Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.
Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.
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DOI：
10.1093/carcin/19.6.979
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发表时间：
1998-06
期刊：
影响因子：
4.7
通讯作者：
R. Rines;N. V. van Orsouw;I. Sigalas;F. Li;C. Eng;J. Vijg
中科院分区：
文献类型：
--
作者：
R. Rines;N. V. van Orsouw;I. Sigalas;F. Li;C. Eng;J. Vijg
设计并评估了基于多重PCR和二维DNA电泳的p53编码区全面突变扫描测试。在两步多重 PCR 中，p53 编码区（外显子 2-11）在第一步中通过长距离 PCR 被扩增为单个 8646 bp 片段。该片段用作随后在第二步中对两个多重组中的各个外显子进行共扩增的模板。然后，首先根据非变性聚丙烯酰胺凝胶中的大小，然后根据变性梯度凝胶电泳 (DGGE) 的序列来分离多重产物。使用最近开发的计算机程序设计了最佳 PCR、熔解行为和二维凝胶分布的引物。由此产生的二维基因扫描 (TDGS) 测试通过以盲法筛选来自 Li-Fraumeni 综合征患者的 29 个编码 DNA 样本进行评估，这些患者之前已发现了种系突变。所有突变均被正确检测到。该测定提供了一种准确、经济高效且非放射性的方法，用于同时对所有 p53 编码外显子进行突变扫描。
A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.