应用焦磷酸测序法检测进展期结直肠癌中K-ras基因点突变

Detection of K-ras mutation in advanced colorectal carcinoma by Pyrosequencing

目的建立基于焦磷酸测序技术(Pyrosequencing)的结直肠癌K-ras基因点突变的检测方法,并应用于进展期结直肠癌及其转移灶中K-ras基因突变的检测。方法提取116例Ⅳ期结直肠癌及11例转移灶中的DNA,进行PCR扩增后应用Pyrosequencing法检测K-ras基因12及13密码子点突变。结果 116例Ⅳ期结直肠癌中有29例检出K-ras基因点突变,突变率为25.0%,在所有29例突变病例中12密码子点突变率为82.8%(24/29),13密码子点突变率为17.2%(5/29),所有11例转移灶的K-ras基因均与其原发病灶相一致。结论 Pyrosequencing能准确、快速、高通量地进行K-ras基因点突变测定,适合大规模临床标本检测,有利于指导结直肠癌尤其是转移性病变的个体化治疗。

Objective To establish a method for detecting K-ras gene point mutation in colorectal carcinoma （ CRC） based on the pyrosequencing. Methods Genomic DNA was extracted from formalin-fixed,paraffin-embedded CRC tissues,including 127 primary CRC and 20 metastasis lension from CRC. K-ras gene point mutations at codon 12 and codon 13 were analyzed by pyrosequencing. Result K-ras gene point mutations was found in 33 CRC cases,mu-tation rate was 26. 0%. Of all 33 mutation cases,point mutation rate at codon 12 and codon 13 was 81. 8% and 18. 2% respectively. The status of K-ras in metastasis sites were accord with their primary CRC in all 20 cases. Con-clusions Pyrosequeccing is a accurate,rapid and of high throughput means detecting K-ras gene point mutation,it may serve as a practical method for fast batch analysis of clinical samples.