Overlap extension PCR cloning: a simple and reliable way to create recombinant plasmids (1)
The first of two PCRs creates a linear insert with plasmid sequences at both ends. These extensions subsequently allow the strands of the PCR product to act as a pair of oversized primers on the vector fragment. After denaturation and annealing, the insert strands hybridize to the vector and extend to form new double- stranded plasmid. This relaxed double- stranded plasmid is then transformed into competent Escherichia coli cells, which seal the nicks with DNA repair enzymes.
The gfp gene was PCR-amplified with the chimeric primers (5′ ends complementary to the pQE30 plasmid; 3′-end complementary to gfp). Overlap extension PCR was performed with five different DNA polymerases. High concentra- tions of the insert and relatively low annealing temperatures in the reaction (5–10°C below the calculated melting temperature of the primer/plasmid complex) are important for efficient overlap extension.
Some of the PCR products correspond to the relaxed form of the desirable vector, as revealed by agarose gel analysis. The number of recombinant clones increased geometri- cally during the first 15 cycles and peaked at 17–18 cycles. Further cycles resulted in a slight (~30%) decrease in the quantity of clones produced, associated with the accumulation of the high–molecular weight DNA products observed in agarose gels. Three different vector:insert ratios (1:5; 1:50 and 1:250) resulted in the appearance of the nicked form of the plasmid. The graph is almost linear, which suggests that 6.7 kb is the upper limit for inserts with this technique.
The stringency of PCR can be controlled by altering reactant concentrations (primers, template), annealing temperature, buffer ingredients (magnesium, pH, DMSO) or the number of temperature cycles.