Construction of semi-randomized gene libraries with weighted oligonucleotide synthesis and PCR

Abstract

Randomized gene libraries may be constructed and screened to find novel candidates with particular functions, and the applications can range widely, from protein engineering to selecting new microRNAs. Here we describe a technique to construct gene libraries using semi-randomized weighted oligonucleotide synthesis and end-to-end ligation. This method makes it possible to search the combinatorial space around a particular nucleotide sequence for a greater number of positions than is possible with fully randomized oligonucleotides. As an alternative to full cassette construction, library mutations can also be introduced through 'round-the-world PCR' approaches. Construction of a randomized gene cassette and cloning can typically be achieved in 2 weeks. Therefore, these are rapid and convenient methods to generate successive generations of libraries for iterative selection and optimization.