USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction

Abstract

USER friendly DNA recombination (USERec) is introduced as a near homology-independent method that allows the simultaneous recombination of an unprecedented number of 10 DNA fragments (approximately 40-400 bp) within a day. The large number of fragments and their ease of preparation enables the creation of libraries of much larger genetic diversity (potentially approximately 10(10)-10(11) sequences) than current alternative methods based on DNA truncation (ITCHY, SCRATCHY and SHIPREC) or type IIb restriction enzymes (SISDC). At the same time, the frequency of frameshifts in the recombined library is low (90% of the recombined sequences are in frame). Compared to overlap extension PCR, USERec also requires much reduced crossover sequence constraints (only a 5'-AN(4-8)T-3' motif) and fewer experimental steps. Based on its simplicity and flexibility, and the accessibility of large and high quality recombined DNA libraries, USERec is established as a convenient alternative for the combinatorial assembly of gene fragments (e.g. exon or domain shuffling) and for a number of applications in gene library construction, such as loop grafting and multi-site-directed or random mutagenesis.