Terminal repeat amplification method

US5665545A · kind A · utility

Assignee

• AKZO NOBEL N.V. · NL

Inventors

• Lawrence T. Malek · Brampton, CA
• Roy Rabindranauth Sooknanan · Beaconsfield, CA

Key dates

• Filing date: Nov 28, 1994
• Grant date: Sep 9, 1997
• Expiry date: Nov 28, 2014

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6865
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

This invention relates to a process for amplifying a specific nucleic acid sequence or its complement at a relatively constant temperature and without serial addition of reagents. The process provides in a single reaction medium an RNA polymerase, DNA polymerase, a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single or double-stranded RNA or DNA, and ribonucleoside and deoxyribonucleoside triphosphates. The process then provides an RNA first template in the reaction medium. The RNA first template comprises a sequence complementary to a specific nucleic acid sequence, minus-sense sequences for a promoter and initiation site that are recognized by the RNA polymerase, and a 5'-terminal sequence that is complementary to at least the minus-sense sequence of the initiation site. Thus, the RNA first template has an inverted repeat sequence which could fold into a 5'-terminal stem-loop structure. The DNA polymerase uses the RNA first template to synthesize a DNA second template that together comprise an RNA-DNA hybrid. The DNA second template has plus-sense sequences of the promoter and the initiation site, and a 3'-terminal priming sequence that is complementa…