Figure 2.

Primer selection for mutagenesis. In the mutagenic primer, the underlined codon includes the italicized substitution that encodes the Thr-37 to Ala mutation. The solid arrows describe the endpoints for the PCR1 reaction product; the dashed arrows describe the endpoints for the PCR2 reaction. The mutagenesis primer contains the desired Thr-37 to Ala mutation with a TGG → CGG substitution in the Thr-37 codon that introduces an analytically useful MscI restriction site (ACC/GGT).