The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis

Abstract

Significant innovations in molecular biology methods have vastly improved the speed and efficiency of traditional restriction site and ligase-based cloning strategies. "Enzyme-free" methods eliminate the need to incorporate constrained sequences or modify Polymerase Chain Reaction (PCR)-generated DNA fragment ends. The Polymerase Incomplete Primer Extension (PIPE) method further condenses cloning and mutagenesis to a very simple two-step protocol with complete design flexibility not possible using related strategies. With this protocol, all major cloning operations are achieved by transforming competent cells with PCR products immediately following amplification. Normal PCRs generate mixtures of incomplete extension products. Using simple primer design rules and PCR, short, overlapping sequences are introduced at the ends of these incomplete extension mixtures which allow complementary strands to anneal and produce hybrid vector/insert combinations. These hybrids are directly transformed into recipient cells without any post-PCR enzymatic manipulations. We have found this method to be very easy and fast as compared to other available methods while retaining high efficiencies. Using this approach, we have cloned thousands of genes in parallel using a minimum of effort. The method is robust and amenable to automation as only a few, simple processing steps are needed.