John M. S. Bartlett.pdf - Bio-Nica.info

518 Jones and Winistorfer
Fig. 1. Diagram illustrating DNA recombination using recombination PCR. The primers are
numbered hemiarrows. The insert is the cross-hatched region. Smooth circles represent the DNA
strands of the donor plasmid. Circles with wavy and jagged portions represent DNA strands of
the recipient plasmid. Reprinted by permission from Biotechniques 10, 62–66.
be removed from the plasmid before transformation to prevent background transformants
arising from the supercoiled plasmid template. PCR product purification is accomplished
either by agarose gel purification followed by glass bead extraction or by adding the
restriction endonuclease DpnI to the PCR mixture. DpnI is a restriction endonuclease that
digests methylated GATC sites. These sites are methylated in the plasmid by strains of
E. coli used routinely in cloning (by dam methylase), but are not methylated in the PCR
products, permitting DpnI to digest the plasmid without cutting the PCR product (10).
2. The two PCR products are combined and used to transform MAX efficiency competent
E. coli (BRL, Life Technologies, Gaithersburg, MD). If each plasmid template is restriction
endonuclease digested outside the region to be amplified prior to PCR amplification,
the two crude PCR products can simply be combined, and the resulting mixture used
to transform E. coli.
In a simple variation of this recombination PCR strategy, the inserted segment can be an
unmodified PCR product. In that case, primers 3 and 4 have 5′ ends that are homologous
to the ends of the PCR fragment to be inserted, and the recipient plasmid is linearized
by restriction endonuclease digestion prior to PCR amplification. We routinely use this
approach to clone any PCR product (4).