Explore Of Forensic DNA Typing For Degraded Sample

Objective In order to increase the success rate of analysis for degraded DNA samples, it is necessary to explore some new methods of forensic DNA typing using current technique and equipments. This study explored some novel methods of analysis for degraded DNA samples.Methods We selected four STR loci that generated lower success rate than other loci with commercial STR kits to assay the degraded DNA samples from forensic casework. Through redesigning the primer binding sites, the amplicons of each STR marker were made as small as possible. This is so called miniSTR. The four miniSTR loci were combined into a multiplexed amplification system. PCR products were detected with ABI Genetic Analyzer for fluorescent system. The study of the concordance, the degraded DNA and the PCR inhibitors were performed using both this multiplex system and a commercial STR kit. A series of validation experiments were done for this multiplex system according to the recommendation of the Technical Work Group DNA Analysis Methods (TWGDAM).We studied five loci of the single nucleotide polymorphisms (SNP). Three SNP markers (rs999842, rs922992 and rs924181) located on autosome. One SNP marker (rs997262) located on X chromosome. Another (m9) located on Y chromosome. The five SNP loci were combined into a multiplexed amplification system. The amplicons with very small size were employed for single base extension reaction. The five reactions of single base extension were combined into a multiplex system. The products of the extension reaction were analyzed for SNP typing with ABI Genetic Analyzer. Population data were collected with the multiplex SNP system. A comparing study of assaying degraded DNA and resisting PCR inhibitors were performed between this multiplex system and the miniSTR system. Some validation experiments and a study for identifying contributor sex were performed in order to assess the technical performance of the multiplex SNP system.An internal PCR control (IPC) DNA fragment was designed. This DNA fragment was amplified by PCR and the amplicon was analyzed by capillary electrophoresis along with a commercial STR typing kit. Through assaying quantity of PCR products from IPC, effect of some PCR inhibitors was evaluated.Results Through analyzing 220 forensic DNA samples with a commercial STR typing kit, we found that D7S820, D18S51, CSF1PO, D2S1338 and FGA loci generated lower success rate than other loci. Because the FGA marker had very long repeat motif, we abandoned it as candidate locus. So, a multiplexed amplification system was established for four miniSTR markers: D7S820, D18S51, CSF1PO and D2S1338. Concordance study showed there were no different DNA profiler between our multiplex system and a commercial STR kit. In DNA typing of the degraded samples or the samples containing some PCR inhibitors, the miniSTR multiplex system was capable of recovering more complete profiles when compared to the larger sized amplicon form the commercial kit. The validation experiments proved that the miniSTR multiplex system was highly reproducible and very useful for forensic casework.A multiplex SNP typing for rs999842, rs922992, rs924181, rs997262 and m9 with the multiplex primers for single base extension reaction (SBE) was established successfully. In DNA typing of the degraded samples or the samples containing some PCR inhibitors, our designed SNP multiplex system was more capable of producing profiles when compared to the miniSTR. The validation experiments showed that this system was highly reproducible and very useful for forensic casework.An internal PCR control (IPC) DNA fragment with 83bp was constructed successfully. It can be amplified and the amplicon can be detected by ABI Genetic Analyzer along with a commercial STR typing kit. There was no influence on sensitivity and species specificity for the commercial STR kit. Through assaying quantity of PCR products from IPC, we can assess effect of some inhibitors for PCR. The validation experiments showed that this system was very useful for forensic casework.Conclusion This study constructed some new markers and established some new methods for degraded DNA typing. They are potential tools for recovering more information from the degraded DNA samples. They can make up commercial STR kits in detecting the degraded DNA of forensic casework.