Nucleic acid amplification process

Claim 1

A process for the amplification of a specific nucleic acid sequence, at a relatively constant temperature and without serial addition of reagents, comprising the steps of: (A) providing a single reaction medium containing reagents comprising (i) a first oligonucleotide primer, (ii) a second oligonucleotide primer comprising an antisense sequence of an RNA polymerase promoter, (iii) a DNA-directed RNA polymerase that recognizes said promoter, (iv) an RNA-directed DNA polymerase, (v) a DNA-directed DNA polymerase, (vi) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single- or double-stranded RNA or DNA, and (vii) ribonucleoside and deoxyribonucleoside triphosphates; then (B) providing in said reaction medium RNA comprising an RNA first template which comprises said specific nucleic acid sequence or a sequence complementary to said specific nucleic acid sequence, under conditions such that a cycle ensues wherein (i) said first oligonucleotide primer hybridizes to said RNA first template, (ii) said RNA-directed DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms an RNA-DNA hybrid intermediate, (iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid intermediate, (iv) said second oligonucleotide primer hybridizes to said DNA second template, (v) said DNA-directed DNA polymerase uses said second oligonucleotide primer as template to synthesize a functional RNA polymerase promoter by extension of said DNA second template; and (vi) said DNA-directed RNA polymerase recognizes said functional promoter and transcribes said DNA second template, thereby providing copies of said RNA first template; and thereafter (C) maintaining said conditions for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.