A Study Of Multiplex PCR For Four Y-STR Loci And Validation In Forensic Science

Objective The short tandem repeat loci on chromosome Y (Y-STR) are very useful markers for human identity testing, including typing the perpetrator of sexual assault cases and tracing paternal lineages for missing person investigations. In order to make Y-STR markers more widely accepted, population studies and robust assays are required. We focus on developing new multiplex PCR for Y-STR loci that can be detected by sliver staining system and fluorescent system. A series of validation experiments and genetic studies should be performed for the Y-STR multiplex system according to the suggestion of the Technical Work Group DNA Analysis Methods (TWGDAM). Method We selected four Y-STR loci, DYS434, DYS438, DYS439, A10 (Y-GATA-A10) and designed two set of tailed primers to improve the efficiency of the multiplex PCR. The parameters of the multiplex PCR were optimized. The multiplex PCR was performed in a GeneAmp PCR System 9600. PCR products were analyzed by PAGE for sliver staining system or detected by ABI PRISM310 Genetic Analyzer for fluorescent system. A total of 100 unrelated male (Chengdu, Sichuan, China) were analyzed by themultiplex PCR. Result A multiplex PCR system of four Y-STR loci (Y-A489-plex) was established. Here, Y-A489 means the selected loci DYS434, DYS438, DYS439, A10. The Y-A489-plex revealed stability, robustness and sensitivity in validation experiments. There was no interference of female for Y-A489-plex typing in mixture samples. For the male/male mixtures, the minor component in the mixture could be identified up to a ratio of 1:9. In male/female DNA mixtures the alleles of Y-A489 could be identified for the highest ratio tested, 0.5ng male DNA in 600ng female DNA. The analysis of population samples showed that 37 haplotypes were found from 100 unrelated males for the four Y-STR loci. The haplotype diversity was 0.9628 and standard error was 0.00429. Furthermore, the home product of Taq DNA polymerase had the same specificity and efficiency compared to the AmpliTaq Gold (PE, USA) in this study. Conclusion This is the first time that the tailed primer design protocol for multiplex PCR system is used for Y-STR loci. The tailed primer design protocol is feasible for Y-STR loci and can be used to develop more multiplex PCR assays of Y-STR loci. The results of validation prove that the Y-A489-plex multiplex PCR system can be accepted for the forensic DNA typing. The Y-A489-plex multiplex PCR is feasible for using home product of Taq DNA polymerase.