Patent ID: 6025134
Filing Date: 2000-02-15
Classification: C12Q

Abstract:
A process for the amplification of a specific DNA sequence in a sample, comprisinga) providing in one reaction vessel reagents comprising(i) a first DNA-directed RNA polymerase wherein said polymerase is capable of non-specifically transcribing a double-stranded DNA template independent of specific promoter sequences;(ii) a double-stranded DNA first template comprising the specific DNA sequence;(iii) ribonucleoside triphosphates;(iv) deoxyribonucleotide triphosphates;(v) a first oligonucleotide primer comprising a plus sequence of a promoter recognized by a second RNA polymerase;(vi) a second oligonucleotide primer;(vii) a second RNA polymerase which is a DNA-directed RNA polymerase that recognizes said promoter;(viii) an RNA-directed DNA polymerase;(ix) a DNA-directed DNA polymerase;(x) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA; andunder conditions such that,(1) said first RNA polymerase uses said DNA first template to synthesize an RNA first template which comprises an RNA sequence which corresponds to said specific DNA sequence or a sequence complementary to said specific DNA sequence,(2) said first oligonucleotide primer hybridizes to said RNA first template,(3) 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,(4) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid intermediate,(5) said second oligonucleotide primer hybridizes to said DNA second template,(6) said DNA-directed DNA polymerase uses said second oligonucleotide primer and said DNA second template to synthesize a functional promoter recognized by said second RNA polymerase, and(7) said second RNA polymerase recognizes said functional promoter and transcribes said DNA second template, thereby providing copies of an RNA second template; andb) maintaining the conditions for a time sufficient such that a cycle ensues to achieve amplification of the specific DNA sequence.