Random Mutagenesis of Short Target DNA Sequences via PCR with Degenerate Oligonucleotides

Abstract

Polymerase chain reaction (PCR) is a powerful technique for the amplification of specific DNA sequences, for the generation of chimeric molecules, and for site-directed mutagenesis. It allows the rapid generation of relatively large quantities of mutated DNA. Drawbacks are the costs of synthesizing oligonucleotide primers and the high error rates of thermostable polymerases relative to other DNA polymerases. In cases where extensive collections of mutations in defined target sequences are desired, it is often more efficient and cost effective to synthesize degenerate oligonucleotides in a single step. One elegant method to create primer libraries with the potential of all possible point mutations is to dope each of the four nucleoside phosphoamides with small amounts of the other three (1). AlternativeIy, primers can also be generated by allowing misincorporation only at selected sites (2). The frequencies and types of mutations in the degenerate oligonucleotides can be easily varied by the selection of doping nucleotide percentages at each step of the synthesis. In the early protocols, the complementary mutagenic oligonucleotides bore cohesive ends (3), or contained flanking restriction sites (4), to allow cloning. These approaches did not have general utility because of the need for conveniently located restriction sites at the ends of the target sequence. In this chapter, we present a method that uses complex oligonucleotide mixtures to generate numerous changes in short target DNA sequences by PCR via splicing by overlap extension (SOE-PCR) (5,6). The use of SOE-PCR makes it possible to use restriction sites distant from the target sequence for easy cloning, and also to minimize the size of the mutagenized PCR fragment compared to other PCR-based strategies in which complete plasmids are generated (7–9). However, it is also possible to use recombinant PCR (R-PCR), in which the entire plasmid is amplified during PCR and recovered by homologous recombination after transformation of Escherichia coli (9). It has recently been described that using alkaline denatured plasmid template results in both reduced background of wild-type clones and higher efficiencies of amplification (10). SOE-and R-PCR with degenerate oligonucleotides provide rapid, simple, and effective means to generate a large number of mutant clones in defined target sequences.