Staggered Extension Process (StEP) In Vitro Recombination

Abstract

In vitro polymerase chain reaction (PCR)-based recombination methods are used to shuffle segments from various homologous DNA sequences to produce highly mosaic chimeric sequences. Genetic variations created in the laboratory or existing in nature can be recombined to generate libraries of molecules containing novel combinations of sequence information from any or all of the parent template sequences. Evolutionary protein design approaches, in which libraries created by in vitro recombination methods are coupled with screening (or selection) strategies, have successfully produced variant proteins with a wide array of modified properties including increased drug resistance (1,2), stability (3, 4, 5, 6), binding affinity (6), improved folding and solubility (7), altered or expanded substrate specificity (8,9), and new catalytic activity (10).

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