Instrument for monitoring nucleic acid amplification

• US 6,814,934 B1
• Filed: 11/12/1997
• Issued: 11/09/2004
• Est. Priority Date: 05/02/1991
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Higuchi, Russell Gene
• Primary Examiner(s): SNAY, JEFFREY R
• Application Number:
  US08/968,208
• Time in Patent Office: 2,554 Days
• Field of Search: 422/67, 422/82.08, 422/307, 250/454.11, 250/458.1
• US Class Current: 422/82.08
• 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. An instrument for use in monitoring a nucleic acid amplification reaction comprising multiple thermal cycles, comprising:

(a) an automated thermal cycler capable of alternately heating and cooling, and adapted to receive, at least one reaction vessel containing an amplification reaction mixture comprising a target nucleic acid, reagents for nucleic acid amplification, and a detectable nucleic acid binding agent; and

(b) a detector operable to detect a fluorescence optical signal while the amplification reaction is in progress and without opening the at least one reaction vessel, which fluorescence optical signal is related to the amount of amplified nucleic acid in the reaction vessel.

2. The instrument of claim 1, wherein the thermal cycler is adapted to receive a plurality of reaction vessels, each containing an amplification reaction mixture.

3. The instrument of claim 1, wherein the detector is operable to detect a fluorescence optical signal at a wavelength at or about 570 nm.

4. The instrument of claim 1, wherein includes a sealed light transmission path between the reaction vessel and the detector.

5. The instrument of claim 4, wherein the sealed light transmission path is a fiber optic cable.

6. The instrument of claim 1, wherein the thermal cycler is computer-controlled.

7. A system for use in monitoring a nucleic acid amplification reaction comprising multiple thermal cycles, comprising:

(a) at least one reaction vessel adapted to contain an amplification reaction mixture comprising a target nucleic acid, reagents for nucleic acid amplification, and a detectable nucleic acid binding agent;

(b) an automated thermal cycler capable of alternately heating and cooling such a reaction vessel, and

(c) a detector operable to detect a fluorescence optical signal while the amplification reaction is in progress and without opening the at least one reaction vessel, which fluorescence optical signal is related to the amount of amplified nucleic acid in the reaction vessel.

8. The system of claim 7, wherein the system comprises a plurality of reaction vessels, each adapted to contain an amplification reaction mixture.

9. The system of claim 7, wherein the detector is operable to detect a fluorescence optical signal at a wavelength at or about 570 nm.

10. The system of claim 7, wherein the at least one reaction vessel includes a clear or translucent cap optically coupled to the detector by a sealed light transmission path.

11. The system of claim 10, wherein the sealed light transmission path is a fiber optic cable.

12. The system of claim 7, wherein the thermal cycler is computer-controlled.