Volume 2, Issue 1 , Pages 83-84, December 2009
Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot® EZ1 Workstation
Article Outline
- Abstract
- 1. Introduction
- 2. Materials and methods
- 3. Results and discussion
- 4. Conclusion
- Conflict of interest
- Role of funding
- References
- Copyright
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 80
pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.
Keywords: FTA Cards, Reference samples, DNA extraction, STR, SNP
1. Introduction
Since 2005, the Danish police have collected more than 50,000 reference samples as buccal cells on FTA Cards (GE Healthcare) for STR typing for the Danish crime prevention DNA database. STR typing is performed by our department and the STR profiles are sent to the police that administrate the crime prevention DNA database. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used (cf. below). Full STR profiles were generated from approximately 95% of the FTA Cards, while partial or no STR profiles were obtained from 5% of the samples. The problem with insufficient DNA profiles from buccal cells on punches of FTA Cards is most likely due to uneven distribution of DNA at the surfaces of the FTA Cards.
The aim of this study was to find a reliable method for extracting sufficient amounts of DNA from the FTA Cards from which complete STR profiles were not generated with the standard punching protocol. The FTA Card extraction method was performed with the Qiagen BioRobot® EZ1 Workstation and the EZ1 DNA Investigator Kit (Qiagen) [1], [2].
2. Materials and methods
2.1. Samples
A total of 29 buccal swab samples on FTA Cards from which complete STR profiles were not generated with the standard punching protocol were selected. Another sample from each individual was made available for repeated DNA typing. The buccal cells were taken with sterile foam tipped applicators (GE Healthcare) and transferred directly onto the FTA Card.
2.2. Extraction and quantification of DNA
One cm2 was cut out of the FTA Card and placed into a 2mL sample tube. A mix of 250μL G2 buffer and 10μL proteinase K from the EZ1 DNA Investigator Kit (Qiagen) was added. The tubes were incubated at 56°C for 15min and at 95°C for 5min. DNA was extracted using the Trace TD protocol on the BioRobot® EZ1 Workstation (Qiagen). The DNA was eluted in 200μL autoclaved Milli-Q water. The DNA concentrations were determined using the Quantifiler® Human DNA Quantification Kit (Applied Biosystems—AB) according to the manufacturer's recommendations.
2.3. STR typing of DNA on punches of FTA Cards
One 1.2mm disk was punched out of the FTA Card directly into a microtitre plate (Eppendorf) using a BSD600-duet puncher (BSD Robotics) and washed as previously described [3], [4]. The DNA was investigated for 10 STRs and Amelogenin using the AmpFℓSTR® SGM Plus™ PCR Amplification Kit (AB) according to the manufacturer's recommendations. Capillary electrophoresis was performed on a AB3130xl Genetic Analyzer (AB) followed by GeneScan® 3.7 and Genotyper® 3.7 (AB) software analysis. Each sample was analyzed at least twice and up to four times in order to obtain two complete and concordant STR profiles.
2.4. STR and SNP typing of extracted DNA
1–2μL of eluted DNA equalling at least 80pg DNA was typed in duplicate for (1) 15 STRs and Amelogenin using the AmpFℓSTR® Identifiler™ PCR Amplification Kit (AB) according to the manufacturer's recommendations and (2) 49 SNPs using the SNPforID 49plex SNP assay as previously described [5], [6]. Capillary electrophoresis was performed on the AB3130xl Genetic Analyzer (AB) followed by GeneScan® 3.7 and Genotyper® 3.7 (AB) software analysis. Each sample was analyzed twice in order to obtain two complete and concordant STR and SNP profiles.
3. Results and discussion
The yield of DNA from 1cm2 FTA Card was 8–468ng (40–2340pg/μl). The lowest amount of DNA that resulted in complete STR and SNP profiles was 80pg. Complete STR and SNP profiles were obtained from extracted DNA from all 29 FTA Cards that previously had given no or incomplete STR profiles with the standard punching protocol. The STR and SNP profiles were identical to those obtained from a new buccal sample on FTA Cards from each individual.
Fig. 1 shows the STR profile of a sample using the standard punching protocol (A) and DNA extracted with the Qiagen BioRobot EZ1 Workstation from the FTA Card (B). For the illustrated sample, STR typing with the standard punching protocol failed to give acceptable results in four of the 15 STR systems, whereas a full profile was generated from extracted DNA. In our hands, the AmpFℓSTR® SGM Plus™ PCR Amplification Kit and the AmpFℓSTR® Identifiler™ PCR Amplification Kit have similar sensitivities. There was no significant correlation between the number of successful STR systems obtained from punches from the FTA Cards and the amount of DNA extracted with the robot (data not shown).
Fig. 1.
STR profiles with an FTA Card punch and extracted DNA. A: STR profile obtained with an FTA Card punch using the AmpFℓSTR® SGM Plus™ PCR Amplification Kit (AB). B: STR profile obtained with 620 pg extracted DNA using the AmpFℓSTR® Identifiler™ PCR Amplification Kit (AB). STR systems that failed with FTA punches are indicated by circles in both electropherograms.
When DNA extraction using the Qiagen BioRobot EZ1 Workstation was routinely performed, complete STR profiles were obtained from 94% of the FTA Cards from which complete STR profiles were not generated with the standard punching protocol. This gave a combined success rate of STR typing on buccal cells on STR Cards of 99.7%.
4. Conclusion
Buccal swab samples on FTA Cards with low amounts of DNA may be successfully typed after DNA extraction using the Qiagen BioRobot® EZ1 Workstation and the EZ1 DNA Investigator kit. Full STR and SNP profiles were obtained from as little as 80pg eluted DNA. The quality of the diluted DNA seemed to be well suited for STR and SNP typing.
Conflict of interest
None.
Role of funding
None.
References
- . Application of the BioRobot EZ1 in a forensic laboratory. Leg. Med. (Tokyo). 2005;7:164–168
- . Optimization of DNA extraction from low-yield and degraded samples using the BioRobot EZ1 and BioRobot M48. J. Forensic Sci. 2006;51(5):1055–1061
- . Semi-automatic preparation of biological database samples for STR and SNP typing. Prog. For. Genet. 11. International Congress Series. 2006;1288:663–665
- . Automated washing of FTA Card punches and PCR setup for reference samples using a LIMS-controlled Sias Xantus Automated Liquid Handler. Forensic Sci. Int. Gene. Suppl. 2009;2:71–73
- . A multiplex assay with 52 single nucleotide polymorphisms for human identification. Electrophoresis. 2006;27(9):1713–1724
- C. Børsting, E. Rockenbauer N. Morling. Validation of a single nucleotide polymorphism (SNP) typing assay with 49 SNPs for forensic genetic testing in a laboratory accredited according to the ISO 17025 standard. Forensic Sci. Int. Genet. (in press).
PII: S1875-1768(09)00165-6
doi:10.1016/j.fsigss.2009.08.152
© 2009 Elsevier Ireland Ltd. All rights reserved.
Volume 2, Issue 1 , Pages 83-84, December 2009
