An error prone PCR method for small amplicons

Abstract

Error-prone PCR (epPCR) is a commonly employed approach in molecular biology, especially in directed evolution, to generate libraries of DNA molecules with broad mutational spectrums. Though commonly applied to mutagenize protein coding sequences of several hundreds or thousands of basepairs, we found that commonly used protocols were not suitable for small (<100 bp) amplicons. Here we report a modified error-prone PCR protocol utilizing a Touchdown approach and employing only commercially available components, that should be broadly useful for the researcher interested in concentrating mutations into a small region of plasmid DNA. It will also be useful for achieving very high mutational loads on a standard-sized amplicon.