A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to...

Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.