An optimized procedure to mutagenize long (>10 kb) plasmids by PCR

Abstract

▼A simple method to mutagenize a DNA sequence that is contained in a plasmid involves the use of two oligonucleotide primers that are located back-to-back on the opposite DNA strands of the sequence to be modified (Ref. 1). PCR amplification thus generates a linear DNA that covers the whole plasmid length, which, upon self-ligation, renders the modified version of the original plasmid in a single step (Fig. 1). Previous reports (Ref. 1) have pointed out that the total length of the plasmid to be manipulated represents a significant limitation of this method, which was found feasible for plasmids of ∼3 kb. Here, we describe an optimized procedure for the application of this technique to plasmids that are >10 kb, which should be of interest in genomic analysis, generation of vectors for Drosophila transformation, transfection and other applications. The first challenge was to obtain enough product of the appropriate length with correct termini. A variety of thermostable polymerases were used (see Fig. 2). Enzymes that are currently used for PCR mutagenesis include the DNA polymerases from the thermophilic bacteria Pyrococcus furiosus (Pfu) and Thermococcus litoralis (Vent). Neither of these enzymes (lanes 4 and 5) nor Taq polymerase (lane 6) were able to generate reasonable amounts of full-length PCR product under a variety of experimental conditions. A mixture of the P. woesei (Pwo) and Taq polymerases generated products that were apparently full-length (lane 7) but that upon closer examination had deletions of 3–25 nucleotides, most likely owing to the 3′ exonuclease activity of the Pwo enzyme under the conditions of limiting concentration of dNTPs in the late cycles of amplification. Full-length products with correct termini were amplified only in reactions that contained either the Thermus thermophilus Tth polymerase (lane 8) or mixtures of Pfu and