Ward R, Hawkins N, O'Grady R, Sheehan C, O'Connor T, Impey H, Roberts N, Fuery C, Todd A
Department of Medical Oncology, St. Vincent's Hospital, Sydney, New South Wales, Australia.
Am J Pathol. 1998 Aug;153(2):373-9. doi: 10.1016/S0002-9440(10)65581-2.
The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.
富集聚合酶链反应（PCR）检测方法已广泛应用于多种人类恶性肿瘤中ras基因突变的检测。尽管它非常灵敏，但仍有许多特性限制了其在常规诊断实验室中的应用。本研究的目的是开发一种新型的富集PCR策略，其中限制性内切酶BstNI和Taq聚合酶的协同活性可使突变型K-ras得到扩增，同时抑制野生型产物的形成。这种限制性内切酶介导的选择性PCR检测方法在反应混合物中使用三组引物以及BstNI，扩增产物通过凝胶电泳进行分析。在包括139例新鲜结直肠癌以及来自80个不同的结肠、直肠、胰腺、乳腺或肾脏肿瘤的113个石蜡包埋块等多种临床样本中，确定了限制性内切酶介导的选择性PCR检测方法检测活化K-ras的可靠性。以快速、灵敏且可重复的方式在新鲜和石蜡包埋肿瘤的DNA中鉴定出了K-ras癌基因的第12密码子突变。在33例（24%）新鲜结直肠癌和16例（20%）石蜡包埋肿瘤中检测到了突变。在同一肿瘤的石蜡块和新鲜标本均可用于分析的病例中，这些结果的一致性为97%。我们得出结论，限制性内切酶介导的选择性PCR是一种用于检测多种临床样本中点突变的灵敏、快速且可靠的检测方法。重要的是，一旦设置好PCR，就无需对样本进行处理，因此污染的可能性显著降低。与先前的检测方法相比，请点击此处查看完整翻译内容，限制性内切酶介导的选择性PCR不需要大量人力，其形式适合在常规诊断实验室中使用。
