Offset recombinant PCR: a simple but effective method for shuffling compact heterologous domains.

ABSTRACT: 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

SUBMITTER: Rozak DA 

PROVIDER: S-EPMC1130489 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature