A New Assay Of In Vitro Genotype Switch Using High Fidelity DNA Polymerase Coupled With Restriction Endonuclease

Objectives: To constitute an in vitro genotype switch through coupling high-fidelity DNA polymerase and restriction endonuclease via specific primers.Methods: Wild-type sequence template of CDH1 gene carrying HaeIII restriction enzyme cutting site and mutation sequence template without the cutting site were constructed. The wild-typed and mutant gene segments were mixed with mutant gene segment according to the proportion of W:T= 10:1, 100:1, 1000:1, and 10000:1 to mimic the clinical situations where circulating tumor DNAs are rare an circulating wild typed DNA are predominant in early state cancer patients. High-fidelity DNA polymerase was adopted for PCR amplification when Hae III enzyme existed. Wild-type gene fragment was largely digested due to the existence of Hae III enzyme, and the amplification efficiency was thus decreased. The mutant fragment has no restriction site to HaeIII and is resistant to the digestion by this endonuclease.Results: The high fidelity DNA polymerase Pfu and HaeIII work well together in one system. The combination of these two enzymes has successfully changed the ratios of the wild typed and mutant gene fragments. For the 10:1 of the CDH1 gene c.67C>T, the in vitro genotypes switch completely reversed it to about 2:3. For the other 3 mixed ratios, the in vitro genotype switch has successfully increased the amount of the mutant gene fragments, making them from undetectable to detectable by Saner’s sequencing.Conclusions: Taking the advantages of high-fidelity DNA polymerase and heat-resistant restriction enzyme, this experiment constitutes an in vitro genotype switch and tested its effectiveness in detecting CDH1 gene c.67C>T single nucleotide mutation of various ratios mixing the wild typed and the mutants. This system possesses immediate potential applications early diagnosis of cancer and in monitoring of cancer recurrence.