Influence of DNA polymerases on quantitative PCR results using TaqMan probe format in the LightCycler instrument.
Real-time fluorescence polymerase chain reaction (PCR) techniques are increasingly used to quantitate target sequences for diagnostic and research purposes. Currently, the so called TaqMan probe chemistry is mostly used as fluorogenic system. This probe format is strictly dependent on the 5'-exonuclease activity of DNA polymerase as fragmentation of the probe during the reaction is essential for this assay. Based on our experience that dramatic differences in quantitative PCR results may be due to different DNA polymerases we performed a detailed comparison of 15 enzymes. We found that clear differences exist between polymerases of different manufacturers. Thus, three out of seven polymerases which were declared to possess 5'-exonuclease activity appeared to be completely unsuitable for this method while the remaining had significantly different reaction efficiencies. We conclude that different DNA polymerases may determine the entire analytical performance of TaqMan assays suggesting that DNA polymerase testing is of special importance when this probe format is used.
Kreuzer
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KA
Abteilung für Innere Medizin und Poliklinik m.S. Hämatologie und Onkologie, Medizinische Fakultät Charité der Humboldt-Universität zu Berlin, Germany.
Bohn
A
A
Lass
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U
Peters
U R
UR
Schmidt
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CA
D002138:Calibration; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005453:Fluorescence; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D019914:Taq Polymerase / Q000378:metabolism
D017931:DNA Primers; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase