Direct cloning of PCR products amplified with Pwo DNA polymerase.

The cloning of polymerase chain reaction (PCR) products provides one with a stable form of the amplified segment and facilitates further manipulation and study of the target molecule. A number of cloning protocols have been developed either based on a restriction endonuclease recognition site built into the primers or by using the template-independent terminal transferase activity (“extendase” activity) of Taq DNA polymerase to add “A” residues at the end of each molecule (basis for T/A cloning) (4,5). It has been shown recently (3) that the extended nucleotide is dependent on the specific nucleotide present at the 3′ end of the molecule. Moreover, different polymerases have different extendase characteristics with regard to which base is added at the 3′ end, and this might significantly complicate the cloning of the corresponding fragments. Blunt-end cloning is an attractive alternative, but it is also hampered by the extendase activity intrinsic to most of the polymerases used for PCR amplifications. The cloning efficiency of Taq-generated PCR products can be greatly improved by “polishing” the ends with polymerases derived from bacteriophage T4 or from Pyrococcus furiosus (Pfu), but this implies that extra manipulations be performed (1,2). In this study we wanted to test the efficiency of the blunting polymerase Pwo (Boehringer Mannheim, Mannheim, Germany) for direct blunt-end cloning of PCR amplification products. Pwo DNA polymerase was originally isolated from the hyperthermophilic archaebacterium Pyrococcus woesei and possesses 3′–5′ exonuclease activity, also known as proofreading activity. In our laboratory we are currently analyzing flower-specific cDNA clones that were obtained after subtractive hybridization. Tertiary screen-positive plaques were eluted in 500 μL of phage dilution buffer (6), and 1 μL of this bacteriophage suspension was used to amplify the cDNA insert with the flanking SP6 and T7 primers in a standard 25-μL PCR containing 10 mM Tris-HCl, pH 8.85, 25 mM KCl, 5 mM (NH4)2SO4, 2 mM MgSO4, 0.2 mM each dATP, dCTP, dGTP, dTTP, 150 ng of each SP6 and T7 primer, and 2.5 units of Pwo DNA polymerase (Boehringer Mannheim). The reaction proceeded for 30 cycles in a Techne PHC-3 thermal cycler (Princeton, NJ, USA) programmed for 30 s at 94°C, 30 s at 55°C and 30 s at 72°C per cycle, followed by an extension step of 10 min at 72°C. After amplification, the PCR products were purified using the QIAquick PCR purification kit (QIAGEN, Chatsworth, CA, USA). Different amounts (ranging from 5 to 50 times in excess over vector concentration) of PCR product were then ligated into 50 ng of SmaI-digested pGem®-3Zf(-) Vector (Promega, Madison, WI, USA), and the complete ligation mixture was used to transform the E. coli XL1-blue strain, which was subsequently plated on LB agar plates containing isopropyl-1-thio-β-D-galactoside (IPTG) and 5-bromo-4-chloro-3indolyl-β-D-galactoside (X-gal) using standard procedures (6). Four different cDNA inserts ranging in size from 500 bp up to 1400 bp were successfully cloned using the above-mentioned protocol. Depending on the experiment, the obtention of recombinant clones for each insert varied between 10% and 15%, resulting in a sufficient number of colonies containing the correct amplification product. Ligations of the same inserts amplified using Taq DNA polymerase (Boehringer Mannheim) or Goldstar (EurogentecSA, Seraing, Belgium) DNA polymerase did not result in the obtention of recombinant clones. Polishing the Taq DNA polymerase or Goldstar amplification products with Klenow DNA polymerase prior to