CA2658290A1 - Monitoring hybridization during pcr using fluorescent dye specific to double-stranded dna - Google Patents

Monitoring hybridization during pcr using fluorescent dye specific to double-stranded dna Download PDF

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Publication number
CA2658290A1
CA2658290A1 CA002658290A CA2658290A CA2658290A1 CA 2658290 A1 CA2658290 A1 CA 2658290A1 CA 002658290 A CA002658290 A CA 002658290A CA 2658290 A CA2658290 A CA 2658290A CA 2658290 A1 CA2658290 A1 CA 2658290A1
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nucleic acid
amplification
acid sequence
sample
temperature
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CA2658290C (en
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Carl T. Wittwer
Kirk M. Ririe
Randy P. Rasmussen
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University of Utah Research Foundation UURF
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University Of Utah Research Foundation
Carl T. Wittwer
Kirk M. Ririe
Randy P. Rasmussen
University Of Utah
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/1844—Means for temperature control using fluid heat transfer medium using fans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6482—Sample cells, cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/0303—Optical path conditioning in cuvettes, e.g. windows; adapted optical elements or systems; path modifying or adjustment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/07—Centrifugal type cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/025—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having a carousel or turntable for reaction cells or cuvettes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]

Abstract

Methods of monitoring hybridization during polymerase chain reaction are disclosed. These methods are achieved with rapid thermal cycling and use of double stranded DNA dyes or specific hybridization probes. A fluorescence resonance energy transfer pair comprises fluorescein and Cy5 or Cy5.5. Methods for quantitating amplified DNA and determining its purity are carried out by analysis of melting and reannealing curves.

Claims (23)

1. A method for detecting a target nucleic acid sequence in a biological sample during amplification comprising the steps of:

(a) adding a thermostable polymerase, a double-strand-specific DNA binding dye and primers configured for amplification of the target nucleic acid sequence to the biological sample;

(b) amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of the dye, the polymerase chain reaction comprising thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles;

(c) illuminating the biological sample comprising the amplified target nucleic acid sequence with light at a wavelength absorbed by the dye; and (d) detecting a fluorescent emission from the dye using a 520-580 nm band pass filter, wherein the fluorescent emission is related to the quantity of the amplified target nucleic acid sequence in the sample.
2. The method of claim 1 wherein the fluorescent emission is monitored in the range of 520-550 nm.
3. The method of claim 1 wherein the illuminating is via light at a wavelength in the range of 450-490 nm.
4. The method of claim 1 wherein the illuminating is via visible light.
5. The method of claim 1 wherein the amplifying step includes using a rapid temperature cycling profile wherein 30 amplification cycles are completed in 10 to 30 minutes.
6. The method of claim 1 wherein the detecting step occurs while the biological sample is heated and further comprises the step of (e) generating a melting curve.
7. A method of analyzing nucleic acid hybridization comprising the steps of (a) providing a mixture comprising a nucleic acid sample to be analyzed and a nucleic acid binding fluorescent entity; and (b) monitoring fluorescence while changing temperature at a rate of >= 0.1°C/second.
8. The method of claim 7 wherein the nucleic acid binding fluorescent entity comprises a pair of oligonucleotide probes wherein one of the probes is labeled with a donor and the other probes is labeled with an acceptor of a fluorogenic resonance energy transfer pair.
9. A method for detecting a target nucleic acid sequence in a biological sample during amplification comprising the steps of:

(a) adding a thermostable polymerase and primers configured for amplification of the target nucleic acid sequence to the biological sample;

(b) amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of a fluorescent dye which is pico green, the polymerase chain reaction comprising thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles using a rapid temperature cycling profile wherein 30 amplification cycles are completed in 10 to 30 minutes;
(c) illuminating the biological sample comprising the amplified target nucleic acid sequence with light at a wavelength absorbed by the fluorescent dye; and (d) detecting a fluorescent emission from the fluorescent dye related to the quantity of the amplified target nucleic acid sequence in the sample.
10. The method of claim 9 wherein during each amplification cycle the sample is held no more than 60 seconds at the elongation temperature.
11. The method of claim 9 wherein during each amplification cycle the sample is held less than 20 seconds at the elongation temperature.
12. The method of any one of claims 9 to 11 wherein during each amplification cycle the sample is held less than 1 second at the denaturation temperature.
13. The method of any one of claims 9 to 12 wherein the sample is illuminated and fluorescence is detected during each amplification cycle.
14. The method of claim 13 wherein a fluorescence value is acquired during an extension or a combined annealing/extension phase at each amplification cycle.
15. The method of any one of claims 9 to 14 wherein the sample is illuminated and fluorescence is detected as the temperature is increased, to generate a melting curve.
16. A method of monitoring the amplification of a nucleic acid in a biological sample during PCR amplification, comprising the steps of:

(a) forming an amplification mixture comprising the biological sample, pico green as a fluorescent entity capable of producing a fluorescent signal related to the amount of nucleic acid present in the sample, a thermostable polymerase, and primers for the nucleic acid;

(b) amplifying the target sequence by thermally cycling the amplification mixture through a plurality of thermal cycles, and (c) illuminating the sample and monitoring the fluorescent signal from the fluorescent entity during amplification.
17. A kit for analysis of a nucleic acid sequence during amplification, the kit comprising: an amplification solution comprising a fluorescent dye which is pico green; a thermostable DNA polymerase; and deoxynucleoside triphosphates.
18. The kit of claim 17 further comprising a pair of primers for amplifying the nucleic acid sequence.
19. A method for detecting a target nucleic acid sequence in a biological sample during amplification comprising the steps of:

(a) adding a thermostable polymerase and primers configured for amplification of the target nucleic acid sequence to the biological sample;

(b) amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of a fluorescent dye which is pico green, the polymerase chain reaction comprising thermally cycling the biological sample between at least a denaturation temperature and an elongation temperature through a plurality of amplification cycles;

(c) illuminating the biological sample comprising the amplified target nucleic acid sequence with light at a wavelength absorbed by the fluorescent dye; and (d) detecting a fluorescent emission from the fluorescent dye related to the quantity of the amplified target nucleic acid sequence in the sample; wherein during each of the plurality of amplification cycles the sample is held no more than 60 seconds at the elongation temperature and held less than 1 second at the denaturation temperature.
20. A method of determining a concentration of an amplified product in a selected polymerase chain reaction mixture comprising:

(a) determining a second order rate constant for the amplified product at a selected temperature and reaction conditions by monitoring rate of hybridization of a known concentration of the amplified product;

(b) determining rate of annealing for an unknown concentration of the amplified product; and (c) calculating the concentration of the amplified product from the rate of annealing and the second order rate constant.
21. The method of claim 20 wherein the rate of annealing is determined after multiple cycles of amplification.
22. A method for determining a second order rate constant for an amplified product to determine the concentration of the amplified product, said method comprising the steps of raising the temperature of a first polymerase chain reaction mixture comprising a known concentration of the amplified product and an effective amount of a double-strand specific fluorescent dye, above the denaturation temperature of the amplified product to result in a denatured amplified product;

rapidly reducing the temperature of the first polymerase chain reaction mixture comprising the known amount of denatured amplified product to a selected temperature below the denaturation temperature of the amplified product while continuously monitoring the fluorescence of the first polymerase chain reaction mixture as a function of time;

plotting fluorescence as a function of time for determining maximum fluorescence, minimum fluorescence, the time at minimum fluorescence, and a second order rate constant for the known concentration of amplified product from the equation wherein F is fluorescence, F max is maximum fluorescence, F min is minimum fluorescence, k is the second order rate constant, t0 is the time at F min, and [DNA] is the known concentration of the amplified product.
23. A fluorescence energy transfer pair comprising a first probe labeled with fluorescein and a second probe labeled with Cy5 or Cy5.5.
CA2658290A 1996-06-04 1997-06-04 Monitoring hybridization during pcr using fluorescent dye specific to double-stranded dna Expired - Lifetime CA2658290C (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US65899396A 1996-06-04 1996-06-04
US08/658,993 1996-06-04
US81826797A 1997-03-17 1997-03-17
US08/818,267 1997-03-17
CA002591550A CA2591550C (en) 1996-06-04 1997-06-04 Monitoring hybridization during pcr

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CA2658290A1 true CA2658290A1 (en) 1997-12-11
CA2658290C CA2658290C (en) 2012-04-10

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CA002257109A Expired - Lifetime CA2257109C (en) 1996-06-04 1997-06-04 Monitoring hybridization during pcr
CA2658290A Expired - Lifetime CA2658290C (en) 1996-06-04 1997-06-04 Monitoring hybridization during pcr using fluorescent dye specific to double-stranded dna
CA002591550A Expired - Lifetime CA2591550C (en) 1996-06-04 1997-06-04 Monitoring hybridization during pcr

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US (9) US6174670B1 (en)
EP (3) EP0912766B2 (en)
JP (4) JP4540754B2 (en)
AT (3) ATE295427T1 (en)
AU (1) AU726501B2 (en)
CA (3) CA2257109C (en)
DE (3) DE69735313T2 (en)
DK (2) DK1179600T3 (en)
ES (2) ES2243393T3 (en)
NZ (2) NZ502323A (en)
PT (2) PT912766E (en)
WO (1) WO1997046714A1 (en)

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