The PCR Polishing Kit is designed to increase the blunt-ended cloning efficiencies associated with polymerase chain reaction (PCR)-generated fragments. Recent studies have shown that many species of DNA polymerases (e.g., T7, modified T7, Taq, VentR and Klenow fragment) exhibit terminal deoxynucleotidyltransferase-like activity (extendase). It has been found that the 3´-end nucleotide extension of PCR products by DNA polymerases is both nucleotide and polymerase specific. Each DNA polymerase, including the Klenow fragment, has characteristic terminal extendase activity with no consistent pattern of 3´-end modification. Therefore, it cannot be assumed that all DNA polymerases create blunt-ended fragments. Fortunately, Pfu DNA polymerase does not exhibit any DNA extendase and can be used to create blunt-ended fragments following PCR. Due to the unique 3´ -> 5´ exonuclease (proofreading) activity of the Pfu DNA polymerase, Taq DNA polymerase-generated PCR products can be polished with Pfu DNA polymerase following temperature cycling to create blunt-ended DNA fragments for use in cloning, mutagenesis and cDNA construction.
The PCR Polishing Kit is designed to polish the ends of the 3´-overhang extensions of polymerase-generated DNA fragments directly from a PCR amplification reaction. The PCR Polishing Kit can also be used to perform complete fill-in of 5´ overhangs to generate blunt ends. Dramatic increase in the population of blunt-ended DNA fragments following polishing treatment results in a drastic increase in overall experimental efficiency associated with procedures utilizing blunt-ended ligation.
For Research Use Only. Not for use in diagnostic procedures