Mitochondrial Dna Coding Region Single Nucleotide Polymorphisms

Objective To explore the new mitochondrial DNA(mtDNA) singlenucleotide polymorphisms(SNP) in coding area;Establishing an effectivetechnique of denaturing high-performance liquid chromatography(dHPLC) toscreen mtDNA SNPs in coding area.To provide a basis for biological chip withthe use of SNP loci.Methods According to the reported polymorphic region ofmtDNA coding area,four segments were determined,including nt8162 ～8483(mtco1),nt13070～13299(mtco2),nt10287～10679(mtco3),nt8507～8805(mtco4),whose primers were designed according to the Anderson'ssequence.The amplification system was optimized so that the different segmentscan be amplified under the same condition.Using PCR-sequencing method todetect SNPs of mtco1 and mtco2 segments in 100 Chinese Han populations.Then 15 samples were selected randomly,whose polymorphisms of mtco3 andmtco4 were determined by sequencing and dHPLC method respectively. ThusdHPLC method can be evaluated and established on the basis of 15 samples'sequencing results of mtco3 and mtco4.The established dHPLC method werethen employed to investigate other sample's polymorphisms of mtco3 andmtco4.Results Among 100 Chinese Han populations,there were 24 mtDNAhaplotypes caused by 21 variations in mtco1 and mtco2 segments,of which thegenetic diversity(h) was found to be 75.11% and the probability of two unrelatedpersons having the same sequence(P) was 25.64%.In mtDNA COⅡ/tRNALysregion of mtco1,1 sample(1%) have 9bp(CCCCCTCTA) insertion and 15samples(15%) have 9bp deletion.In mtco3 and mtco4 segments,23 haplotypeswere identified,which were caused by 23 variations.The h and P value were84.14% and 16.70% respectively.In combination of the four segments,a total of42 haplotypes were identified,which were caused by 44 variations,of whichnt10400 and nt8701 were the most(57%),then the nt10398(38%),nt8584(20%),nt8414(19%).The h and P value of the combined four segments were 94.79% and 6.16% respectively.Conclusions The extended detecting area of mtDNA should be applied to Forensic science to increase the discriminational power.The established dHPLC method was proved to be effective to apply in detecting mutations.And the detected SNP sites have provided a theretical basis for biological chip.