Gradient PCR screening and sequence analysis of the transformants generated by SDM without Dpn I digestion. A. Gradient PCR screening of multiple-site-mutant D112K. Five transformants were examined with the WT template as criteria under a set of gradient annealing temperatures from 64°C to 69°C. The identified Tc was 66°C at which the WT template lost detectable products. B. Sequence analysis of the same transformants, which shows that three transformants (D112K-2, 4 and 5) still detectable at annealing temperatures over Tc were desired mutants. C. Gradient PCR screening of single-site-mutant D112G. The identified Tc was 68°C. D. Sequence analysis of the same transformants showing that two transformants (D112G-2 and 4) still detectable at annealing temperatures over Tc were desired mutants.