×

Nucleic acid amplification process

  • US 5,409,818 A
  • Filed: 06/24/1988
  • Issued: 04/25/1995
  • Est. Priority Date: 02/24/1988
  • Status: Expired due to Term
First Claim
Patent Images

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.

View all claims
  • 3 Assignments
Timeline View
Assignment View
    ×
    ×