US 5,409,818 AGranted

Nucleic acid amplification process

AssigneeCangene Corporation

InventorsCheryl Davey, Lawrence T. Malek

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Abstract

This invention relates to a process for amplifying a specific nucleic acid sequence. The process involves synthesizing single-stranded RNA, single-stranded DNA and double-stranded DNA. The single-stranded RNA is a first template for a first primer, the single-stranded DNA is a second template for a second primer, and the double stranded DNA is a third template for synthesis of a plurality of copies of the first template. A sequence of the first primer or the second primer is complementary to a sequence of the specific nucleic acid and a sequence of the first primer or the second primer is homologous to a sequence of the specific nucleic acid. The amplification process may be used to increase the quantity of the specific nucleic acid sequence to allow detection, or to increase the purity of the specific nucleic acid sequence as a substitute for conventional cloning methodology.

Claims40

Dependent claims

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.

16 more elements

Dependencies are inferred from the claim text.