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发展了一种可用于快速检测胰腺癌中K-ras癌基因点突变的电化学发光-聚合酶链式反应(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;再采用限制性内切酶MvaI对扩增产物进行酶切。由于野生型样品和突变型样品间存在酶切位点的变化,其中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片段收集到检测池中,进行电化学发光检测。采用该法对13例胰腺癌组织中的K-ras癌基因第12位密码子进行点突变分析,只需要10μL样品、20min孵育时间和30s采集时间,就可得出其中有12例存在点突变,点突变率为92.3%。本方法操作简便、安全、快速、灵敏,可用于检测任何一种导致限制性内切酶位点改变的基因点突变。
An electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for the rapid detection of point mutations in K-ras oncogene in pancreatic cancer has been developed. The method uses the ternary pyridine ruthenium labeled upstream primer and the biotin labeled downstream primer to amplify the target fragment by PCR; and then uses the MvaI restriction enzyme to digest the amplification product. Since there was a change in restriction sites between the wild-type and mutant samples, only the wild-type sample could be cleaved; biotin-labeled DNA fragments were collected by biotin-streptavidin-coated magnetic beads To the detection pool, the electrochemical luminescence detection. Using this method, point mutation analysis of the 12th codon of K-ras oncogene in 13 pancreatic cancer tissues was carried out. Only 10μL sample, 20min incubation time and 30s acquisition time were needed to obtain point mutations in 12 of them , The point mutation rate was 92.3%. The method is simple, safe, fast and sensitive, and can be used for detecting any point mutation of a gene which causes the change of a restriction enzyme site.