Detection of KRAS rare mutation of codon 12 blue/white colony screening coupled with de-phosphorylation of enzymatic products
Aim To study the feasibility of detecting rare mutations in the 12 th 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∶1 000,or 1∶3 000,followed by restriction enzyme digestion and blue-white screening.In the experimental group,plasmids containing MUT/WT 12 were mixed at ra-tios of 0∶1,1∶3 000,1∶10 000,or 1∶30 000.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∶30 000,and the se-quences of the inserted fragments were verified through first-generation sequencing.Restriction enzyme digestion combined with de-phosphorylation 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∶3 000 ratio,there were 17 white clones and 2 329 blue clones,resulting in a white-to-blue clone ratio of 1∶137.In the experimental group with a 1∶30 000 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∶30 000 experimental group was significantly higher than that in the 1∶3 000 control group.Three positive clones were identified by enzymatic digestion of five positive clones on a 1∶30 000 culture dish in the experimental group.Restriction enzyme digestion combined with dephosphorylation and blue-white screening suc-cessfully identified 12 th codon mutations in KRAS gene in 2 out of 26 cases of lung cancer.Conclusion The use of restriction en-zyme digestion combined with dephosphorylation and blue-white screening allows for the detection sensitivity of rare KRAS gene muta-tions as 1∶30 000.
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