US 5,766,849 AGranted

Methods of amplifying nucleic acids using promoter-containing primer sequence

AssigneeGen-Probe Incorporated

InventorsSherrol H. McDonough, Daniel L. Kacian, Nanibhushan Dattagupta, Diane L. McAllister, Philip W. Hammond, Thomas B. Ryder

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Abstract

A method, composition and kit for synthesizing multiple copies of a target nucleic acid sequence autocatalytically under conditions of substantially constant temperature, ionic strength, and pH are provided in which multiple RNA copies of the target sequence autocatalytically generate additional copies using a mixture of blocked and unblocked primers and/or promoter-primers to initiate DNA and RNA synthesis, preferably with reduced non-specific product formation. The invention is useful for generating copies of a nucleic acid target sequence for purposes that include assays to quantitate specific nucleic acid sequences in clinical, environmental, forensic and similar samples, cloning and generating probes.

Claims53

Dependent claims

A method for producing multiple copies of a target nucleic acid comprising the steps of:

a) incubating, under conditions in which an oligonucleotide/target sequence complex is formed and DNA and RNA synthesis occurs, a mixture comprising:
a nucleic acid comprising a (+) target sequence,
a first oligonucleotide comprising a primer sequence able to hybridize at or near the 3'-end of said (+) target sequence, a 5' promoter sequence, and a modification at or near the 3' end of said first oligonucleotide primer sequence which reduces or blocks extension of said first oligonucleotide primer sequence by a polymerase compared to said first oligonucleotide primer sequence not having said modification,
a second oligonucleotide comprising a primer sequence able to hybridize at or near said 3'-end of said (+) target sequence, a 5' promoter sequence, and an optionally present modification at or near the 3' end of said second oligonucleotide primer sequence which reduces or blocks extension of said second oligonucleotide primer sequence by a polymerase compared to said second oligonucleotide primer sequence not having said modification, wherein said second oligonucleotide hybridizes to said (+) target sequence in effectively the same position as said first oligonucleotide and said second oligonucleotide modification, if present, is different than said first oligonucleotide modification,
a third oligonucleotide comprising a primer sequence able to hybridize to the 3'-end of a (-) target nucleic acid sequence which is produced during said method and which is the complement of said (+) target sequence, an optionally present 5' promoter sequence, and an optionally present modification at or near the 3' end of said third oligonucleotide primer sequence able to reduce or block extension of said third oligonucleotide primer sequence by a polymerase compared to said third oligonucleotide primer sequence not having said modification,
at least one enzyme activity selected from the group consisting of:
DNA dependent DNA polymerase and RNA dependent DNA polymerase, and
one or more RNA polymerases able to recognize said first and said second oligonucleotide 5' promoter sequences,
wherein under said incubating conditions said third oligonucleotide facilitates production of said (+) target sequence using said (-) target sequence as a template, and
b) producing said multiple copies of said target sequence.

7 more elements

A method for producing multiple copies of a target nucleic acid sequence comprising the steps of:

a) incubating, under conditions in which an oligonucleotide/target sequence complex is formed and DNA and RNA synthesis occurs, a mixture comprising:
a nucleic acid comprising a (+) target sequence,
a first oligonucleotide comprising a primer sequence able to hybridize to the 3'-end of said (+) target sequence, an optionally present 5' promoter sequence, and an optionally present modification at or near the 3' end of said first oligonucleotide primer sequence able to reduce or block extension of said first oligonucleotide primer sequence by a polymerase compared to said first oligonucleotide primer sequence not having said modification,
a second oligonucleotide comprising a primer sequence able to hybridize at or near the 3'-end of a (-) target nucleic acid sequence which is produced during said method and which is the complement of said (+) target sequence, a 5' promoter sequence, and a modification at or near the 3' end of said second oligonucleotide primer sequence which reduces or blocks extension of said second oligonucleotide primer sequence by a polymerase compared to said second oligonucleotide primer sequence not having said modification,
a third oligonucleotide comprising a primer sequence able to hybridize at or near said 3'-end of said (-) target sequence, a 5' promoter sequence, and an optionally present modification at or near the 3' end of said third oligonucleotide primer sequence which reduces or blocks extension of said third oligonucleotide primer sequence by a polymerase compared to said third oligonucleotide primer sequence not having said modification, wherein said third oligonucleotide hybridizes to said (-) target sequence in effectively the same position as said second oligonucleotide and said third oligonucleotide modification, if present, is different than said second oligonucleotide modification,
at least one enzyme activity selected from the group consisting of:
DNA dependent DNA polymerase and RNA dependent DNA polymerase, and
one or more RNA polymerases able to recognize said second and said third oligonucleotide 5' promoter sequences,
wherein under said incubating conditions said first oligonucleotide facilitates production of said (+) target sequence using said (-) target sequence as a template, and
b) producing said multiple copies of said target sequence.

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 08/345,861Filed 28 Nov 1994 · continuation
Priority: 11 Jul 1989
Granted: 16 Jun 1998
Term ends: Not estimatedThe source records don’t support an estimate.

Classification7 codes

C12Q 1/6865
Nucleic acid amplification reactions: promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence…
C07K 14/35
From bacteria: from Mycobacteriaceae
C12N 15/1096
Processes for the isolation, preparation or purification of DNA or RNA: cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
C12Q 1/689
For detection or identification of organisms: for bacteria

Family

Serial379,501Parent of this continuation-in-part · filed 11 Jul 1989
Serial550,837Parent of this continuation-in-part · filed 10 Jul 1990
Serial855,732Parent of this continuation-in-part · filed 19 Mar 1992
Serial925,405Parent of this continuation · filed 4 Aug 1992
US 5,480,784 AParent of this continuation-in-part
US 5,399,491 AParent of this continuation-in-part
US 5,766,849 AThis patent

From the US filing history. Foreign family members aren’t listed.