Monitoring hybridization during PCR using SYBR™ Green I

• US 6,569,627 B2
• Filed: 03/05/2001
• Issued: 05/27/2003
• Est. Priority Date: 06/04/1996
• Status: Expired due to Term

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Ririe, Kirk M., Rasmussen, Randy P., Wittwer, Carl T.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/799,160
• Publication Number:
  US 20020058258A1
• Time in Patent Office: 813 Days
• Field of Search: 435/6, 435/91.2, 435/810
• US Class Current: 435/6.12
• 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 a method and kits for monitoring a nucleic acid during amplification. More particularly, the present invention relates to a method wherein the nucleic acid is monitored during polymerase chain reaction using a double-stranded nucleic acid binding dye capable of producing a fluorescent signal related to the amount of the nucleic acid present in a sample, wherien the dye is selected from the group consisting of SYBR™ Green I and pico green.

Claims

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

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

amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of a fluorescent dye selected from the group consisting of SYBR™

Green I and 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;

illuminating the biological sample comprising the amplified target nucleic acid sequence with light at a wavelength absorbed by the fluorescent dye; and

detecting a fluorescent emission from the fluorescent dye related to the quantity of the amplified target nucleic acid sequence in the sample.

2. The method of claim 1 wherein during each amplification cycle the sample is held no more than 60 seconds at the elongation temperature.

3. The method of claim 1 wherein during each amplification cycle the sample is held less than 20 seconds at the elongation temperature.

4. The method of claim 1 wherein during each amplification cycle the sample is held less than 1 second at the denaturation temperature.

5. The method of claim 1 wherein the sample is illuminated and fluorescence is detected during each amplification cycle.

6. The method of claim 5 wherein a fluorescence value is acquired during an extension or a combined annealing/extension phase at each amplification cycle.

7. The method of claim 1 wherein the sample is illuminated and fluorescence is detected as the temperature is increased, to generate a melting curve.

8. A method of real time monitoring of amplification of a target nucleic acid sequence in a biological sample, said method comprising the steps of:

amplifying the target sequence by polymerase chain reaction in the presence of a quantity of SYBR™

Green I, said polymerase chain reaction comprising the steps of adding the SYBR™

Green I, a thermostable polymerase, and primers for the target nucleic acid sequence to the biological sample to create an amplification mixture and thermally cycling the amplification mixture between at least a denaturation temperature and an elongation temperature during a plurality of amplification cycles;

illuminating the mixture with light at a wavelength absorbed by the SYBR™

Green I in at least a portion of the plurality of amplification cycles;

and detecting a fluorescent emission from the SYBR™

Green I following sample illumination, said fluorescent emission being related to the quantity of amplified target nucleic acid in the sample.

9. The method of claim 8 wherein the fluorescent emission is obtained during an extension phase of each of the plurality of amplification cycles.

10. The method of claim 8 wherein the fluorescent emission is obtained during a combined annealing/extension phase of each of the plurality of amplification cycles.

11. A method of real time monitoring of amplification of a target nucleic acid sequence in a biological sample, said method comprising the steps of:

amplifying the target sequence by polymerase chain reaction in the presence of SYBR™

Green I, said polymerase chain reaction comprising the steps of adding SYBR™

Green I, a thermostable polymerase, and primers for the target nucleic acid sequence to the biological sample to create an amplification mixture and thermally cycling the amplification mixture between at least a denaturation temperature and an elongation temperature during a plurality of amplification cycles under conditions wherein the SYBR™

Green I retains the ability to produce a fluorescent signal related to the quantity of the nucleic acid sequence;

illuminating the sample with light at a wavelength absorbed by the SYBR™

Green I, subsequent to at least a portion of the plurality of amplification cycles;

and monitoring fluorescent emission from the SYBR™

Green I in the sample as a function of sample temperature to generate a melting curve for the amplified target sequence.

12. In a method of monitoring the amplification of a nucleic acid in a biological sample during PCR amplification, comprising the steps of

forming an amplification mixture comprising the biological sample, 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,

amplifying the target sequence by thermally cycling the amplification mixture through a plurality of thermal cycles, and

illuminating the sample and monitoring the fluorescent signal from the fluorescent entity during amplification,

the improvement comprising the step of selecting the fluorescent entity from the group consisting of SYBR™

Green I and pico green.

13. A PCR reaction product mixture comprising an amplified nucleic acid product and SYBR™

Green I in an amount capable of providing a fluorescence signal indicative of the concentration of the amplified nucleic acid product in said mixture, said product mixture prepared by subjecting a PCR amplification mixture comprising the target nucleic acid to be amplified, oligonucleotide primers, a thermostable polymerase, and the SYBR™

Green I, to sufficient thermal cycles to amplify the target nucleic acid.

14. A kit for analysis of a nucleic acid sequence during amplification, the kit comprising:

an amplification solution comprising

a fluorescent dye selected from the group consisting of SYBR™

Green I and pico green;

a thermostable DNA polymerase; and

deoxynucleoside triphosphates.

15. The kit of claim 14 further comprising a pair of primers for amplifying the nucleic acid sequence.

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

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

amplifying the target nucleic acid sequence by polymerase chain reaction in the presence of a fluorescent dye selected from the group consisting of SYBR™

Green I and 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;

illuminating the biological sample comprising the amplified target nucleic acid sequence with tight at a wavelength absorbed by the fluorescent dye; and

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.