Figure 4.

Scheme of the approach used to produce templates containing unnatural nucleotides isodG and 5-Me-isodC. (A) Pairs of primers complementary to each other, so that the forward primer carried 5-Me-isodC in the second position of the V29 codon, and the reverse primer carried isodG, and vice versa. (B) In the first round of PCR, two fragments were amplified using primers flanking both ends of the template and mutant primers carrying unnatural nucleotides. (C) In the second round of amplification, these PCR products were mixed without adding primers flanking ends of the template and performed 10 amplification cycles without using unnatural nucleoside triphosphates. (D) Single-stranded DNA molecules and unreacted fragments remaining in the reaction were removed by agarose gel electrophoresis.