PLEASE SUPPLY SHORT TITLE 201 MOLECULAR BIOTECHNOLOGY Volume 28, 2004 HINTS AND TIPS 201 Molecular Biotechnology © 2004 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2004/28:3/201–204/$25.00 *Author to whom all correspondence and reprint requests should be addressed. 1 Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK; 2 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. E-mail: J.Davey@Warwick.ac.uk. Abstract An Efficient Bipartite PCR Technique to Introduce Specific Changes in Large Plasmids Kevin Davis, 1 Graham Ladds, 2 Anamika Das, 2 Alan Goddard, 2 and John Davey* ,2 Amplifying an entire double-stranded plasmid by an inverse polymerase chain reaction (PCR) using a pair of tail-to-tail primers is a particularly efficient approach for introducing changes into DNA sequences. However, the approach generally works best for plasmids less than 5 Kb and it can be difficult to amplify the large multicomponent vectors that are used for protein expression in various eukaryotic cells. We have therefore adopted an alternative approach in which two smaller PCR products are generated and then ligated to produce the complete plasmid. A mutagenic primer is used to introduce the desired change and each reaction includes one of a pair of tail-to-tail primers from within an antibiotic resistance gene contained on the plasmid so that the two PCR products contain complementing parts of the complete gene. Ligating the two products generates various combinations but only the correctly ligated molecules recreate the antibiotic resistance gene and are able to replicate in Escherichia coli. When combined with methods to minimize the carryover of template plasmid, this can be an efficient way of introducing mutations into large plasmids. Index Entries: Site-directed mutagenesis; PCR; large plasmid; antibiotic resistance. Site-directed mutagenesis is a powerful ap- proach for exploring protein structure and func- tion, and many different techniques have been developed to introduce specific changes in the amino acid residues of a wild-type protein (1). Most approaches involve a polymerase chain re- action (PCR) in which a mutagenic primer is used to amplify a particular section of DNA. The prod- uct, incorporating the changes directed by the mutagenic primer, is then used to generate a mu- tated construct. For target sequences available in plasmids, a particularly efficient method is to use inverse PCR (2,3). In this approach a pair of tail- to-tail primers is made from the site of the de- sired mutation and used to amplify the entire double-stranded plasmid containing the desired gene. To introduce mutations, one of the two primers used is mutagenic. The resulting linear double-stranded PCR product is then circularized by ligating the two blunt ends and introduced into Escherichia coli for propagation and analysis. Inverse PCR is simple and effective and has been used to generate mutant plasmids as large as 11 Kb (2). However, it generally works best for plasmids less than 5 Kb and it can be difficult to amplify large plasmids. The technique is therefore unsuitable for many of the multicomponent vectors used for protein expression in various systems. For example, yeast expression vectors contain elements that allow replication and selection in both bacteria and yeast, as well as the transcriptional and trans- lational elements required for expression of the cloned gene. Plant and mammalian expression vectors contain the equivalent elements. Because inverse PCR can be very inefficient on such large templates, we have developed an alternative ap- proach in which two smaller PCR products are generated and then ligated to produce the complete