A Study Of Multiplex MiniSTR With Three Autosomal Loci And Mt-SNP Genotyping By Minisequencing

Objective Analysis of degraded DNA samples is a great challenge for forensicgenetics. MiniSTR assays and mitochondrial DNA (mtDNA) testing, the current hotresearch field in forensic genetics, are the potential access to solve this problem.Though miniSTR can help recovering information from degraded DNA samples, itstill can not be 100ï¼…success because its amplified length is restricted by that of STRrepeats. Currently mtDNA has been reported as a useful marker for the analysis ofdegraded materials and samples containing little or no genomic DNA. However, thetwo routinely analyzed mtDNA hypervariable regions (HVS-â… /â…¡) provide limitedpower of discrimination in a forensic context. The purpose of this study is to establisha new 3plex-miniSTR multiplex miniSTR system and to establish a new mt-SNPsmultiplex system for testing degraded DNA samples.Methods Potential miniSTR loci were obtained from GenBank. Thepopulation genetic research of these selected miniSTR loci were performed. Using thefluorescent primers, a multiplex miniSTR system including D1S1676, D6S1274 and D17S1299 was constructed. PCR products of the multiplex miniSTR system weredetected by ABI 310 Genetic Analyzer. A series of validation experiments wereperformed for the miniSTR multiplex system according to the recommendation of theTechnical Work Group DNA Analysis Methods (TWGDAM). Nine mt-SNP loci,including position 709, 6455, 8020, 8964, 10398, 12705, 12771, 14569 and 15043,were selected for the population genetic research by fragment length discrepant allelespecific PCR(FLDAS-PCR). PCR primers were designed according to the RevisedAnderson's sequence. 6 mt-SNP loci from above-mentioned 9 mt-SNP loci wereselected out for their polymorphy in Chengdu Han population. They were studied bymultiplex PCR and followed by minisequencirlg. The products of minisequencingreaction were detected by ABI 310 Genetic Analyzer.Results 3plex-miniSTR with D1S1676, D6S1274 and D17S1299 loci wassuccessfully constructed. The sensitivity of this system was 0.5 ng template DNA. Itsspecies specificity was good. After testing the aged bloodstains and the degraded DNAmode, it was demonstrated that this miniplex system had a high successful rate foranalysis of degraded DNA samples. Nine mt-SNP loci were detected 12 differenthaplotypes in Chengdu Han population samples of 140 unrelated volunteer donors.The haplotypes diversity was calculated as 0.7638. A 6-multiplex mt-SNP typing for709, 6455, 10398, 12705, 14569 and 15043 was established successfully.Conclusion We successfully established the miniSTR multiplex system withthree autosomal miniSTR loci. This miniplex system can be successfully used in theABI-310 detection platform. This system was proved to be reliable. The miniSTRmultiplex system could offer a new potential tool for recovering useful informationfrom degraded samples and be applied for forensic DNA typing.Six mt-SNP loci, including 709, 6455, 10398, 12705, 14569 and 15043, which were observed to be polymorphic in our population, together with a total of 12different haplotypes in our samples were detected by FLDAS-PCR assay. Theminisequencing method for testing multiplex mt-SNP with SNaPshot kit wassuccessfully established. This system can increase the power of discrimination ofmtDNA and be used to detect mtDNA with efficiency and high-throughput.