Offset recombinant PCR: a simple but effective method for shuffling compact heterologous domains.
DNA shuffling and other in vitro recombination strategies have proven highly effective at generating complex libraries for mutagenesis studies. While most recombination techniques employ DNA polymerases in part of a multi-step process, few seek to exploit the natural recombinogenic tendencies and exponential amplification rates of PCR. Here, we characterize a simple but effective method for using standard PCR to promote high recombination frequencies among compact heterologous domains by locating the domains near one end of the template. In a typical amplification reaction, Pfu polymerase generated chimeric crossover events in 13% of the population when markers were separated by only 70 nt. The fraction of recombinant sequences reached 42% after six consecutive rounds of PCR, a value close to the 50% expected from a fully shuffled population. When homology within the recombinant region was reduced to 82%, the recombination frequency dropped by nearly half for a single amplification reaction and crossover events were clustered toward one end of the domain. Surprisingly, recombination frequencies for template populations with high and low sequence homologies converged after just four rounds of PCR, suggesting that the exponential accumulation of chimeric molecules in the PCR mixture serves to promote recombination within heterologous domains.
Rozak
David A
DA
Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850, USA. rozak@umbi.umd.edu
Bryan
Philip N
PN
D032621:DNA Shuffling / Q000379:methods*; D017930:Genes, Reporter; D005819:Genetic Markers; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011995:Recombination, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D005819:Genetic Markers; D001616:beta-Galactosidase