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A sensitive and high-resolution method for detection of mutations in the p53 gene using multiple fluorescence-based symmetric PCR-SSCP analysis

机译：基于多重荧光对称PCR-SSCP分析的p53基因突变的灵敏且高分辨率检测方法

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摘要

Mutations of the p53 gene are an important feature of neoplastic progression in humans. The presence of such mutations has been detected in exons 5 through 8, which contain 86% of all mutations reported for the p53 gene. We have developed a simple and rapid method, multiple fluorescence-based symmetric polymerase chain reaction in a single tube and single-strand conformation polymorphism analysis in one lane (MF-SPCR-SSCP) for detection of mutations in exons 5, 6, 7 and 8 of the p53 gene. These four exons were amplified by MF-SPCR technique in a single tube using mixed four-color fluorescence-labeled sense and antisense primers. This technique enabled labeling of each exon of the product with a unique fluorescent. The MF-SPCR products were heat-denatured and applied to 7% polyacrylamide gel containing 5% glycerol set on an automated DNA sequencer with a gel temperature-controlling system. We analyzed 18 specimens of lung cancer tissue with mutations in exons 5 through 7 using MF-SPCR-SSCP method. These mutations were detected even with use of only one PCR and one set of conditions for electrophoresis.
机译：p53基因的突变是人类肿瘤进展的重要特征。在第5至第8外显子中检测到此类突变的存在，该外显子占p53基因报道的所有突变的86％。我们已经开发了一种简单而快速的方法，在单管中进行基于多个荧光的对称聚合酶链反应，并在一个泳道中进行单链构象多态性分析（MF-SPCR-SSCP），以检测外显子5、6、7和5的突变。 p53基因的8个。使用混合的四色荧光标记的有义和反义引物，通过MF-SPCR技术在单个试管中扩增这四个外显子。这项技术可以用独特的荧光标记产品的每个外显子。将MF-SPCR产物加热变性，然后将其施加到含有5％甘油的7％聚丙烯酰胺凝胶上，该凝胶在带有凝胶温度控制系统的自动DNA测序仪上设置。我们使用MF-SPCR-SSCP方法分析了18个肺癌组织标本，其外显子5到7有突变。即使仅使用一种PCR和一套电泳条件，也可以检测到这些突变。

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