SINGLE NUCLEOTIDE PRIMER EXTENSION TO DETECT GENETIC-DISEASES - EXPERIMENTAL APPLICATION TO HEMOPHILIA-B (FACTOR-IX) AND CYSTIC-FIBROSIS GENES
SINGLE NUCLEOTIDE PRIMER EXTENSION TO DETECT GENETIC-DISEASES - EXPERIMENTAL APPLICATION TO HEMOPHILIA-B (FACTOR-IX) AND CYSTIC-FIBROSIS GENES
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DOI：
10.1073/pnas.88.4.1143
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发表时间：
1991-02-01
影响因子：
11.1
通讯作者：
BAJAJ, SP
中科院分区：
文献类型：
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作者：
KUPPUSWAMY, MN;HOFFMANN, JW;BAJAJ, SP
在本报告中，我们描述了一种检测异常等位基因存在的方法，用于检测相同突变发生频率高的遗传疾病（例如，囊性纤维化和镰状细胞病），以及其他多种突变导致疾病且已知某一特定家族受影响成员序列变异的遗传疾病（例如血友病B）。最初，从每个受试者中，含有假定突变位点的DNA片段通过聚合酶链反应扩增。然后为每个片段制备两种反应混合物。每一个都包含扩增片段，一个引物（18-mer或更长），其序列与突变位点5'端的正常基因编码序列相同，以及与突变位点正常编码序列对应的α - p -32标记的核苷酸或与突变序列对应的α - p -32标记的核苷酸。然后通过变性聚丙烯酰胺凝胶电泳和放射自显影进行单核苷酸引物延伸和分析。正如沃森-克里克碱基对规则所预测的那样，在野生型中只有正常碱基，在受影响的成员中只有突变碱基，在载体中正常碱基和突变碱基都被纳入引物。因此，本方法的一个基本特征是，与模板结合的引物直接3'的碱基是突变体中改变的碱基之一，因为以这种方式，引物延长一个碱基将获得突变体或野生型的扩展分子特征。该方法快速，可用于已知序列变异的所有遗传疾病的携带者检测和产前诊断。
In this report, we describe an approach to detect the presence of abnormal alleles in those genetic diseases in which frequency of occurrence of the same mutation is high (e.g., cystic fibrosis and sickle cell disease), and in others in which multiple mutations cause the disease and the sequence variation in an affected member of a given family is known (e.g., hemophilia B). Initially, from each subject, the DNA fragment containing the putative mutation site is amplified by the polymerase chain reaction. For each fragment two reaction mixtures are then prepared. Each contains the amplified fragment, a primer (18-mer or longer) whose sequence is identical to the coding sequence of the normal gene immediately flanking the 5' end of the mutation site, and either an alpha-P-32-labeled nucleotide corresponding to the normal coding sequence at the mutation site or an alpha-P-32-labeled nucleotide corresponding to the mutant sequence. Single nucleotide primer extensions are then carried out and analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography. As predicted by the Watson-Crick base-pair rule, in the wild type only the normal base, in an affected member only the mutant base, and in carriers both the normal and the mutant base are incorporated into the primer. Thus, an essential feature of the present methodology is that the base immediately 3' to the template-bound primer is one of those altered in the mutant, since in this way an extension of the primer by a single base will give an extended molecule characteristic of either the mutant or the wild type. The method is rapid and should be useful in carrier detection and prenatal diagnosis of every genetic disease with a known sequence variation.