An efficient bipartite PCR technique to introduce specific changes in large plasmids

Davis, Kevin and Ladds, Graham and Das, Anamika and Goddard, Alan and Davey, John (2004) An efficient bipartite PCR technique to introduce specific changes in large plasmids. Molecular Biotechnology, 28 (3). pp. 201-204. ISSN 1073-6085

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Abstract

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.

Item Type:Article
Additional Information: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.
Keywords:Antibiotic resistance, Large plasmid, PCR, Site-directed mutagenesis
Subjects:C Biological Sciences > C700 Molecular Biology, Biophysics and Biochemistry
Divisions:College of Science > School of Life Sciences
ID Code:6824
Deposited By: Alan Goddard
Deposited On:15 Nov 2012 16:26
Last Modified:15 Nov 2012 16:26

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