Saiki R K, Bugawan T L, Horn G T, Mullis K B, Erlich H A
Nature. 1986;324(6093):163-6. doi: 10.1038/324163a0.
Allelic sequence variation has been analysed by synthetic oligonucleotide hybridization probes which can detect single base substitutions in human genomic DNA. An allele-specific oligonucleotide (ASO) will only anneal to sequences that match it perfectly, a single mismatch being sufficient to prevent hybridization under appropriate conditions. To improve the sensitivity, specificity and simplicity of this approach, we used the polymerase chain reaction (PCR) procedure to enzymatically amplify a specific segment of the beta-globin or HLA-DQ alpha gene in human genomic DNA before hybridization with ASOs. This in vitro amplification method, which produces a greater than 10(5)-fold increase in the amount of target sequence, permits the analysis of allelic variation with as little as 1 ng of genomic DNA and the use of a simple 'dot blot' for probe hybridization. As a further simplification, PCR amplification has been performed directly on crude cell lysates, eliminating the need for DNA purification.
等位基因序列变异已通过合成寡核苷酸杂交探针进行分析，这些探针可检测人类基因组DNA中的单碱基替换。等位基因特异性寡核苷酸（ASO）仅与与其完全匹配的序列退火，单个错配就足以在适当条件下阻止杂交。为了提高这种方法的灵敏度、特异性和简便性，我们在与ASO杂交之前，使用聚合酶链反应（PCR）程序酶促扩增人类基因组DNA中β-珠蛋白或HLA-DQα基因的特定片段。这种体外扩增方法可使靶序列的量增加超过10^5倍，允许使用低至1 ng的基因组DNA分析等位基因变异，并使用简单的“点杂交”进行探针杂交。作为进一步的简化，已直接对粗细胞裂解物进行PCR扩增，无需进行DNA纯化。
