Cha R S, Zarbl H, Keohavong P, Thilly W G
Center for Environmental Health Sciences, Whitaker College of Health Science and Technology, Massachusetts Institute of Technology, Cambridge 02139.
PCR Methods Appl. 1992 Aug;2(1):14-20. doi: 10.1101/gr.2.1.14.
We have found that under appropriate conditions, an allele-specific polymerase chain reaction (PCR) can achieve a sensitivity suitable for measuring specific, infrequent mutations in single cell systems or in animal tissues. Using the 12th codon GC-to-AT mutation in the rat c-Ha-ras gene as a model system, we have defined conditions that allow for measurement of mutations present at frequencies as low as one in 10(5) gene copies. Our approach involved the use of PCR primers that created a single mismatch with the mutated allele (GAA) but created a double mismatch with the wild-type allele (GGA). Five out of the six such double-mismatch primers we tested permitted amplification of the mutant allele (GAA) with a high degree of specificity. The specificity of the assay was further enhanced by using a two-step PCR cycle consisting of a denaturation step (1 min incubation at 94 degrees C) and an annealing/extension step (1 min incubation at 50 degrees C) in the presence of 10% (vol/vol) glycerol. Reconstruction experiments using genomic DNA demonstrate that this procedure cna measure the presence of 30 copies of the transforming ras allele present amongst 3 x 10(6) copies of the wild-type allele.
我们发现，在适当条件下，等位基因特异性聚合酶链反应（PCR）能够达到适合在单细胞系统或动物组织中检测特定的、罕见突变的灵敏度。以大鼠c-Ha-ras基因第12密码子由GC突变为AT作为模型系统，我们确定了能检测出频率低至10⁵个基因拷贝中有一个的突变的条件。我们的方法是使用与突变等位基因（GAA）产生单个错配但与野生型等位基因（GGA）产生双错配的PCR引物。我们测试的六种此类双错配引物中有五种能以高度特异性扩增突变等位基因（GAA）。通过在10%（体积/体积）甘油存在下使用由变性步骤（94℃孵育1分钟）和退火/延伸步骤（50℃孵育1分钟）组成的两步PCR循环，进一步提高了检测的特异性。使用基因组DNA进行的重建实验表明，该方法能够检测出在3×10⁶个野生型等位基因拷贝中存在的30个转化型ras等位基因拷贝。
