限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测KRAS基因第12位稀有突变

Detection of KRAS rare mutation of codon 12 blue/white colony screening coupled with dephosphorylation of enzymatic products

目的研究限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测KRAS基因第12位密码子稀有突变的可行性。方法将采用限制性内切酶酶切后去磷酸化联合蓝白斑筛选检测方法作为实验组,限制性内切酶酶切联合蓝白斑筛选检测方法作为对照组。对照组将KRAS基因第12位密码子突变型(MUT)/野生型(WT)(MUT/WT 12)质粒按0∶1、1∶300、1∶1000、1∶3000的比例混合,限制性内切酶酶切PCR产物,酶切后产物进行蓝白斑筛选。实验组将MUT/WT 12质粒按0∶1、1∶3000、1∶10000、1∶30000的比例混合,在对照组方法的基础上酶切产物去磷酸化后再蓝白斑筛选。比较两组蓝白斑克隆数。限制性内切酶酶切鉴定实验组MUT/WT 12为1∶30000的阳性克隆,一代测序阳性克隆验证插入片段的序列。采用限制性内切酶酶切后去磷酸化联合蓝白斑筛选验证26例肺癌病例游离DNA的KRAS基因第12位稀有突变。结果对照组1∶3000的培养皿上白色克隆17个,蓝色克隆2329个,白色/蓝色克隆为1/137;而实验组1∶30000的培养皿上白色克隆23个,蓝色克隆394个,白色/蓝色克隆为1/17,1∶30000实验组白色/蓝色克隆比例明显高于1∶3000对照组。实验组1∶30000培养血上5个阳性克隆酶切鉴定出3个阳性,其插入片段为KRAS第12位突变片段。限制性内切酶酶切后去磷酸化联合蓝白斑筛选26例肺癌病例可检测出2例为KRAS基因第12位密码子突变。结论采用限制性内切酶酶切后去磷酸化联合蓝白斑筛选可检测出1∶30000 KRAS基因稀有突变。

Aim To study the feasibility of detecting rare mutations in the 12th codon of the KRAS gene through restriction endonuclease digestion followed by dephosphorylation combined with blue and white spot screening.Methods The experimental group employed restriction enzyme digestion,dephosphorylation,combined with blue-white screening,while the control group utilized restriction enzyme digestion followed by blue-white screening.In the control group,plasmids containing KRAS gene codon 12 mutant(MUT)and wild type(WT)(MUT/WT 12)sequences were mixed at ratios of 0∶1,1∶300,1∶1000,or 1∶3000,followed by restriction enzyme digestion and blue-white screening.In the experimental group,plasmids containing MUT/WT 12 were mixed at ratios of 0∶1,1∶3000,1∶10000,or 1∶30000.In addition to the methods used in the control group,the digested products in the experimental group were subjected to dephosphorylation before blue-white screening.The number of blue-white clones was compared between the two groups.The experimental group was identified as positive clones with a MUT/WT 12 ratio of 1∶30000,and the sequences of the inserted fragments were verified through first-generation sequencing.Restriction enzyme digestion combined with dephosphorylation and blue-white screening were used to validate 12th codon mutations in rare KRAS gene in 26 cases of lung cancer.Results In the control group with a 1∶3000 ratio,there were 17 white clones and 2329 blue clones,resulting in a white-to-blue clone ratio of 1∶137.In the experimental group with a 1∶30000 ratio,there were 23 white clones and 394 blue clones,resulting in a white-to-blue clone ratio of 1∶17.The white/blue clone ratio in the 1∶30000 experimental group was significantly higher than that in the 1∶3000 control group.Three positive clones were identified by enzymatic digestion of five positive clones on a 1∶30000 culture dish in the experimental group.Restriction enzyme digestion combined with dephosphorylation and blue-white screening successfully identified 12th codon mutations in KRAS gene in 2 out of 26 cases of lung cancer.Conclusion The use of restriction enzyme digestion combined with dephosphorylation and blue-white screening allows for the detection sensitivity of rare KRAS gene mutations as 1∶30000.