Primer Extension-DHPLC检测线粒体DNA 12SrRNA常见耳聋相关突变方法的建立~△

Establishment of a Common Deafness-Related Mutation Method for Detection of Mitochondrial DNA 12SrRNA by Primer Extension-DHPLC

目的建立针对线粒体DNA 12SrRNA基因常见耳聋相关突变的Primer Extension-DHPLC(PE-DHPLC)基因诊断新方法。方法将包含线粒体DNA 12SrRNA 961insC、T1095C、C1494T、A1555G 4种不同突变的4例外周血DNA样本和4例正常对照标本经测序验证,PCR扩增靶序列,设计延伸引物,延伸后得到线粒体DNA 12SrRNA基因961insC、T1095C、C1494T、A1555G 4个常见突变的特异性延伸片段,用全变性高效液相色谱分析延伸片段混合物,分离图谱鉴定被检样本的基因型。结果 4例包含线粒体DNA 12SrRNA基因4种耳聋相关突变的DNA样本均扩增出包含4种突变的975 bp特征性条带,PE-DHPLC检测结果显示突变者有引物峰和突变峰两个峰,完全可以鉴别出4种突变,而4例正常对照标本仅显示单个引物峰。结论本实验建立了Primer Extension-DHPLC的线粒体相关耳聋突变分析新方法,可用于耳聋的基因诊断。

Objective To develop a primer-extension method in combination with denaturing high-performance liquid chromatography (PE-DHPLC)-based assay for the genetic diagnosis of the four most common mutations. Methods The human mtDNA 12SrRNA gene fragment was amplified by PCR, followed by a multiple PE reaction specific for the four mutations of 12SrRNA 961insC,T1095C, C1494T,and A1555G. Then the PE product mixtures were separated for the genotyping of mtDNA 12SrRNA gene mutations using fully-DHPLC analysis. Results DNA samples were amplified by PCR, and the 975 bp characteristic bands were obtained. Overall, by using the PE-DHPLC analysis, the authors could identify the genotypes involving the four mutations and normal alleles and actually make final decisions for genetic diagnosis. Conclusion The PE-DHPLC protocol can be a simple, rapid, and highly accurate assay in the genetic detection of common mtDNA 12SrRNA gene mutations.