title: A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis.
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.
authors.affiliation: Department of Physiology, School of Basic Medical Sciences, Wuhan University, Hubei, 430071, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Ting
authors.initials: T
authors.lastname: Wu
authors.forename: Jian
authors.initials: J
authors.lastname: Song
authors.forename: Xuelian
authors.initials: X
authors.lastname: Chen
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yu
authors.initials: Y
authors.lastname: Wan
mesh_terms: D000818:Animals; D020137:Base Pair Mismatch; D004247:DNA / Q000032:analysis*; D017931:DNA Primers / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D013006:Growth Hormone / Q000235:genetics; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011986:Receptors, Somatotropin / Q000235:genetics; D013552:Swine; D013696:Temperature
chemical_list: D017931:DNA Primers; D011986:Receptors, Somatotropin; D013006:Growth Hormone; D004247:DNA; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific