Specific inhibition of the polymerase chain reaction using a non-extendable oligonucleotide blocker

US5849497A · kind A · utility

Assignee

• The Research Foundation of the State University of New York · US

Inventor

• Charles Steinman · Setauket, US

Key dates

• Filing date: Apr 3, 1997
• Grant date: Dec 15, 1998
• Expiry date: Apr 3, 2017

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/686
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A process is disclosed for inhibiting the amplification of a DNA template by subjecting a sample of biological material containing nucleic acid to the polymerase chain reaction (PCR) using a DNA polymerase deficient in 5' exonuclease activity. The method comprises forming a PCR admixture comprising the DNA template, first and second oligonucleotide primers which are complementary to separated regions of the nucleic acid template, a non-extendable oligonucleotide blocker which is complementary to the inter-primer region of the DNA, and the DNA polymerase lacking 5' exonuclease activity, and subjecting the PCR admixture to at least one PCR thermocycle. The DNA polymerase lacking 5' exonuclease activity is incapable of excising the non-extendable blocker which anneals to the DNA template during the PCR, thereby inhibiting amplification which would otherwise occur during the PCR. Preferably, the DNA polymerase lacking 5' exonuclease activity is the Stoffel fragment of Taq polymerase. The method may be adapted for detecting whether DNA from specific pathogens is present in a sample material.