RAPID DEOXYRIBONUCLEIC-ACID ANALYSIS BY ALLELE-SPECIFIC POLYMERASE CHAIN-REACTION FOR DETECTION OF MUTATIONS IN THE STEROID 21-HYDROXYLASE GENE
RAPID DEOXYRIBONUCLEIC-ACID ANALYSIS BY ALLELE-SPECIFIC POLYMERASE CHAIN-REACTION FOR DETECTION OF MUTATIONS IN THE STEROID 21-HYDROXYLASE GENE
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DOI：
10.1210/jc.80.5.1635
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发表时间：
1995-05-01
影响因子：
5.8
通讯作者：
NEW, MI
中科院分区：
文献类型：
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作者：
WILSON, RC;WEI, JQ;NEW, MI
利用突变位点特异性引物，基于等位基因特异性聚合酶链反应（PCR）的快速DNA分析被开发出来，用于检测已知导致类固醇21-羟化酶缺乏症的CYP21基因突变。与之前的方法不同，该方法对基因组DNA进行PCR后，用放射性探针进行点印迹分析，对8个最常见的突变位点进行多轮剥离和重新检测，而新方法在用溴化乙啶染色琼脂糖凝胶电泳进行PCR后，结果立即可见。使用等位基因特异性PCR，在160个测试的受影响染色体中鉴定出148个突变。虽然这些患者中只有11人在一条染色体上发现了突变，但他们的父母在DNA分析中显示出一致的模式。唯一的例外是，在一个家庭中，父母都有可检测到的突变，在患者的一个等位基因上检测到突变。最有可能的是，患者的其他等位基因中有一个突变，可能是从头出现的，或者是从父母那里遗传来的，但在传递父母的表型中并不明显。与点印迹法相比，等位基因特异性PCR更快，劳动强度更低，并且避免了放射性的使用。
Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radioactive probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient's other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent's phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity.