Figure 3

Recombination and oligonucleotide-mediated mutagenesis using ssDNAs. (A) SSCP-PAGE gel of phosphorylated (P) and hydroxylated (O) recombination cassettes in all four combinations (OO, OP, PO, PP) with (+) or without (-) Redα in vitro digestion. The lower bands show undenatured dsDNA (ds) and the upper bands show the slower migrating secondary structures of the single-strands after heat denaturation (up and low). M - size markers. (B) Schematic representations of the recombination reactions, which either replaced the ampicillin resistance gene (bla) in pBelo-BAC11 with the 500 nt ssDNA products shown in A, which contain the blasticidin resistance gene (bsd; left side) or repaired a 4 nt mutation in the kanamycin resistance gene (neo) using 100 nt single-stranded oligonucleotides (right side). Only Redβ was expressed in these experiments. The oligonucleotides were either complementary to the leading (Ld) or lagging (Lg) strands. (C, D) Quantification of recombinant colonies using the experimental designs shown in (B) and comparison of the wild type (wt) to either mutS (C) or sbcB (D) strains.