Alexander Volkov

Directed evolution of biocatalysts.

Directed evolution is being used increasingly in academic and industrial laboratories to modify and improve important biocatalysts. Significant advances during this period of review include compartmentalization of genes and the in vitro translation apparatus in emulsions, as well as several impressive demonstrations of catalyst improvement. Shuffling of homologous genes offers a new way to utilize natural diversity in the evolution of novel catalysts.

[26] Methods for in vitro DNA recombination and random chimeragenesis

Publisher Summary In vitro polymerase chain reaction (PCR)-based methods for recombining homologous deoxyribonucleic acid (DNA) sequences are capable of creating highly mosaic chimeric sequences. Several different methods have been reported for in vitro recombination or DNA shuffling: for example, the original Stemmer method of DNase I fragmentation and reassembly, the staggered extension process (STEP), and random priming recombination. Slight variations in the shuffling protocols can affect the outcome of the experiment. Different gene sequences recombine most efficiently under different conditions. This chapter provides protocols that are designed to give a high likelihood of success. The protocols are known to work for recombining sequences of ∼>85% identity.

[27] Random chimeragenesis by heteroduplex recombination

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.