Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids

Abstract

Here we describe a straightforward, efficient, and reliable way to clone an insert of choice into a plasmid of choice without restriction endonucleases or T4 DNA ligase. Chimeric primers containing plasmid sequence at the 5' ends and insert sequence at the 3' ends were used to PCR-amplify insertion sequences of various sizes, namely the genes for GFP (gfp), beta-d-glucuronidase (gusA), and beta-galactosidase (lacZ), as well as the entire luxABCDE operon. These inserts were employed as mega-primers in a second PCR with a circular plasmid template. The original plasmid templates were then destroyed in restriction digests with DpnI, and the overlap extension PCR products were used to transform competent Escherichia coli cells. Phusion DNA polymerase was used for the amplification and fusion reactions, so both reactions were easy to monitor and optimize.

Figure 1. An outline of the overlap extension PCR cloning

(A) First, the insert is PCR-amplified with the chimeric primers so that the final PCR product has overlapping regions with the vector. (B) Then, vector and insert are mixed, denatured and annealed; the hybridized insert then is extended by Phusion DNA polymerase using vector as a template until polymerase reaches 5′ end of the insert. After several PCR cycles, the new plasmid with two nicks (one on each strand) gets accumulated as a product. (C) The new plasmid can be transformed into E. coli after the parental plasmid is destroyed by DpnI digest.

Figure 2. Analysis of the overlap extension PCR cloning reaction

(A) Products of the overlap extension PCR cloning reaction after 0, 5, 10, 15, 20, 25, and 30 cycles by agarose gel electrophoresis. Three nanograms of pQE30 vector were mixed with 175 ng insert (250 molar excess) in 10 μL total volume; a 4-μL aliquot of reaction was separated on a 0.8% agarose gel. M2, 1 kb DNA ladder; M1, assembled plasmid in closed circular and relaxed circular forms. (B) Overlap extension PCR cloning efficiency of a gfp gene as a function of the number of PCR cycles. Twenty microliters of competent E. coli cells were transformed with 1 μL pQE30/insert overlap extension PCR. The number of green colonies was plotted against the number of PCR cycles for each plate. (C) Overlap extension PCR cloning efficiency as a function of the insert length. Phusion DNA polymerase was used to PCR-amplify products of various sizes: GFP (gfp) gene, β-D-glucuronidase (gusA) gene, β-galactosidase (lacZ) gene, and the luxABCDE operon from the carrying pIMBB plasmid. These products were gel-purified and used in the overlap extension PCR reaction with pQE30 vector. Three nanograms of pQE30 vector were mixed with 175–500 ng insert in a total reaction volume of 10 μL and subjected to 18 cycles of PCR. After DpnI treatment, the overlap extension PCR products were used to transform competent E. coli cells. The number of colonies per plate was plotted against the size of the insert.