STIMULATION AND SUPPRESSION OF PCR-MEDIATED RECOMBINATION

Recombination, or chimera formation, is known to occur between related template sequences present in a single PCR amplification. To characte rize the conditions under which such recombinant amplification product s form we monitored the exchange of sequence between two homologous te mplates carrying different restriction sites separated by 282 bp. Usin g a typical cycling program the rates of recombination between the two restriction sites were 1 and 7% using Tag and Vent polymerases respec tively over 12 doublings, However, by using long elongation times and cycling only to the mid-point of the amplification recombination could be suppressed below visual detection with both polymerases, Conversel y, cycling programs designed to promote incomplete primer elongation a nd subsequent template strand exchange stimulated recombination to > 2 0%.