Establishment Of Fluorescence Labeled24Y-STRs Loci Multiplex PCR System And Its Application In Forensic Medicine

Y chromatosome is sex-chromosome, it is presence on normal male, when the Y-chromosome is inheritance from father to son, the gene is Constant excluded the pseudoautosomal region (PAR) unless variation is occurrence, in theory, a patrilineal family have same Y chromatosome. Therefore, Y chromosome is potentially useful in forensic studies and paternity testing, At the same time, Compared with others, because of it’s the male specificity, Y chromosome genetic markers has some unparalleled properties. Where male assailant and female victim DNA are mixed, as in rape cases, Y chromosome genetic markers will give specific information on the assailant. In conclusion, Y chromosome is very important research object in forensic medicine.The fluorescence labeled multiplex PCR testing of short tandem repeats (STR), that is the second generation of Y chromatosome genetic marker, is the most useful and ripe method test technology of Y chromatosome, it possesses the qualities of simply operate, high diversity information and digital result. Base on this technology, several kinds of commercial kits had been produced, and those kits have been widely used in forensic medicine DNA field. In2003, the Promega company put out the PowerPlexTM Y kit, it can test12Y-STRs loci in one reaction, include MHL(minimal haplotypes loci)9Y-STRs loci of Europe that is congnized by ISFH (International Society of Forensic Haemogenetics),and11Y-STRs loci that is recommended by SWGDAM (Scientific Working Group On DNA Analysis Methods). In2004, the ABI company put out the AmpFISTR Y filerTM kit, it can test17Y-STRs loci in one reaction. But these existing commercial kits are primarily according to the population statistics of Europe when selecting the loci, some of which polymorphism in Chinese population is relatively low, for example the polymorphism of DYS391in China population is less than0.4. plus the number of loci is small, as a result, the discrimination power of the whole system is low, there will be difficulty in forensic identification of actual application, especially in recent years as the development of DNA database and increasing number of comparing sample, Y-STR as a main index of families identify, the insufficiency of existing Y-STR detection system recognition rate is increasingly highlight, it makes bad effect on the comparison effectiveness of database, sometimes it appears that the Y-STR haplotype of suspects and multiple different genealogies match, giving a fail condition of troubleshooting. Or misjudgment caused by Y-STR mutation.Therefore, developing a Y-STR detection system which contains high polymorphism loci for Chinese group and more loci can be detected at the same time. This detection system become a urgent need of forensic material evidence examination and identification. The detection system as a complement to existing commercial kits for detection methods, and combined with existing commonly used Y-STR Kit, can significantly improve the cumulative discrimination power and cumulative excluding probability of paternity, this meet the technical requirements for DNA testing. According to this article under a dozen years of research on Y-STR for Chinese people at home and abroad in the past, we selected some Y-STR loci of high GD for China male and established a compound amplification system containing24Y-STRs loci labeled with fluorescence, effectively improved the capability and efficiency on China crowd and solve low identified power problem of the existing Y-STR fluorescence test kits.Through a literature review, we select DYS531, DYS630, DYS622, DYS552, DYS510, DYS449, DYS459a/b, DYS446, DYS443, DYS587, DYS527a/b, DYS460, Y-GATA-A10, DYS520. DYS557, DYS522, DYS481, DYS570, DYS444, all of which are not in existing commercial kits and have high gene diverstity (GD>0.6), and have small allele span, considering the needs of combining existing commercial kits we add DYS635and DYS385a/b three loci, and we use the most advanced5color fluorescence mark system, separate24loci into four groups, marked with6-FAM, and HEX, and TAMRA, and ROX, Molecular weight of internal standard mark with the fived color fluorescence dye SIZ (Orange fluorescence, Wuxi AGCU company), comprising24y-str loci amplification system of review. Then we optimize each loci on their own amplification condition, base on the success of establishing single loci amplification conditions, we test24loci multiplex PCR reaction conditions, by experiment we determined cycle parameter, and annealing temperature, and buffer liquid ionic strength, and enzyme volume, and reaction volume and fluorescently-labeled primer concentration, all parameter, makes amplification output reach a balance, and specific requirements, established stability, and balanced multiple amplification system, achieving simultaneous amplification of24Y-STR loci.In this study, we used the24Y-STRs loci multi-PCR system to test146independent male people that living in Guangdong province., and tested parallel by AmpFISTR YfilerTM Kit, and got the genotyping data of Y-STRs. GD(gene diversity) and HD(haplotype diversity) is calculated directly by the equation:h=n(1-∑Pi2)/(n-1),n is the sample number, Pi is the allele frequencies. TGD(total gene diversity, such as CPE in evaluating system of euchromosome) is calculated by the equation:TGD=1-(1-GD1)(1-GD2)(1-GD3)(1-GD4)...(1-GDn), GDn is the GD of ever loci. By24Y-STRs loci multi-PCR system we totally got145haplotype,144haplotype appears once,1haplotype appears twice, loci GD is from0.55(DYS531) to0.96(DYS385a/b) HD is0.99972, TGD is1-1.88E-14. By AmpFISTR YfilerTM it we totally got143haplotype,141haplotype appears once,1haplotype appears twice,1haplotype appears thrice, loci GD is from0.36(DYS389)-0.96(DYS385a/b, HD is0.99858, TGD is1-4.97E-9. If unite these two test systems, we got145haplotype,144haplotype appears once,1haplotype appears twice, HD is0.99972, TGD is1-1.066E-20. Through the study.24y-str loci amplification system established by our research is stable and efficient, can meet the needs of daily Y-STR forensic evidence testing, at the same time target on Chinese group, recognition system rate is higher than the commonly used imported Y-STR kits, can be a good solution to the current test kit for less information and low individual identification rate. As Y-STR inspection system, using fluorescent capillary electrophoresis technology, can be applied to most of laboratories of forensic evidence, have a wide range of applications in forensic evidence case、paternity test and foundation of Y-STR database.