变性高效液相色谱技术在线粒体基因突变检测中的应用

目的以变性高效液相色谱(DHPLC)技术分析检测线粒体肌病患者线粒体基因突变,以明确诊断。方法收集4例临床诊断为线粒体肌病的患者,以50例健康体检者作为对照,提取患者肌肉组织及对照组外周血细胞DNA,用聚合酶链反应(po lym erase cha in reaction,PCR)扩增线粒体22个tRNA基因,利用变性高效液相色谱分析技术(denaturing h igh-per-form ance liqu id chrom atography,DHPLC)对PCR产物进行突变筛选,出现异常峰型的tRNA基因进行核苷酸序列测定,明确突变位点。结果4例线粒体肌病患者均检测出线粒体基因突变,突变分别为:例1tRNA-V a l基因发生A 1625G纯合突变,例2 tRNA-V a l基因发生A 1625G/A杂合突变,例3 RNA-A rg基因发生A 10411C/A杂合突变,例4 RNA-T rp基因发生T 5553C纯合突变。结论DHPLC能有效地用于线粒体基因突变的检测,该方法简便,结果稳定,可作为大样本筛查突变位点的一种便捷可靠手段。

Application of Denaturing High-performance Liquid Chromatography for Detecting Mutations of Mitochondrial DNA

Objective To establish and evaluate denaturing high-performance liquid chromatography(DHPLC)as a rapid and efficient technique of detecting mutations of mitochondrion gene.Methods Collected four mitochondrial myopathy patients and 50 healthy individuals as control.The genome DNA was extracted.The whole tRNA gene of mitochondria was amplified by polymerase chain reaction(PCR).The PCR products were analyzed by denaturing high-performance liquid chromatography(DHPLC).The samples with abnormal profiles were sequenced.Results The examinations revealed that patient l had a homoplasmic A1625G mutation in tRNA-Val gene,patient 2 had a heteroplasmic A1625G/A mutation in tRNA-Val gene,patient 3 had a heteroplasmic A10411C/A mutation in tRNA-Arg gene and patient 4 had a homoplasmic T5553C mutation in tRNATrp gene.Conclusion The DHPLC technique provides an efficient,sensitive and rapid method for detecting mutations of mitochondrial DNA.