In vitro DNA recombination by random priming.

1. Introduction Variation coupled to selection is the hallmark of natural evolution. Although there is no full agreement concerning the best way to create variation , mutation or recombination (1), computational simulation studies have demonstrated the importance of homologous recombination in the evolution of biological systems (2,3). As compared to random mutagenesis, recombi-nation may be advantageous in combining beneficial mutations that have arisen independently and may be synergistic, while simultaneously removing deleterious mutations. The principles of natural evolution may be equally applied to the molecular evolution directed by an experimenter. For example, natural random mutagen-esis is commonly simulated by the use of error prone PCR, while natural recombination is simulated by various in vitro recombination-based methods. These directed evolution methods have been extremely effective in engineering proteins, metabolic pathways, and whole genomes with improved or novel functions (4–6). The power of in vitro recombination was first demonstrated by Stemmer in 1994 with the technique of DNA shuffling or sexual PCR (7,8), in which DNA fragments generated by random digestion of parental genes with DNase I are combined and reassembled into full-length chimerical progeny genes in a PCR-like process. Since then, a number of in vitro recombina-tion methods have been developed, including the staggered extension process (StEP) recombination (9), random-priming in vitro recombination (RPR) (10) and random chimeragenesis on transient templates (RACHITT) (11). Here, we describe the method and protocol of random-priming in vitro recombination (RPR). As illustrated in Fig. 1, RPR relies on the reassembly of DNA fragments that are created by extension of random primers rather than by