Mitochondrial Dna Single Nucleotide Polymorphisms (snps), Micro-sequencing Detection Method

Objective: The aim of this study is to establish multiplex testing method of 21 SNP located in the control and coding region of mitochondrial DNA with minisequencing. And according to this study,we can offer an preference for quickly multiplex analysis and relative result interpretation of mitochondrial DNA.Method: We have selected high variable SNP from Chinese ethnic group-Han,Yao,Zang and so on, frequency of all these SNPs is more than 0.2, according to the previous research on mitochondrial DNA. Based on that ,we established a rapid and robust assay to simultaneously genotype these SNP loci by the minisequencing with PCR multiplexes in order to improve the power of mtDNA analysis in forensic science. We performed two mutiplexes. One set covered 10 loci which is nt8414, nt199, nt10400, nt10398, nt489, nt16183, ntl6234, ntl52, nt150 and nt8701. The other covered 11 loci which is ntl6261, ntl6207, ntl6223, nt8584, ntl6327, ntl6362, nt231, ntl89, nt473, ntl95 and nt581.Then,this assay were validated for forensic purpose. We collected 26 tissue samples from two individuals (each provides 13 ,blood,heart,liver,spleen,lung,kidney,pancreas,brain,skelecton muscle,skin,rib,nail and hair were included.);two unrelated individuals; nine samples from daily forensic casework and ten animal muscles samples,separately from camel,cat,cow,horse,chick, goat,fish,dog and deer. We have detected all samples with optimized multiplex PCR and minisequencing method.At the same time,we have sequenced all samples and compared the sequencing and miniquencing result finally.Result: In this study, sequencing and minisequencing have the same result.And minisequencing method has the characteristic of forensic specility.Conclusion: The minisequencing method can quickly detect mitochondrial DNA SNPs ,it is easy to use,economic, sensitivite and automatic,so can be used as a convenience way for mitochondrial DNA SNPs detecting on forensic science.