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

Abstract

Abstract Background Distinguishing 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 (T c)-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 T c-PCR with the same mutagenic primers was performed without Dpn I digestion. The T c for each pair of the primers was identified by gradient PCR. The relationship between PCR-identified T c and T m of the primers was analyzed and modeled with correlation and regression. Results Gradient PCR identified a T c for each of 14 tested mutagenic primers, which could discriminate mismatched parental molecules and undesired mutants from desired mutants. The PCR-identified T c was correlated to the primer’s T m (r = 0.804, P<0.0001). Thus, in practical applications, the T c can be easily calculated with a regression equation, T c = 48.81 + 0.253*T m. Conclusions The new protocol introduced a novel T c-PCR method for mutant screening which can more efficiently and accurately select against parental molecules and undesired mutations in mutagenic sequence segments.