EP0500224A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Exonuclease mediated strand displacement amplification
Assignee: Becton Dickinson and Co
•
Filing: 1992-01-29
•
Granted: —
•
Expires: 2012-01-29
Abstract
Abstract This invention relates a nucleic acid target amplification method which operates at a single temperature and makes use of a polymerase in conjunction with an exonuclease that will digest the primer portion of the polymerized strand such that the polymerase will displace the partially digested strand without digestion of the target strand while generating a newly polymerized strand.
Inventors
- 👤 George T. Walker
- 👤 James L. Schram
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-31 00:11
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
116 words
A method for amplifying a target nucleic acid sequence in a sample comprising the steps of: a) isolating nucleic acid fragments from a sample; b) preparing single stranded target nucleic acids; c) adding to the target sequence sample a reaction mixture comprising an excess of deoxynucleosidetriphosphates, at least one of which is substituted, a DNA polymerase lacking 5' to 3' exonuclease activity, one or more primers that are complementary to the single strands of the target sequence, and a double strand specific 5'→3' exonuclease; d) allowing the reaction mixture to react with the single stranded fragments for a period of time sufficient to generate reaction products; and e) detecting the presence of the reaction products produced.
Claim 2
Independent Claim
116 words
A method for amplifying a target nucleic acid sequence in a sample comprising the steps of: a) isolating nucleic acid fragments from a sample; b) preparing single stranded target nucleic acids; c) adding to the target sequence sample a reaction mixture comprising an excess of deoxynucleosidetriphosphates, at least one of which is substituted, a DNA polymerase lacking 5' to 3' exonuclease activity, one or more primers that are complementary to the single strands of the target sequence, and a double strand specific 5'→3' exonuclease; d) allowing the reaction mixture to react with the single stranded fragments for a period of time sufficient to generate reaction products; and e) detecting the presence of the reaction products produced.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein a restriction enzyme is used to create nucleic acid fragments.
Claim 4
Dependent Claim
28 words
The method of claim 1 wherein the sample is derived from animal material comprising blood, bone marrow, lymph, hard tissue, urine, feces and sputum, plant material and soil.
Claim 5
Dependent Claim
18 words
The method of claim 1 wherein the nucleic and fragments are rendered single stranded by heating the sample.
Claim 6
Independent Claim
117 words
A method for amplifying a target nucleic acid sequence comprising the steps of: a) isolating nucleic acids from a sample wherein the sample is derived from human biological material; b) generating single stranded nucleic acids by heating the sample; c) adding a reaction mixture comprising an excess of deoxynucleosidetriphosphates at least one of which is substituted, a DNA polymerase lacking 5' to 3' exonuclease activity, one or more primers that are complementary to the single strands of the target sequence, and a double strand specific 5'→3' exonuclease; d) allowing the reaction mixture to react with the single stranded nucleic acids for a period of time sufficient to generate reaction products; and e) detecting the reaction products produced
Claim 7
Independent Claim
117 words
A method for amplifying a target nucleic acid sequence comprising the steps of: a) isolating nucleic acids from a sample wherein the sample is derived from human biological material; b) generating single stranded nucleic acids by heating the sample; c) adding a reaction mixture comprising an excess of deoxynucleosidetriphosphates at least one of which is substituted, a DNA polymerase lacking 5' to 3' exonuclease activity, one or more primers that are complementary to the single strands of the target sequence, and a double strand specific 5'→3' exonuclease; d) allowing the reaction mixture to react with the single stranded nucleic acids for a period of time sufficient to generate reaction products; and e) detecting the reaction products produced
Claim 8
Dependent Claim
38 words
The method of claim 5 wherein the polymerase is selected from the group consisting of the klenow fragment of DNA polymerase I, the exonuclease deficient klenow fragment of DNA polymerase I and the klenow fragment of Bst polymerase.
Claim 9
Dependent Claim
16 words
The method of claim 6 wherein the polymerase is the klenow fragment of DNA polymerase I.
Claim 10
Dependent Claim
13 words
The method of claim 5 wherein the exonuclease is T7 gene 6 exonuclease.
Claim 11
Dependent Claim
35 words
The method of claim 5 wherein detection of the reaction products is carried out by tagging the primer with a label selected from the group consisting of a radiolabel, an enzyme and a fluorescent dye.
Claim 12
Independent Claim
20 words
The method of any of claims 1 and 5 wherein the probes are specific for a viral nucleic acid sequence.
Claim 13
Independent Claim
20 words
The method of any of claims 1 and 5 wherein the probes are specific for a bacterial nucleic acid sequence.
Claim 14
Independent Claim
20 words
The method of any of claims 1 and 5 wherein the probes are specific for an oncogene nucleic acid sequence.
Claim 15
Independent Claim
25 words
The method of any of claims 1 and 5 wherein the probes are specific for a nucleic acid sequence involved in a single point mutation.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.