Influence of DNA polymerases on quantitative PCR results using TaqMan (TM)probe format in the LightCycler (TM) instrument

Real-time fluorescence polymerase chain reaction (PCR) techniques are incre asingly used to quantitate target sequences for diagnostic and research pur poses. Currently the so called TaqMan(TM) probe chemistry is mostly used as fluorogenic system. This probe format is strictly dependent on the 5'-exon uclease activity of DNA polymerase as fragmentation of the probe during the reaction is essential for this assay. Based on our experience that dramati c differences in quantitative PCR results may be due to different DNA polym erases we performed a detailed comparison of 15 enzymes. We found that clea r differences exist between polymerases of different manufacturers. Thus, t hree out of seven polymerases which were declared to posses 5'-exonuclease activity appeared to be completely unsuitable for this method while the rem aining had significantly different reaction efficiencies. We conclude that different DNA polymerases may determine the entire analytical performance o f TaqMan(TM) assays suggesting that DNA polymerase testing is of special im portance when this probe format is used. (C) 2000 Academic Press.