Establishment Of MtDNA SNPs Multiple System And Its Application In Forensic Medicine

Biological samples such as corrupted tissues,old bones were vulnerable to external environment factors and have always been a difficult problem which the forensic experts confronted. Theses samples have had been degraded in the complex environment and the integrity of the DNA sequence had been damaged obviously, the appraisal conclusion was influenced subsequently. STR genotyping system was used widely in the forensic laboratories. It usually apply to conventional samples, but trace DNA samples and degraded samples, non-nuclear materials detection were often lost because of null alleles, unspecific amplification, or e failure detection. Forensic biology has not yet been fully to solve this problem.Human mitochondrial DNA (human mitochondrial DNA, mtDNA) is located outside the nucleus of genomic DNA. The genetic manner mainly follow maternal inheritance, mitochondrial DNA of mother and child, brothers and sisters is theoretically consistent, a individual have only the mitochondrial DNA does not exist. More than 16,000 mtDNA bases are composed by the closed ring double-stranded covalent structure, surrounded by envelope, so that mtDNA are less susceptible to the influence of nucleic acids enzyme in the external environment. Compared to the nuclear DNA genetic marker, every cell has only two copies, every cell of mtDNA has thousands of copies, the mutation rate of mtDNA is at least 5～10 times more than nuclear DNA. mtDNA is more preserved in the external environment and easy to be amplified, it has a good application prospect to forensic screening for archaeology of individual identification, relationship recognizance and failured nuclear genetic markers typing specimens.Single-nucleotide polymorphisms (SNPs) was defined as a single nucleotide variation caused by DNA sequence polymorphism in the genome. At least the frequency of SNPs was not less than 1% and SNPs were characteristic of high density, genetic stability, easy automation etc. It regarded as the most common genetic diversity and make up about 80% of all human genetic variation. Due to the smaller fragments with analysis (45-150bp), genetic stability, lower mutation rate, capability of high-throughput through the fluorescent marker in single base extention (SBE) technique detection and automation, SNPs have rapidly become a hotspot in forensic medicine aimed at the degraded samples. In the SNPs application of forensic medicine, the main purpose is a lot of synchronous detection of SNPs loci and accumulative multiple sites genotyping results, which is important in individual identification and QinQuan appraisal.There were a lot of researches of mitochondrial SNPs about human evolution, gene mutations and expression, pharmacokinetic, epigenetics, oncology and other diseases such as Parkinson's and Alzheimer's, obesity, diabetes and other relevant researches.But there had less researches of high-throughput.MtDNA is characteristic of maternal genetics, high mutation rate, hardly reorganization, fast evolution, now a lot of researches is mainly based on disease related to mtDNA, epigenetics etc. And also in the forensic research in China, but there is still no effective detection of the crowd of composite system of genetic markers.We developed a highly sensitive multiplex mtSNPs typing system based on multiplex PCR amplification, single base extension (SBE) and capillary electrophoresis technology. The eatablished mtDNA SNPs system with high sensitivity, simple operation, high accuracy, and also for the trace, degraded, nuclear-free DNA samples.The system provided a new type of high-throughput means and methods. We gained the population genetics data in Guangdong area and discussed its application in forensic cases.According to the dbSNPs database (http://www.ncbi.nlm.nih.gov/sites/dbSnp NCBI) and nucleic acid data to find rCRS (revised Cambridge Reference Sequence), mitoMAP database(A human mitochondrial genome database), mtDB database (Human Mitochondrial Genome Database),we primarily selected 33 candidates mtDNA SNPs with highly polymorphisms among in Asia population, DNA region ssurrounding the substitution site and flank sequence of SNPs sites were stable. We choosed some SNPs sites for aims at detecting whether existing polymorphisms in China population. All we selected SNPs including 8392,5178,8414,10211,14470,13626,15217,709,1719,15607,13928,7028,10398,10400,6392,9090,5843,2092,16519,4833,5250,12438,4529,4580,11719,477,4793,11914,12633,12858,10873,13263,7202。According to the above sites, PCR primers were designed and single site of SNPs was amplified, after that, we used the native polyacrylamide gel electrophoresis (nPAGE) and silver stain to detect products of SNPs. The lengths of template specific parts of the single base extension primers ranged from 18 to 88 nucleotides,the desired length of a primer was adjusted at the 5'end by addition of a piece of a 'neutral'sequence and/or, if necessary, a poly-C/T tail. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. 2092,6392,9090,5843 were removed because of no products in the multiple PCR reaction; 8392 was removed due to dimerization with other primers; 10400,8414,5178,10211 were removed because they were not detected in the multiple SBE reaction.13626,15217,14470 were removed because of non-specific products. Through orthogonal method for PCR components and cycle parameters adjustment of complex system,considering the interaction between primers, amplification efficiency and stability of the multiplex system, we finally established a stable 21-pelx mtDNA SNPs multiplex detection system. A total 236 samples from Guangdong area were typed with 21-pelx system and all the allele frequencies were determined. All of the mtDNA SNPs are polymorphism except for 477,12438.The genetic diversity (GD) of mtDNA SNPs ranged from 0.0085～0.4980 and the haplotype diversity(HD) was eatimated to be 0.9705.90 haplogroups were found.The electropherograms with the best peak balance were obtained with 0.1～10 ng input DNA, but acceptable results were also obtained with less to 50 pg DNA. Without specific amplification products were detected from animal blood samples such as cattle, pigs, geese, rats and rabbits, chicken, sheep, dogs, cats. muscles and skin, liver, spleen, hair, nail, bone and cartilage, bloods from a same individual were genotyped by the 21-pelx system obtained the same results.We have typed 30 family trios(grandmother-female-son/daughter),20 brother-pairs and sister-pair and five maternal cousins. No mutation event had be found.Trace, degraded of 50 samples we collected from crime scene were also typed. In some cases, when only 1-5 STR loci could be detected with common STR multiplex system, there were more than 18 mtSNPs loci could be detected.Conclusion:we have established 19-plex mtDNA SNPs multiple system based on single base extension and capillary electrophoresis fluorescence detection technology. The multiple system is polymorphic in Chinese Guangdong Han population. The markers included 709,1719,15607,13928,7028,10398,16519,4833,5250,12438,4529,4580,7202,11719,477,4793,11914,12633,12858,10873,13263. Established 19-plex mtDNA SNPs was high sensitivity, high accuracy, high-throughput, high species-specific, reproductivity, genetic stability and was particularly suit for trace, degraded samples, maternal relationship identification etc. The results of hair, bone, muscles and degraded samples detected by the system were satisfactory. The research based on forensic DNA laboratory platform and the conventional equipment directly. The prospect of multiple system would be very well.