Development Of A Multiplex AS-PCR System Of Mitochondrial SNPs And Its Forensic Application

With the high development of molecular biology, the DNA identification plays an increasingly important role in forensic science from DNA fingerprint which was called the first generation of genetic marker, and PCR-STR the mainstream technology today, to SNP which is called the third generation of genetic marker. In forensic application most of the cases can be resolved by detecting the short tandem repeats (STR) of autosome, which inherit in accordance with mendel’s law, and detecting the STR of Y chromosome which is paternally inherited. However, this routine STR system makes no function in situations of sparse nuclear DNA and comparison between maternal relatives. At this time, detecting mitochondrial DNA (mtDNA) which is maternally inherited is of great significance.The mtDNA, a closed circular double-stranded covalent molecular with 16569bp, is an important part of human genome. It can be segmented into control region and coding region, the former one contains a hypervariable region I (HVI) and a HVII, and the latter one contains many sporadic single nucleotide polymorphisms (SNP). Compared to the nuclear DNA, mtDNA is maternally inherited without recombination events and has a high copy number, which make it an important tool in forensic science, population genetics, human ecology, molecular evolutionary and archaeology. The routinely method for mtDNA analysis in forensic science is sequencing the control region, especially the HVI and HVII. However, this method, with cumbersome steps, needs a high quality of DNA template and gives a limited power of discrimination.In recent years, a large number of whole mtDNA data are reported with the study of anthropogenesis and population genetics. According to these data, a phylogenetic tree (PhyloTree) of mtDNA was constructed and haplotypes were defined, which lays a foundation to studies of forensic science and human evolutionary. Detecting haplotype-defining SNP according to PhyloTree can be the tendency of mtDNA detecting.Currently, several typing methods for mtDNA SNP have been established such as PCR-RPLP, DHPLC, pyrosequencing, SNaPshot and allele-specific PCR (AS-PCR). Compared to PCR-RFLP, DHPLC, SNaPshot, the AS-PCR technique which is based on the 3’end of primers to distinguish different alleles has been proved to be rapid, simple, economical and reliable. In the present study, using the knowledge of the worldwide mtDNA phylogeny, we developed a multiplex AS-PCR system targeting 60 mtDNA SNP that defined most haplotypes of the Chinese population. We demonstrated that this system was rapid, simple, and could be used in forensic application.Objective:In order to establish a rapid, simple and reliable method for detecting mtSNP,60 haplotype-defining SNP that specific to Chinese population were selected and analysed by using AS-PCR and CE. The feasibility of this system for forensic and population genetic analyses were evaluated by testing the species specificity and resistant ability, genotyping 400 samples from two populations of China (200 of Han and 200 of Uyghur) and several samples from practical cases.Methods:60 mtSNP were selected and devided into 3 groups. Primers were designed based on the principle of AS-PCR. The 3’end of AS primers of different length was matched to the corresponding allele. Each generic PCR primer was labeled with fluorescence:the first group was labeled with fluorochrome 6-FAM (blue), the second HEXTM (green) and the third TAMRATM (yellow). The final three multiplex PCR systems were established by orthogonal test. An allelic ladder was created to ensure the repeatability and accuracy of this method. And this assay was verified by direct sequencing.200 samples of Chinese Han population and 200 of Uyghur were genotyped by using this AS-PCR system. For each population, haplotype frequencies were calculated and genetic comparisons with other published data were performed by Arlequin v3.1 software package. Sensitivity experiments were performed on serial dilutions of DNA of known quantity of total human genomic DNA. Species specificity experiments were performed by genotyping the DNA of pig, duck, chicken, fish, sheep and Ecoli. Detect DNA added with PCR inhibitors to test the resistant ability of this assay. Several samples with sparse nuclear DNA were analysed to explore its application value.Results:Using the AS-PCR technique followed by CE, we have successfully developed three multiplex PCR systems targeting on 60 mtSNP which makes the mtSNP high throughput detecting come true.60 SNP were successfully amplified. The results of direct sequencing showed a complete agreement with those obtained by multiplex AS-PCR.200 samples of Han population can be ascribed into 72 haplotypes with a haplotype diversity of 0.978 and DP of 97.28%.200 samples of Uyghur population can be ascribed into 61 haplotypes and the HD is 0.972 with a DP of 96.69%. The AMOVA showed difference between the two populations. Different results were obtaind for different groups:group FAM and HEX were 0.1pg/μL, while group TAMRA was 0.5pg/μL. Excellent results could be obtained when the DNA concentration ranged from 0.5pg/μL to 2 pg/μL.Genotyping the DNA of pig, duck, chicken, fish, sheep and Ecoli showed no result. This assay can be used to detect samples with sparse nuclear DNA.Conclusion:The multiplex AS-PCR system developed in present study is simple, reliable, economical and sensitive which can make it a promising complementary tool for forensic purposes. By combining the AS-PCR with CE, the system can easily be popularized for it can be performed on a platform commin in most forensic laboratories. The 60 SNP were haplotype-defining sites and showed high polymorphism in both Han and Uyghur populations. The success of genotyping hair shaft and high degraded skeleton indicated that this system can be used in forensic application.