Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction.
We demonstrate the utility of antibodies to the DNA polymerase from Thermus aquaticus (TaqPol) as thermolabile inhibitors of TaqPol activity. One of the limitations of the polymerase chain reaction (PCR) is the co-amplification of non-specific products caused by TaqPol activity on low stringency templates present in the initial cycle of PCR. We have used anti-TaqPol antibodies as thermolabile switches that inhibit TaqPol activity at low temperatures (20-40 degrees C) and release fully active TaqPol when they are inactivated by elevated temperatures in the PCR thermal cycling (70-98 degrees C). Several in a set of high affinity anti-TaqPol monoclonal antibodies fully inhibited TaqPol activity at 37 degrees C. The capacity for inhibition was ablated by incubation at temperatures high enough to denature antibodies but not sufficiently high to significantly reduce TaqPol activity. In a PCR model system, preincubation of TaqPol with these antibodies yielded PCR product consisting entirely of the intended product and the absence or significant reduction of non-specific products and primer dimers. In evaluation of clinical samples such antibody triggering yielded defined PCR product and higher sensitivity because of the absence of non-specific products.
Sharkey
D J
DJ
Clinical Diagnostics Research Laboratories, Eastman Kodak Company, Rochester, NY 14650-2117.
Scalice
E R
ER
Christy
K G
KG
Atwood
S M
SM
Daiss
J L
JL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D004259:DNA-Directed DNA Polymerase / Q000276:immunology*; D006358:Hot Temperature; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D051379:Mice; D019384:Nucleic Acid Synthesis Inhibitors; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D019384:Nucleic Acid Synthesis Inhibitors; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase