Oligonucleotide inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR.
A random sequence library of single stranded DNA was screened to isolate sequences with high affinity for Thermus aquaticus DNA polymerase (Taq pol), a thermostable enzyme commonly used in the polymerase chain reaction (PCR). Selected oligonucleotide sequences bound Taq pol with dissociation constants in the low picomolar range, and efficiently inhibited polymerase activity at room temperature (20 to 25 degrees C), but did not inhibit at temperatures above 40 degrees C. Moreover, inhibition was thermally reversible. A process called "hot start" PCR is commonly used to prevent non-specific PCR products in amplification of low copy number targets. We show that the addition of oligonucleotide inhibitors eliminated the need for "hot start" conditions and improved the efficiency of detection of a low copy number target in PCR.
Dang
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NeXstar Pharmaceuticals Inc., Boulder, CO 80301, USA.
Jayasena
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D004279:DNA, Viral / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase; D018628:Gene Dosage; D015498:HIV-2 / Q000235:genetics*; D006361:Heating; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013379:Substrate Specificity; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology
D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D019914:Taq Polymerase; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase