论文部分内容阅读
发展了一种可用于快速检测K-ras癌基因点突变的电化学发光PCR(ECL-PCR)分析方法,该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;随后,采用限制性内切酶MvaI对扩增产物进行酶切,由于突变导致酶切位点的丢失,所以只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到反应池中进行电化学发光检测。采用该法对20例结肠癌组织中的K-ras癌基因第12位密码子进行点突变分析,得出其中有9例存在点突变,点突变率为45%。该方法操作简便、安全、快速、灵敏,可用于快速检测K-ras癌基因点突变。
An electrochemiluminescence PCR (ECL-PCR) method for the rapid detection of point mutations in K-ras oncogene has been developed. The method uses a ternary bipyridine ruthenium-labeled upstream primer and a biotin-labeled downstream primer to perform PCR Followed by digestion of the amplified product with the restriction enzyme MvaI, only the wild-type sample can be cleaved due to the mutation resulting in the loss of the cleavage site; biotin and streptavidin coating Of the magnetic beads connected to the biotin-labeled DNA fragments collected in the reaction cell for electrochemical luminescence detection. Point mutation analysis of the 12th codon of K-ras oncogene in 20 cases of colon cancer was carried out by this method. It was found that 9 of them had point mutation and the point mutation rate was 45%. The method is simple, safe, fast and sensitive, and can be used for rapid detection of K-ras oncogene point mutations.