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 characterize the conditions under which such recombinant amplification products form we monitored the exchange of sequence between two homologous templates carrying different restriction sites separated by 282 bp. Using a typical cycling program the rates of recombination between the two restriction sites were 1 and 7% using Taq and Vent polymerases respectively 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. Conversely, cycling programs designed to promote incomplete primer elongation and subsequent template strand exchange stimulated recombination to >20%.
Judo
M S
MS
Department of Biology and Center for the Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.
Wedel
A B
AB
Wilson
C
C
D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D015183:Restriction Mapping; D019914:Taq Polymerase; D013698:Templates, Genetic
D017931:DNA Primers; D019914:Taq Polymerase; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase