Methods and devices for hemogeneous nucleic acid amplification and detector

• US 6,171,785 B1
• Filed: 06/06/1995
• Issued: 01/09/2001
• Est. Priority Date: 05/02/1991
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Higuchi, Russell G.
• Primary Examiner(s): Prouty, Rebecca E.
• Application Number:
  US08/470,532
• Time in Patent Office: 2,044 Days
• Field of Search: 435/6, 435/91.2, 436/63, 436/94
• US Class Current: 435/6.12
• 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 the presence of a target nucleic acid in a sample by amplification, said method comprising:

(a) providing a nucleic acid amplification reaction mixture that comprises said sample and a fluorescent dye, whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid, wherein said reaction mixture is contained within a container which container is operably linked to a device suitable for inputting excitation light into said reaction mixture and conveying fluorescent light emitted by said reaction mixture to a detector;

(b) measuring said emitted light produced by the mixture of step (a);

(c) treating said mixture under conditions for amplifying said target nucleic acid to produce amplified double-stranded nucleic acid;

(d) measuring said emitted light produced by said mixture of step (c); and

(e) determining if amplification has occurred.

2. The method of claim 1, wherein at step (e) an increase in fluorescence indicates that amplification has occurred.

3. The method of claim 2, wherein at steps (b) and (d) the amount of emitted light produced by exposing said mixture to UV light is determined, and at step (e) the relative amount of emitted light produced at steps (b) and (d) is compared to determine if amplification has occurred.

4. The method of claim 3, wherein said fluorescent dye is ethidium bromide.

5. The method of claim 3, wherein the amount of target DNA in said sample, prior to amplification, is quantitated by determining the increase in fluorescence during amplification.

16. The method of claim 2, wherein said measurement according to step (d) is carried out during step (c).

17. The method of claim 16, wherein said measurement according to step (d) is continuously carried out during step (c).

18. The method of claim 1 wherein said device suitable for inputting excitation light into said reaction mixture is a fiber optic device.

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

(a) amplifying a region of said target nucleic acid by polymerase chain reaction method in the presence of a fluorescent pigment whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid;

which method comprises locating said sample in a container, which container is suitably located for inputting excitation light into said sample and conveying emission light to a detector for measuring said emissions, and wherein said method comprises the steps of;

(b) monitoring the fluorescence intensity of said fluorescent pigment during said polymerase chain reaction,

(c) determining the amount of fluorescence emission that is emitted by said sample.

7. The method of claim 6 wherein said monitoring is continuous during said amplification reaction.

8. The method of claim 7 wherein said container is contained in a vessel to be heated and cooled.

9. The method of claim 8 wherein said vessel is in contact with a block capable of being heated and cooled.

10. The method of claim 9 wherein said block is contained within a thermal cycler.

11. The method of claim 6, further comprising the step of calculating the change in fluorescence of said fluorescent pigment.

19. The method of claim 6, wherein a fiber optic device provides means for inputting said excitation light, said detector is operably linked to said fiber optic device, and said fluorescence emission is carried through said fiber optic device and detected by said detection means.

12. A method for detecting a target nucleic acid in a sample, comprising the steps of:

(a) amplifying a region of said target nucleic acid by a polymerase chain reaction method in the presence of a fluorescent pigment whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid; and

(b) measuring the fluorescence intensity of said fluorescent pigment before and after said polymerase chain reaction by means of a device operably linked to a measuring devise, whereby an increase in fluorescence intensity is indicative of the presence of said target nucleic acid.

20. The method of claim 12 wherein said device is a fiber optic device.

22. The method of claim 20 wherein said polymerase chain reaction is contained within a container which container is operably linked to a fiber optic device suitable for transmitting excitation light to said container and conveying emission light to said measuring devise.

13. A method for quantifying a target nucleic acid in a test sample, comprising the steps of:

(a) amplifying a region of said target nucleic acid in a test reaction by the polymerase chain reaction in the presence of a fluorescent pigment whose fluorescence intensity is increased upon intercalation into a double-stranded nucleic acid;

(b) monitoring the fluorescence intensity of said fluorescent pigment during said polymerase chain reaction by use of a device operably linked to a container containing said polymerase chain reaction and to a detector suitable for measuring emitted light; and

(c) determining the quantity of said target nucleic acid in said sample.

14. The method of claim 13, wherein said test sample comprises an unknown amount of target nucleic acid and said method further comprises the steps of:

(a) performing a control experiment comprising repeating said amplifying and said monitoring steps on a control reaction which comprises a known amount of said target nucleic acid; and

(b) comparing the change in fluorescence of said fluorescent pigment in said test reaction during said reaction to the change in fluorescence of said fluorescent pigment in said control reaction during said control experiment.

15. The method of claim 14 wherein said amplifying step further comprises the step of measuring the fluorescence of said fluorescent pigment after each cycle of PCR, and said method further comprises the step of determining the number of cycles at which said fluorescence increases in said method relative to said control experiment.

21. The method of claim 13 wherein said device is a fiber optic device.