用dHPLC技术检测线粒体DNA编码区单核苷酸多态性

Detecting the Polymorphisms in Mitochondrial DNA Coding Area using Denaturing High-Performance Liquid Chromatography(dHPLC)

目的研究线粒体DNA(m tDNA)编码区单核苷酸多态性,建立检测m tDNA编码区单核苷酸多态性(SNP)的变性高效液相色谱(dHPLC)方法。方法设计针对线粒体DNA编码区nt10287-10679及nt8507-8805引物,应用dHPLC技术检测其序列多态性。结果100例中国汉族无关个体中,m tDNA nt10287-10679检出13个SNP位点,13种单倍型,基因多样性(H)为70.79%,偶合概率(P)为29.92%;m tDNA nt8507-8805检出10个SNP位点,12种单倍型,H为70.42%,P为30.28%;两段序列联合起来共检出23个SNP位点,23种单倍型,H为84.14%,P为16.70%。结论所建立的dHPLC方法可用于快速、准确地检测m tDNA编码区序列多态性;m tDNA编码区多态性位点作为m tDNA控制区多态性位点的补充,联合应用可以提高m tDNA的个体识别能力。

Objective To analyze mitochondrial DNA （mtDNA） single nucleotide polymorphisms （SNP） in coding area and establish an effective technique of dHPLC to screen mtDNA SNPs. Methods The primers were designed based on （nt10287 -10679 and nt8507 -8805 ） segments from mtDNA coding region. Using denaturing high -performance liquid chromatography （dHPLC） method, we detect the two segments＇ SNPs. Results Among 100 Chinese Han populations, there were 13 different haplotypes caused by 13 variations in mtDNA nt10287 -10679 segment, of which the gene diversity（H） was found to be 70. 79% and the probability of two unrelated persons having the same sequence（P） was 29.92%. In mtDNA nt8507 -8805 segment, 12 haplotypes caused by 10 variations were found, of which the H and P value were 70.42% and 30.28% , respectively. In combination of the two segments, a total of 23 haplotypes were identified, of which the H and P value were 84.14% and 16.70% , respectively. Conclusion The established dHPLC method can be used for screening mtDNA polymorphisms. The polymorphic sites of mtDNA coding area in combination with that of mtDNA control region can be applied to forensic science so as to increase the discriminational power.