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

作者：Liu, Ying; Wu, Ting; Song, Jian; Chen, Xuelian; Zhang, Yu; Wan, Yu^*

来源：BMC Biotechnology, 2013, 13(1): 21.

DOI：10.1186/1472-6750-13-21

• Mutant screening
• Dpn I digestion
• Critical annealing temperature-PCR
• Site-directed mutagenesis

Background: Distinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange (TM) 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.

• 出版日期2013-3-11
• 单位武汉大学