Random Mutagenesis Using Error-Prone DNA Polymerases

"Random mutagenesis" is a technique that allows researchers to develop large libraries of variants of a particular DNA sequence. Once developed, these libraries can then be used for several purposes, including structure-function and directed evolution studies. Random mutagenesis is different from other mutagenesis techniques in that it does not require the researcher to have any prior knowledge about the structural properties of the DNA sequence being targeted, thus allowing for the unbiased discovery of novel or beneficial mutations. For this reason, random mutagenesis is especially useful for protein evolution studies. This protocol describes mutagenic replication in vitro by a low-fidelity DNA polymerase followed by selective polymerase chain reaction (PCR) amplification of the newly mutated sequences. The initial mutagenic DNA replication step is accomplished by heat-denaturing the template DNA and annealing primers possessing 5' extensions that are not complementary to the template. The purpose of the noncomplementary extensions on the primers is to allow for future selection of only the mutant strands. DNA replication is then performed by a low-fidelity DNA polymerase of choice (polymerase β, η, or ι, or any combination of the three). After mutations have been incorporated into the template, the mutagenized strands are then selectively amplified using PCR. Selective amplification of the mutant strands is accomplished by performing a PCR procedure consisting of a first cycle with a low hybridization temperature followed by subsequent selection cycles under higher hybridization temperatures that do not allow amplification of the original unmutagenized template.