Figure 3.

(a) A schematic outline of the mutagenesis protocol: Step 1, a single-stranded mutagenic DNA (smDNA) was amplified with the high T_m mutagenic primer and a high annealing temperature of 72°C, at which the activities of two low T_m flanking primers is wholly restrained. Step 2, the megaprimer is synthesized using the smDNA template and one of the flanking primers such as F₂ at a low annealing temperature (usually 36–46°C) and a low denaturing temperature (i.e. the T_ra), where the wild-type DNA template restores its double-stranded status. Step 3, by running the two procedures above in the same system, an entire mutagenic single-stranded template DNA can be first produced with the megaprimer at a high annealing temperature (usually 72°C) and then the desired mutagenic DNA will be obtained by using the other low T_m flanking primer such as R1 under the conditions of Step 2. Electrophoretic analysis of mutagenesis products using this protocol is shown on a 1.2% agarose gel. For instance (b) lane M, Marker DL2000; lane 1, PCR product during CNB/K134H mutagenesis (554 bp) with its megaprimer (151 bp); (c) and (d) lane M, Marker DL2000; lane 1, PCR product during CNA/RY112-3LT mutagenesis (1577 bp) with its megaprimer (365 bp).