Wong L J, Senadheera D
Molecular Diagnostics Laboratory, Children's Hospital Los Angeles, CA 90027, USA.
Clin Chem. 1997 Oct;43(10):1857-61.
Mitochondrial defects can be caused by mutations in nuclear or mitochondrial DNA. Large deletion/duplication and point mutations are the two major types of mitochondrial DNA (mtDNA) mutations. Comprehensive molecular diagnosis requires the analysis of multiple point mutations. We developed an effective multiplex PCR/allele-specific oligonucleotide (ASO) method to simultaneously screen multiple point mutations in mtDNA. The system involved three pairs of primers to amplify mutation "hot spots" at tRNA(leu(UUR)), tRNA(lys)/ATPase, and ND4 regions, followed by detection of point mutations with ASO probes. Over 2000 specimens were analyzed and the results were compared with those from previous studies with the PCR/restriction fragment length polymorphism method. Our data demonstrate that the multiplex PCR/ASO method is much more sensitive in the detection of low mutant heteroplasmy. It is simple and cost effective, especially if a large number of samples are to be screened for multiple point mutations.
线粒体缺陷可由核DNA或线粒体DNA的突变引起。大片段缺失/重复和点突变是线粒体DNA（mtDNA）突变的两种主要类型。全面的分子诊断需要分析多个点突变。我们开发了一种有效的多重PCR/等位基因特异性寡核苷酸（ASO）方法，用于同时筛查mtDNA中的多个点突变。该系统涉及三对引物，用于扩增tRNA(leu(UUR))、tRNA(lys)/ATPase和ND4区域的突变“热点”，随后用ASO探针检测点突变。分析了2000多个样本，并将结果与之前使用PCR/限制性片段长度多态性方法的研究结果进行了比较。我们的数据表明，多重PCR/ASO方法在检测低突变异质性方面更为敏感。它简单且成本效益高，特别是在要筛查大量样本的多个点突变时。
