EP0512334A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Methods for detecting a target nucleic acid in a sample
Assignee: F Hoffmann La Roche AG
•
Filing: 1992-04-24
•
Granted: —
•
Expires: 2012-04-24
Abstract
Abstract This invention relates to improved methods for nucleic acid detection using amplification methods such as the polymerase chain reaction (PCR). More specifically, the invention provides methods for simultaneous amplification and detection to enhance the speed and accuracy of prior methods. The methods involve the introduction of detectable DNA binding agents into the amplification reaction, which agents produce a detectable signal that is enhanced upon binding double-stranded DNA. In a preferred embodiment, the binding agent is a fluorescent dye. The methods also provide means for monitoring the increase in product DNA during an amplification reaction.
Inventors
- 👤 Russell G. Higuchi
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-31 00:07
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C12Q1/6844; C12Q1/6851; C12Q1/686; C12Q1/6876; C12Q1/6879; C12Q1/70; C12Q1/701; C12Q1/702; C12Q1/703; B; B01; B01L; B01L7/00; B01L7/52; Y; Y10; Y10S; Y10S435/00; Y10S435/81; Y10T; Y10T436/00; Y10T436/14; Y10T436/142222; Y10T436/143333
Claims Analysis
Total Claims: 24
Claim 1
Independent Claim
113 words
A method for detecting a target nucleic acid in a sample, said method comprises the steps of: (a) providing an amplification reaction mixture that comprises said sample, a DNA binding agent, wherein said agent is characterized as providing a detectable signal when bound to double-stranded nucleic acid which signal is distinguishable from the signal provided by said agent when it is unbound, and reagents for amplification; (b) determining the amount of said signal produced by the mixture of step (a); (c) treating said mixture under conditions for amplifying said target nucleic acid; (d) determining the amount of said signal produced by said mixture of step (c); and (e) determining if amplification has occurred.
Claim 2
Dependent Claim
44 words
Method of claim 1, wherein said DNA binding agent is further characterized as providing an amount of detectable signal when said agent is bound to double-stranded nucleic acids that is greater than the amount of said detectable signal produced when said agent is unbound.
Claim 3
Dependent Claim
15 words
Method of claim 1 or 2, wherein said DNA binding agent is an intercalating agent.
Claim 4
Dependent Claim
12 words
Method of claim 3, wherein said intercalating agent is a fluorescent dye.
Claim 5
Dependent Claim
17 words
Method of claim 4, wherein at step (e) an increase in fluorescence indicates that amplification has occurred.
Claim 6
Dependent Claim
46 words
Method of claim 5, wherein at steps (b) and (d) the amount of signal produced is determined by exposing said mixture to UV light, and at step (e) comparing the relative amount of signal produced at steps (b) and (d) to determine if amplification has occurred.
Claim 7
Dependent Claim
14 words
Method of any one of claims 4-6, wherein said fluorescent dye is ethidium bromide.
Claim 8
Dependent Claim
19 words
Method of any one of claims 4-7, wherein the amount of signal produced is determined using a spectra fluorometer.
Claim 9
Dependent Claim
31 words
Method of any one of claims 5-8, wherein the amount of target DNA in said sample, prior to amplification, is quantitated by determining the increase in fluorescence before and after amplification.
Claim 10
Dependent Claim
20 words
Method of any one of claims 1-9, wherein said target nucleic acid is indicative of a genetic or infectious disease.
Claim 11
Independent Claim
114 words
A method for monitoring the increase in double-stranded DNA during amplification of a target nucleic acid in a sample, said method comprises the steps of: (a) providing an amplification reaction mixture that comprises said sample and a DNA binding agent, wherein said agent is characterized as providing a detectable signal when bound to double-stranded nucleic acid which signal is distinguishable from the signal provided by said agent when it is unbound; (b) determining the amount of said signal produced by the mixture of step (a); (c) treating said mixture under conditions for amplifying said target nucleic acid; and (d) determining the amount of said signal produced by said mixture during said treating step (c).
Claim 12
Dependent Claim
29 words
Method of claim 11, wherein at steps (b) and (d), an optic fiber and spectra fluorometer are used to determine the amount of signal produced during said treating step.
Claim 13
Dependent Claim
20 words
Method of claim 11 or 12, wherein at step (d) the amount of signal is determined continuously throughout amplification reaction.
Claim 14
Dependent Claim
16 words
Method of any one of claims 11-13, wherein said DNA binding agent is an intercalating agent.
Claim 15
Dependent Claim
12 words
Method of claim 14, wherein said intercalating agent is a fluorescent dye.
Claim 16
Dependent Claim
11 words
Method of claim 15, wherein said fluorescent dye is ethidium bromide.
Claim 17
Dependent Claim
17 words
Method of any one of claims 1-16, wherein said amplification reaction is a polymerase chain reaction (PCR).
Claim 18
Dependent Claim
21 words
A kit for amplifying a target nucleic acid, that comprises a buffer for an amplification reaction that comprises an intercalating agent.
Claim 19
Dependent Claim
12 words
Kit of claim 18, wherein said intercalating agent is a fluorescent dye.
Claim 20
Dependent Claim
11 words
Kit of claim 19, wherein said fluorescent dye is ethidium bromide.
Claim 21
Dependent Claim
14 words
Kit of any one of claims 18-20, wherein said buffer is a PCR buffer.
Claim 22
Dependent Claim
27 words
Kit of claim 21, wherein said ethidium bromide is present at a concentration suitable to provide between 0.15 µM and 40.6 µM dye in a PCR reaction.
Claim 23
Dependent Claim
31 words
Kit of claim 21 or 22, wherein said buffer also comprises Tris-HCl, pH 8.08.3 and KCl, each present in a concentration suitable for amplifying a target nucleic acid in a PCR.
Claim 24
Dependent Claim
16 words
Kit of any one of claims 18-22 that also comprises a DNA polymerase, MgCl₂, and dNTPs.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
Maintenance schedule is calculated for granted US utility patents.