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Recombinant PCR 517
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Recombination and Site-Directed Mutagenesis
Using Recombination PCR
Douglas H. Jones and Stanley C. Winistorfer
1. Introduction
The polymerase chain reaction (PCR) (1) provides a rapid means for the recombination
and site-directed mutagenesis of DNA (2). DNA modification can occur during
PCR because the primers are incorporated into the ends of the PCR product. The
simplest PCR-based method for site-directed mutagenesis and DNA recombination
is recombination PCR.
Recombination PCR uses in vivo recombination in Escherichia coli to generate sitedirected
mutants and recombinant constructs (3,4). In the recombination PCR method,
PCR adds homologous ends to DNA. These homologous ends mediate recombination
between these linear PCR products in E. coli, resulting in the formation of DNA joints
in vivo. If two PCR products have homologous ends that can recombine to form a circle,
and if this circle constitutes a selectable plasmid, E. coli can be readily transformed
by the linear PCR products. Recombination PCR has almost no steps apart from PCR
amplification and transformation of E. coli, and this method works well in Rec A minus
E. coli strains used routinely in cloning. Since the introduction of this method in 1991,
it has been used by numerous investigators (5–9). One example of DNA recombination
using recombination PCR is illustrated in Fig. 1, which shows a protocol for amplifying
a portion of a donor plasmid and inserting it in a recipient plasmid at a defined position
and orientation. The donor plasmid is shown on the left side and the recipient plasmid is
on the right side. The steps corresponding to this figure are briefly outlined below:
1. The DNA segment that is to be inserted into the recipient plasmid is amplified from
the donor plasmid using primers 1 and 2. In a separate PCR amplification, the recipient
plasmid is amplified with primers 3 and 4. The 5′ regions of primers 1 and 2 that do not
anneal to the donor plasmid are complementary to primers 4 and 3, respectively (or 3
and 4, depending on the orientation of the insert desired in the recombinant construct).
Frequently, a plasmid template can be linearized outside the region to be amplified by
restriction endonuclease digestion before PCR amplification. When this can be done,
the PCR product does not need to be purified, because linearized plasmids transform E. coli
inefficiently. If a plasmid template cannot be linearized by restriction endonuclease digestion
outside the region to be amplified prior to PCR amplification, the PCR product must
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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