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World J Microbiol Biotechnol (2011) 27:727730 DOI 10.1007/s11274-010-0498-0
SHORT COMMUNICATION
Reliable fusion PCR using Taq polymerases and pGEM-T easy vectors
Ruisheng An Xiaodong Bai Parwinder S. Grewal
Received: 28 May 2010 / Accepted: 28 June 2010 / Published online: 14 July 2010 Springer Science+Business Media B.V. 2010
Abstract We describe a reliable method for the production of fusion PCR products that can be used to transform the wild-type bacteria to replace target genes for muta-genesis studies. The relevant gene fragments and selective cassette are rst amplied separately by PCR using primers that produce overlapping ends. As economic Taq DNA polymerase is disappointed in producing overlap ends due to adding an extra 30-end A base which potentially blocks the successful fusion of the amplied fragments, we use a new primer design strategy to overcome this disadvantage by introducing an additional A base in the overlap primers. The amplied gene fragments were then separately cloned into a pGEM-T easy vector and re-amplied with the aid of a universal primer T7/SP6. This procedure enables performing nested PCR with the outmost primers in the fusion reaction to reliably splice the gene fragments into a single molecule with all sequences in the desired order.
Keywords Gene fusion Overlap extension PCR
Taq polymerase Primer design
Introduction
The Red-mediated recombination procedure which has been used to inactivate chromosomal genes in various bacteria consists of electroporating a PCR fragment
carrying an antibiotic marker anked by a region homologous to the target locus into a strain that expresses the Red recombination system (Lesic and Rahme 2008). Overlap extension PCR has been previously described as a method for generating such PCR product by inserting a selective marker into the target gene without need of restriction sites (Horton et al. 1989). In general, the marker and two adjacent gene fragments are prepared separately using appropriate primers which include four overlap primers derived partly from the marker and partly from the target gene and two outermost primers. An overlap extension PCR is then carried out to combine three fragments using the outermost primer pair. The spliced gene fragment can be further introduced into wild-type bacterial cells for homologous recombination to replace the target genes.
This technique was originally claimed to have 100% efciency for splicing gene fragments...