Open AccessComparison between Taq DNA Polymerase and Its Stoffel Fragment for Quantitative Real-Time PCR with Hybridization Probes
Author(s) -
Jochen Wilhelm,
Alfred Pingoud,
Meinhard Hahn
Publication year - 2001
Publication title -
biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/01305rr04
Subject(s) - taqman , taq polymerase , microbiology and biotechnology , primer dimer , real time polymerase chain reaction , polymerase chain reaction , oligonucleotide , polymerase , multiple displacement amplification , hybridization probe , biology , dna , polymerase chain reaction optimization , dna polymerase , chemistry , thermus aquaticus , multiplex polymerase chain reaction , biochemistry , dna extraction , gene
In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan ® probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in Light-Cycler ® assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart ® antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.
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