蓝白斑联合Sanger测序技术检测KRAS基因12位密码子突变  

Analysis of 12 codon mutation in KRAS gene by blue-white spot and Sanger's sequencing technique

作　　者：温博雅 彭翠英 徐畅 周翠兰 WEN Boya;PENG Cuiying;XU Chang;ZHOU Cuilan(Hengyang Medical College,University of South China,Hengyang 421001,Hunan,China;Clinical Anatomy and Reproductive Medicine Application Institute,Hengyang Medical College,University of South China,Hengyang 421001,Hunan,China;The Key Laboratory of Ecological Environment and Critical Human Diseases Prevention of Hunan Province,Department of Education,Hengyang 421001,Hunan,China;The Key Laboratory of Hengyang City on Biological Toxicology and Ecological Restoration,Hengyang 421001,Hunan,China;The Key Laboratory of Hengyang City on Ecological Impedance Technology of Heavy Metal Pollution in Cultivated Soil of Nonferrous Metal Mining Area,Hengyang 421001,Hunan,China)

机构地区：[1]南华大学衡阳医学院,湖南衡阳421001 [2]南华大学衡阳医学院应用解剖学与生殖医学研究所,湖南衡阳421001 [3]生态健康与人类重要疾病防控湖南省高校重点实验室,湖南衡阳421001 [4]生物毒理与生态修复衡阳市重点实验室,湖南衡阳421001 [5]有色金属矿区耕地重金属污染生态阻抗技术研究衡阳市重点实验室,湖南衡阳421001

出　　处：《中南医学科学杂志》2020年第6期647-650,667,共5页Medical Science Journal of Central South China

基　　金：湖南省教育厅重点项目(15A168);湖南省自然科学基金(2018JJ2325,2017JJ3280,2020JJ4536);湖南省教育厅一般项目(17C1385);衡阳市科技局项目(2018KJ111,2016KF14);大学生创新课题(S201910555128);博士科研启动金(2018XQD12).

摘　　要：为研究蓝白斑联合Sanger测序检测KRAS基因12位密码子突变的可行性,将M12突变型质粒与WT野生型质粒按1∶30,1∶100,1∶300,1∶1000的比例混合以模拟游离DNA(cDNA),利用蓝白斑技术联合Sanger测序对KRAS基因稀有突变进行检测。结果显示,蓝白斑技术联合Sanger测序,可以检测出达到0.1%的KRAS基因稀有突变,无假阳性。通过改变阅读框架,使密码子能转变为终止密码子TAA,TGA,TAG的体细胞突变,可采用蓝白斑技术联合Sanger测序技术进行检测。to study the feasibility of detecting the 12 codon hot spot point mutation in KRAS gene by the blue/white screening in combination with Sanger's sequencing technology.Wild type plasmid was mixed with M12 mutant plasmid at ratios of 1∶30,1∶100,1∶300 and 1∶1000 and the final concentration of wild type plasmid was 2 ng/μL.The introduction of designed restriction sites had been developed with white clones,which comes from mutant versus wild-type at ratio of 1∶1000.A clone was performed by the protocol.Then the white clones were identified by PCR and sequencing techniques.Blue/white screening in combination with Sanger's sequencing could successfully detect rare mutants in KRAS gene in mixtures at the ratio as high as 0.1‰.No false positive was found.Somatic mutations that could be transformed into termination codons TAA,TGA and TAG by changing the reading framework might be detected by the blue/white screening in combination with Sanger's sequencing technology.

关 键 词：KRAS基因 蓝白斑技术 Sanger测序 PCR 

分 类 号：Q782[生物学—分子生物学]