Homogeneous methods for nucleic acid amplification and detection

• US 5,994,056 A
• Filed: 05/02/1991
• Issued: 11/30/1999
• Est. Priority Date: 05/02/1991
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Higuchi, Russell G.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): PROUTY, REBECCA E
• Application Number:
  US07/695,201
• Time in Patent Office: 3,134 Days
• Field of Search: 435/6, 435/91, 435/810, 435/91.2, 436/63, 436/94, 935/17, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2545/10 the purpose being quantitative analysis
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to improved methods for nucleic acid detection using 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.

Claims

1. A method for detecting a target nucleic acid in a sample, said method comprising:

(a) providing a DNA 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 DNA which signal is greater than the amount of said signal provided by said agent when it is unbound, and wherein said agent does not significantly inhibit the rate of nucleic acid amplification 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 to produce amplified double-stranded DNA;

(d) determining the amount of said signal produced by said mixture of step (c); and

(e) determining if amplification has occurred.

2. The method of claim 1, wherein said DNA binding agent is an intercalating agent.

3. The method of claim 2, wherein said intercalating agent is a fluorescent dye.

4. The method of claim 3, wherein at step (e) an increase in fluorescence indicates that amplification has occurred.

5. The method of claim 4, 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.

6. The method of claim 4, wherein said fluorescent dye is ethidium bromide.

7. The method of claim 4, wherein the amount of signal produced is determined using a spectra fluorometer.

8. The method of claim 4, wherein said target nucleic acid is indicative of a genetic or infectious disease.

9. The method of claim 4, wherein the amount of target DNA in said sample, prior to amplification, is quantitated by determining the increase in fluorescence during amplification.

10. A method for monitoring the increase in double-stranded DNA during amplification of a target nucleic acid in a sample, wherein said method comprises the steps of:

(a) providing a mixture that comprises all components necessary for the selective amplification of said target nucleic acid by polymerase chain reaction (PCR) containing 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 greater than the amount of said 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).

11. The method of claim 10, 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.

12. The method of claim 11, wherein said DNA binding agent is an intercalating agent.

13. The method of claim 11, wherein at step (d) the amount of signal is determined continuously throughout the amplification reaction.

14. The method of claim 12, wherein said intercalating agent is a fluorescent dye.

15. The method of claim 14, wherein said fluorescent dye is ethidium bromide.

16. A kit for amplifying a target nucleic acid, that comprises a PCR buffer that comprises an intercalating age wherein said intercalating agent is characterized as providing a detectable signal when bound to double stranded DNA, which signal is greater than the signal provided by said intercalating agent when it is unbound and at least one pair of PCR amplification primers.

17. The kit of claim 16, wherein said intercalating agent is a fluorescent dye.

18. The kit of claim 17, wherein said fluorescent dye is ethidium bromide.

19. The kit of claim 18, wherein said ethidium bromide is present at a concentration suitable to provide between 0.15 μ

M and 40.6 μ

M dye in a PCR reaction.

20. The kit of claim 19, wherein said buffer also comprises Tris-HCl, pH 8.0-8.3 and KCl, each present in a concentration suitable for amplifying a target nucleic acid in a PCR.

21. The kit of claim 19 that also comprises a DNA polymerase, MgCl₂, and dNTPs.