System for fluorescence monitoring

• US 7,670,832 B2
• Filed: 08/15/2005
• Issued: 03/02/2010
• Est. Priority Date: 06/04/1996
• Status: Expired due to Fees

• Current Assignee: Idaho Technology, University of Utah Research Foundation (State of Utah)
• Original Assignee: Idaho Technology, University of Utah Research Foundation (State of Utah)
• Inventors: Ririe, Kirk M., Wittwer, Carl T.
• Primary Examiner(s): Babic; Christopher M.
• Application Number:
  US11/203,947
• Publication Number:
  US 20060029965A1
• Time in Patent Office: 1,660 Days
• Field of Search: None
• US Class Current: 435/283.1
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 3/5082 Test tubes per se
• 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 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6482 Sample cells, cuvettes
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 21/0303 Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment G01N21/031 - G01N21/15 take precedence
• CPC Class Codes: G01N 21/07 Centrifugal type cuvettes G01N21/09 takes precedence
• CPC Class Codes: G01N 21/253 for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• 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: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 35/025 : having a carousel or turntable for reaction cells or cuvettes
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to devices for performing PCR and monitoring the reaction of a sample comprising a nucleic acid and a fluorescent dye. Illustrative devices comprise a heat exchange component for heating and cooling the sample, a control device for repeatedly operating the heat exchange component to subject the sample to thermal cycling, an excitation source for optically exciting the sample to cause the sample to fluoresce, a photodetector for detecting temperature-dependent fluorescence levels from the sample, and a processor configured to record and process emissions from the fluorescent dye.

Claims

1. A device for performing PCR and monitoring the reaction of a sample comprising a nucleic acid and a fluorescent dye comprising:

a reaction vessel with the sample therein, wherein the sample further comprises a polymerase and a pair of primers configured for amplifying a locus of the nucleic acid,

a heat exchange component for heating and cooling the sample,

a control device programmed for repeatedly operating the heat exchange component to subject the sample to thermal cycling to generate an amplification product,

an excitation source for optically exciting the sample to cause the sample to fluoresce,

a photodetector for detecting temperature-dependent fluorescence levels from the sample, and

a processor programmed to generate a melting curve of the amplification product contained within the reaction vessel during or subsequent to amplification.

2. The device of claim 1 wherein the device further comprises a chamber in thermal communication with the heat exchange component, and the chamber is further configured for receiving the reaction vessel.

3. The device of claim 1 wherein the heat exchange component further comprises a heater and a fan for heating and cooling forced air which is in thermal contact with the reaction vessel.

4. The device of claim 1 wherein the heat exchange component comprises a heat block device.

5. The device of claim 1 wherein the control device is programmed to repeatedly operate the heat exchange component between an annealing temperature and a denaturation temperature, where the denaturation temperature is sufficient to denature the amplification product, and the fluorescent dye is SYBR®

Green I

6. The device of claim 5 wherein the processor is further programmed to generate an amplification curve.

7. The device of claim 1 wherein the photodetector is configured to detect temperature-dependent fluorescence levels from the sample during amplification.

8. The device of claim 1 wherein the melting curve is a derivative plot.

9. The device of claim 1 wherein the fluorescent dye is a double-stranded nucleic acid binding dye.

10. The device of claim 1 wherein the control device operates the heat exchange device to heat the sample at a rate of ≧

0.1°

C./second.

13. The device of claim 5, wherein the denaturation temperature is in excess of 90°

C.

11. A device for performing PCR and monitoring the reaction of a sample comprising a nucleic acid and a double-stranded nucleic acid binding dye comprising:

a heat exchange component for heating and cooling the sample,

a control device programmed for repeatedly operating the heat exchange component to subject the sample to thermal cycling,

an excitation source for optically exciting the sample to cause the sample to fluoresce,

a photodetector for detecting temperature-dependent fluorescence levels from the sample,

a processor programmed to generate a melting curve during or subsequent to amplification, and

further comprising a reaction vessel with the sample therein, wherein the sample further comprises a polymerase and a pair of primers configured for amplifying a locus of the nucleic acid.

12. The device of claim 11 wherein the dye is SYBR®

Green I

14. A device for performing PCR and monitoring the reaction of a sample comprising a nucleic acid and a fluorescent dye comprising:

a reaction vessel with the sample therein, wherein the sample further comprises a polymerase and a pair of primers configured for amplifying a locus of the nucleic acid,

a heat exchange component for heating and cooling the sample,

a control device programmed for repeatedly operating the heat exchange component to subject the sample to thermal cycling to generate an amplification product,

an excitation source for optically exciting the sample to cause the sample to fluoresce,

a photodetector for detecting temperature-dependent fluorescence levels from the sample,

a processor programmed to generate a melting curve of the amplification product contained within the reaction vessel during or subsequent to amplification; and

wherein the sample further comprises at least one nucleic acid probe labeled with the fluorescent dye.