Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis
Detection of mitochondrial DNA mutations using temporal temperature gradient gel electrophoresis
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DOI：
10.1002/elps.200406016
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发表时间：
2004-08
期刊：
影响因子：
2.9
通讯作者：
L. Wong;Tian-jian Chen;D. Tan
中科院分区：
文献类型：
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作者：
L. Wong;Tian-jian Chen;D. Tan
线粒体疾病是一组临床和遗传异质性疾病。常见的复发性线粒体DNA（mtDNA）点突变占一小部分患者的分子缺陷。为了鉴定mtDNA突变，需要对整个线粒体基因组进行全面的突变分析。我们发展了时间温度梯度凝胶电泳（TTGE）方法来筛选mtDNA突变。使用32对重叠引物扩增整个线粒体基因组，然后对DNA片段进行TTGE分析。TTGE方法首先在200个含有已知突变或多态性的DNA片段上进行了验证。在TTGE上，同质性核苷酸取代显示单一条带移位，异质性突变显示多条带型。正确鉴定已知突变或多态性。然后使用TTGE来筛选线粒体基因组中的未知突变。DNA带型偏离野生型，提示同质或异质突变，随后进行直接DNA测序以鉴定突变。检测到许多突变和多态性。结果表明，TTGE检测和区分异质性突变从同质性多态性。它还检测异质性变化的背景下同质多态性。TTGE是一种简便、快速、灵敏、有效的突变检测方法。
Mitochondrial disorders are a group of clinically and genetically heterogeneous diseases. Common recurrent mitochondrial DNA (mtDNA) point mutations account for the molecular defects of a small proportion of patients. In order to identify mtDNA mutations, comprehensive mutational analysis of the entire mitochondrial genome is necessary. We developed the temporal temperature gradient gel electrophoresis (TTGE) method to screen for mutations in mtDNA. The entire mitochondrial genome was amplified using 32 pairs of overlapping primers followed by TTGE analysis of the DNA fragments. TTGE method was first validated on 200 DNA fragments containing known mutations or polymorphisms. On TTGE, homoplasmic nucleotide substitutions show a single band shift and heteroplasmic mutations show multiple banding patterns. The known mutations or polymorphisms were correctly identified. TTGE was then used to screen for unknown mutations in the mitochondrial genome. DNA banding patterns, deviated from wild‐type, suggestive of either homoplasmic or heteroplasmic mutations, were followed by direct DNA sequencing to identify the mutations. Numerous mutations and polymorphisms were detected. The results demonstrated that TTGE detects and distinguishes heteroplasmic mutations from homoplasmic polymorphisms. It also detects heteroplasmic changes in the background of a homoplasmic polymorphism. Overall, TTGE was proven to be a simple, rapid, sensitive, and effective mutation detection method.