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EP0201184A2 Expired / Lapsed EP ⏱️ 0.0 years remaining

Process for amplifying nucleic acid sequences

Assignee: F Hoffmann La Roche AG
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Filing: 1986-03-27
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Granted: —
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Expires: 2006-03-27
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Abstract

Abstract The present invention is directed to a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.

Inventors

  • 👤 Kary Banks Mullis

Bibliographic Details

Application Number —
Jurisdiction EP
Data Source google_patents
Last Synchronized 2026-11-06 11:03
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; B; B01; B01L; B01L7/00; B01L7/52; C07; C07K; C07K14/00; C07K14/795; C07K14/805; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6827; C12Q1/6844; C12Q1/6853; C12Q1/6858; C12Q1/686; B01J; B01J2219/00; B01J2219/00274; B01J2219/00277; B01J2219/00495; B01J2219/00583; B01J2219/0059; B01J2219/00718; B01J2219/0072; B01J2219/00722; C12Q1/6876; C12Q1/6883; C12Q1/6886; C12Q1/6888; C12Q1/689; C40; C40B; C40B40/00; C40B40/04; C40B40/06; C40B60/00; C40B60/14

Claims Analysis

Total Claims: 11
Claim 1
Independent Claim 187 words
A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process comprises: (a) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions, such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to different strands of each specific sequence such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template.
Claim 2
Dependent Claim 187 words
A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process comprises: (a) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions, such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to different strands of each specific sequence such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template.
Claim 3
Dependent Claim 18 words
A process according to claim 1 characterized in that steps (b) and (c) are repeated at least once.
Claim 4
Dependent Claim 41 words
A process according to claim 1 or 2 characterized in that step (b) is accomplished by denaturing or by using the enzyme helicase and steps - (a) and (c) are accomplished using an inducing agent for polymerization and four different nucleotides.
Claim 5
Dependent Claim 48 words
A. process according to any one of claims 1-3 characterized in that stpes (a) and (c) are accomplished using an inducing agent for polymerization selected from E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, a heat-stable enzyme, or reverse transcriptase.
Claim 6
Dependent Claim 22 words
A process according to any one of claims 1-4 characterized in that the nucleic acid is DNA and the primers are oligodeoxyribonucleotides.
Claim 7
Dependent Claim 39 words
A process according to any one of claims 1-4 characterized in that the nucleic acid is messenger RNA and a collection of primers is employed for each complementary strand, one of which is 100% homologous with the complementary strand.
Claim 8
Dependent Claim 33 words
A process according to any one of claims 1-6 characterized in that the mixture of nucleic acids used in step (a) is the product of a previous amplification process produced by step (c).
Claim 9
Dependent Claim 23 words
A process according to claim 7 characterized in that the primers employed are different from the primers employed in the previous amplification process.
Claim 10
Dependent Claim 30 words
A process according to any one of claim 1-8 characterized in that one primer contains at least one nucleotide which is not complementary to the specific sequence to be amplified.
Claim 11
Dependent Claim 34 words
A process according to any one of claims 1-9 characterized in that the two primers in steps - (a) and (c) are each present in a molar ratio of at least 1000:1 primer:complementary strand.

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