27] Random chimeragenesis by heteroduplex recombination

Abstract

Publisher Summary Genetic variations existing in nature or created in the laboratory can be recombined to generate libraries of molecules containing novel combinations of sequence information from any or all of the parent sequences. By combining beneficial mutations and removing deleterious ones, recombination may help to accelerate the evolution of single molecules toward a specified function. Novel chimeric sequences generated by the recombination of homologous, naturally occurring genes also provide an extremely rich source of genetic diversity for directed evolution. This chapter describes a simple method for creating libraries of chimeric deoxyribonucleic acid (DNA) sequences derived from homologous parental sequences that uses in vivo repair of heteroduplexes for recombination. This heteroduplex recombination approach relies on the mismatch repair system of the host cells to repair regions of nonidentity in the heteroduplex and creates a library of new sequences composed of elements from each parent. This method combines the advantages of in vitro and in vivo recombination methods and avoids some of their drawbacks. Heteroduplex recombination does not require polymerase chain reaction (PCR) amplification and should be useful for the recombination of large DNA sequences.