A Study Of Multiplex Assay For Y-STR And Y-SNP

Objective Genetic markers in Y chromosome for forensic medicine included both short tandem repeats (STR) and single nucleotide polymorphisms (SNP). In order to increase the efficiency of Y chromosome markers for personal identification and paternity study, it is necessary to develop both Y-STR multiplex system and high-throughput Y-SNP genotyping technology. This study explored a Y-STR multiplex system with nine loci and a multiplex Y-SNP typing assay with single nucleotide primer extension (SNuPE).Methods We selected nine Y-STR loci, DYS434, GATA-A10, DYS438, DYS439, DYS531, DYS557, DYS448, DYS456 and DYS444, and designed three sets of chimeric primers to improve the efficiency of the multiplex PCR. PCR products were detected by ABI PRISM 310 Genetic Analyzer for fluorescent system. A series of validation experiments and genetic studies were performed for the multiplex system according to the recommendation of the Technical Work Group DNA Analysis Methods (TWGDAM). We also selected three Y-SNP loci, M98, M35 and M9, and designed their PCR primers and extension primers. After multiplex PCR and multiplex SNuPEsteps, the products of extension reaction for 3 Y-SNP loci were analyzed by denaturing high-performance liquid chromatography. Some validation experiments and genetic studies were performed in order to assess the technical performance of the multiplex SNP system.Results A multiplex PCR system with nine Y-STR loci was constructed as HY-9-plex. A complete DNA profile with 9 loci was obtained by this system with both simulated and real casework samples. Using HY-9-plex the minor component could be identified by testing different admixtures of DNA up to a ratio of 1:9 from two male donors. A total of 105 different haplotypes were detected in a population sample of 120 unrelated males. The haplotype diversity was calculated as 0.9968. A multiplex Y-SNP typing for M98, M35 and M9 with a multiplex primer extension reaction followed by DHPLC analysis was established successfully. The multiplex Y-SNP typing proved to be useful for forensic casework. The validation experiments proved that the multiplex SNuPE was highly reproducible. A total of four haplotypes for three Y-SNP loci were observed in 105 unrelated males in Chengdu. The haplotype diversity was calculated as 0.5104.Conclusion A multiplex PCR system of nine Y-STR loci (HY-9-plex) was successfully established. The HY-9-plex system was proved to be robust and reliable. The study for three Y-SNP loci showed that suitable thermostable DNA polymerase and the proportion of template and primers were the key to multiplex SNuPE. A genotyping method for multiplex SNP loci was developed, that combined the accuracy of multiplexing primer extension and the efficiency of DHPLC. The results of our study implicate that multiplex-SNuPE with DHPLC analysis is an effective way to increasethe efficiency of SNP detection for forensic medicine.