Primer-directed mutagenesis of an intact plasmid by using Pwo DNA polymerase in long distance inverse PCR.

Oligonucleotide-directed mutagenesis is an important method for modifying specific bases of a DNA sequence (8). Current approaches include combining inverse polymerase chain reaction (PCR) with long PCR to introduce mutations directly into an intact plasmid template (1,5,10). However, the use of Taq DNA polymerase in these protocols results in a high rate of base mis-incorporation (3,7). Moreover, the inherent terminal transferase-like activity of the enzyme results in 3′ overhangs that must be blunt-ended before ligation (2). Screening of the clones is also laborious, if direct selection of the desired mutations is not possible. Our approach to this technique substitutes Pwo DNA Polymerase (Boehringer Mannheim, Laval, QC, Canada) for Taq polymerase and uses the mutagenic primers, not only in long distance inverse PCR, but also as probes for differential screening of transformed colonies. Pwo DNA polymerase has a highly processive 5′→3′ DNA polymerase activity. Unlike Taq, Pwo has 3′→5′ exonuclease “proofreading” activity and generates blunt-ended amplification products that can be directly phosphorylated and ligated. The enzyme also has a longer half-life at high temperatures as compared to Taq. Thus, enzyme stabilizing agents, such as glycerol, are not necessary. We successfully used Pwo DNA polymerase in a long distance (6.2 kb), inverse PCR as outlined below, to eliminate splice acceptor sites in the cloned E6/E7 open reading frame (ORF) of human papillomavirus type 16 (HPV-16). For long distance inverse PCR, the primer pairs are designed to anneal “back to back” on the double-stranded template. A separate pair of primers is required to alter each of the two splice acceptor sites located at nucleotide 409 and nucleotide 526 in the HPV-16 E6/E7 ORF. The mutagenic primer of each pair changes the last nucleotide at the 3′ end of the first intron and the first nucleotide at the 5′ end of the second exon, destroying the splice consensus sequence. Since the 3′→5′exonuclease activity of Pwo will target singlestranded DNA, the mutagenic bases must be centrally located in the PCR primer. The second primer of each pair is complementary to the wild-type template (Table 1). The PCR mixture contains 100 pmol of plasmid template DNA, 25 mM KCl, 10 mM Tris-HCl (pH 8.85), 5 mM (NH4)2SO4, 2.0 mM MgSO4, 5% dimethyl sulfoxide (DMSO) (Fisher Scientific, Winnipeg, MB, Canada), 200 μM each of dGTP, dCTP, dATP and dTTP (Pharmacia Biotech, Baie D’Urfe, QC, Canada), 5 U Pwo DNA polymerase (added at 80°C) and 1 μM primers, in a final reaction volume of 100 μL. Pwo DNA polymerase requires a slightly more alkaline buffer than Taq. The higher pH can affect the stability of the deoxynucleotides (dNTPs), thus these are added to the PCR immediately before the final addition of Pwo. To promote efficient primer annealing and decrease template “snap-back”, DMSO is included in the PCR as a destabilizing agent. The amplification cycle consists of an initial denaturation step at 94°C for 4 min, followed by 40 cycles of amplification with denaturation at 94°C for 1 min, primer annealing at 46°C for 1 min for the nucleotide 409 mutation and at 44°C for 1 min for the nucleotide 526 mutation and primer extension at 72°C for 6 min. The annealing temperature for the mutagenic primers is lowered from the temperature of dissociation (Td) of the primer by 6°C for each base mismatch. The final extension step is carried out for 10 min. The PCR products are purified by using a CHROMA SPIN-100 column (CLONTECH-Bio/Can Scientific, Mississauga, ON, Canada) to remove excess primers, nucleotides and incomplete amplification products of less than 100 bp in size. While incomplete amplification products remain, these molecules are not viable in a bacterial host. The purified amplification