A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis

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BackgroundDistinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange™ mutagenesis. Although Dpn I digestion can eliminate methylated parental (WT) DNA, the efficiency is not satisfying due to the existence of hemi-methylated DNA in the PCR products, which is resistant to Dpn I. The present study designed a novel critical annealing temperature (Tc)-PCR to replace Dpn I digestion for more perfect mutant distinguishing, in which part-overlapping primers containing mutation(s) were used to reduce initial concentration of template DNA in mutagenic PCR. A Tc-PCR with the same mutagenic primers was performed without Dpn I digestion. The Tc for each pair of the primers was identified by gradient PCR. The relationship between PCR-identified Tc and Tm of the primers was analyzed and modeled with correlation and regression.ResultsGradient PCR identified a Tc for each of 14 tested mutagenic primers, which could discriminate mismatched parental molecules and undesired mutants from desired mutants. The PCR-identified Tc was correlated to the primer’s Tm (r = 0.804, P<0.0001). Thus, in practical applications, the Tc can be easily calculated with a regression equation, Tc = 48.81 + 0.253*Tm.ConclusionsThe new protocol introduced a novel Tc-PCR method for mutant screening which can more efficiently and accurately select against parental molecules and undesired mutations in mutagenic sequence segments.