method we narrowed to one the list of suspects. This example proves that DIP–STR markers can bring more evidences to criminal investigations by resolving unbalanced DNA mixtures of two individuals.
To conclude, the method we propose is advantageous over the previous techniques for the following reasons: first, the DIP allele specific primers allow the detection of the minor DNA with sensitivity higher than standard autosomal STRs; second, the genomewide location of DIP–STR allows their application to all DNA mixtures irrespective of the gender of the DNA donors. Third, unlike Y-STRs, DIP-STRs allow to access individuals rather than male lineages.
Future developments will include the validation of a large panel of DIP–STR markers (we are currently testing rs11277790–D10S530, rs60194384–D15S1514, rs67842608–D5S468, rs66679498–D2S342, rs10564579–D3S1282, rs77979422–D14S262, rs35708668–D5S2045) which will compensate for the occurrence of uninformative genotype assortment (when the major and minor contributors are homozygous for the same DIP allele or when the major DNA is heterozygous at the DIP locus) and the development of the statistical framework for the evaluation of this new type of evidence. Two patent applications were filled in on March 2010 and April 2011.
Role of funding
No financial support has been received for this work.
Conflict of interest
None.
Acknowledgement
The authors wish to thank N. Deriaz for technical support.
References
- AmpFl STR® Profiler PLUS (short tandem repeat DNA analysis of casework samples, mixture samples, and nonhuman DNA samples amplified under reduced PCR volume conditions (25μL). Journal of Forensic Sciences. 2003;48:1014–1034
- . The Evaluation of Eight Commercially Available STR Kits. 2008;http://www.nfstc.org/?dl_id=27
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- Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms. Forensic Science International-Genetics. 2009;3:205–213
- Human diallelic insertion/deletion polymorphisms. American Journal of Human Genetics. 2002;71:854–862
- Insertion-deletion polymorphisms (indels) as genetic markers in natural populations. BMC Genetics. 2008;9:1–8
- A new multiplex for human identification using insertion/deletion polymorphisms. Electrophoresis. 2009;30:3682–3690
- . Efficient human paternity testing with a panel of 40 short insertion–deletion polymorphisms. Genetics and Molecular Research. 2010;9:601–607
PII: S1875-1768(11)00066-7
doi:10.1016/j.fsigss.2011.08.065
© 2011 Elsevier Ireland Ltd. All rights reserved.
Volume 3, Issue 1 , Pages e1-e2, December 2011
