Fauser S, Wissinger B
University Eye Hospital, Tübingen, Germany.
Biotechniques. 1997 May;22(5):964-8. doi: 10.2144/97225rr05.
We report the development of a method for the simultaneous genotyping of several distinct nucleotide positions by means of fluorescence-coupled competitive primer extension. We demonstrate the application of this method for the simultaneous detection of three point mutations in the human mitochondrial genome, at nucleotide positions 3460, 11778 and 14484, which account for about 90% of cases with Leber's hereditary optic neuropathy. mtDNA fragments encompassing these nucleotide positions are initially amplified in a multiplex PCR assay. Genotyping is then carried out by a simultaneous primer extension assay using wild-type-specific (FAM-labeled) and mutant-specific (JOE-labeled) oligonucleotides. Primer extension products are separated on a 6% polyacrylamide/8 M urea gel on a fluorescence DNA sequencer. Patients' genotypes can be derived from the peak color of the different-sized extension products. As little as 10% mutant DNA can be detected in heteroplasmic mixtures of wild-type and mutant mtDNA, a degree that is sufficient for routine clinical practice.
我们报告了一种通过荧光偶联竞争引物延伸同时对几个不同核苷酸位点进行基因分型的方法的开发。我们展示了该方法在同时检测人类线粒体基因组中3460、11778和14484核苷酸位点的三个点突变中的应用，这些突变约占Leber遗传性视神经病变病例的90%。包含这些核苷酸位点的线粒体DNA片段首先在多重PCR分析中进行扩增。然后通过使用野生型特异性（FAM标记）和突变型特异性（JOE标记）寡核苷酸的同时引物延伸分析进行基因分型。引物延伸产物在荧光DNA测序仪上的6%聚丙烯酰胺/8M尿素凝胶上分离。患者的基因型可从不同大小延伸产物的峰颜色中得出。在野生型和突变型线粒体DNA的异质性混合物中，低至10%的突变DNA即可被检测到，这一程度足以满足常规临床实践的需要。
