To Develop New Assays For The Detection Of CtDNA Rare Mutations By Targeting The C.4012 Hotspot Mutation Of APC Gene

Objective: To develop new assays for the detection of rare mutations in clinical samples mixed with predominant amount of wild typed DNAs.Methods: By targeting the c.4012 hotspot mutation of APC gene, the present study has documented the feasibility of a new strategy applicable in rare mutation analysis. The wild typed fragments contain a Pst I restriction site while the mutant fragments do not have this restriction site, which allows a discrimination by the restrictive endonuclease of PstI. This study tested the possibilities of applying restriction enzyme digestion combined with blue/white screening technology in analyzing a hotpot point mutation in APC gene. Furthermore, an in vitro genotype switch has been developed to detect this APC hotspot mutation.Results: In combination with restriction endonuclease digestion, PCR, blue/white screening, and Sanger’s sequencing technologies, the present study tested the feasibility of using the restriction nuclease in human somatic mutation of the hotspot mutation at APC gene. Our data have demonstrated that the strategy of integrating three conventional technologies in one assay has higher sensitivity than any single method involved in this new strategy. The features of high sensitivity and low or no false positives render this new strategy clinically applicable in ctDNA assay to hotspot mutations suitable for this new strategy.Conclusion: The strategy tested in the present study has provided a highly efficient, sensitive, and specific assay for somatic mutation analysis targeting c.4012 at APC gene.