Multiplex Q-PCR Arrays

• US 20080176757A1
• Filed: 07/27/2007
• Published: 07/24/2008
• Est. Priority Date: 07/28/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Vikalo, Haris, Hassibi, Babak, Hassibi, Arjang
• Granted Patent: US 8,048,626 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• 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

Abstract

This invention provides methods and systems for measuring the concentration of multiple nucleic acid sequences in a sample. The nucleic acid sequences in the sample are simultaneously amplified, for example, using polymerase chain reaction (PCR) in the presence of an array of nucleic acid probes. The amount of amplicon corresponding to the multiple nucleic acid sequences can be measured in real-time during or after each cycle using a real-time microarray. The measured amount of amplicon produced can be used to determine the original amount of the nucleic acid sequences in the sample.

Claims

1. A method comprising;

(a) performing a nucleic acid amplification on two or more nucleotide sequences to produce two or more amplicons in a fluid wherein the array comprises a solid surface with a plurality of nucleic acid probes at independently addressable locations; and

(b) measuring the hybridization of the amplicons to the two or more nucleic acid probes while the fluid is in contact with the array to obtain an amplicon hybridization measurement.

2. The method of claim 1 further comprising using the amplicon hybridization measurement to determine the concentration of the amplicons in the fluid.

3. The method of claim 2 further comprising using the amplicon hybridization measurement to determine the original amount of nucleotide sequences.

4. The method of claim 3 wherein the concentration of amplicon is measured during or after some, but not all of the amplification cycles.

5. The method of claim 3 wherein the concentration of amplicon is measured during or after each of the amplification cycles.

6. The method of claim 3 wherein the concentration of amplicon is measured during on average every 2, 3, 4, 5, 6, 7, 8, 9 or 10 amplification cycles.

7. The method of claim 3 wherein the nucleic acid amplification is polymerase chain reaction (PCR), and an amplification cycle corresponds with a temperature cycle.

8. The method of claim 7 wherein at least some temperature cycles comprise (a) a probe hybridization phase at one temperature, and (b) primer annealing phase at a higher temperature than the probe hybridization temperature.

9. The method of claim 7 wherein at least some temperature cycles comprise 4 or more temperature phases, wherein one or more of the phases is a probe hybridization phase wherein the hybridization of amplicons to the nucleic acid probes is measured.

10. The method of claim 9 wherein the temperature is changed during probe hybridization phase.

11. The method of claim 9 comprising two or more hybridization phases, carried out at different temperatures.

12. The method of claim 7 wherein at least some temperature cycles comprise a first denaturing phase, a probe hybridization phase, a second denaturing phase, a primer annealing phase, and an extension phase.

13. The method of claim 1 wherein the array is in contact with the fluid during the amplification.

14. The method of claim 1 wherein the two or more nucleotide sequences are not only two complementary nucleotide sequences.

15. The method of claim 1 wherein the amplification is an isothermal amplification.

16. The method of claim 1 wherein the amplification is a linear amplification.

17. The method of claim 1 wherein the measuring of hybridization comprises measuring the kinetics of hybridization of the amplicons to the nucleic acid probes.

18. The method of claim 15 wherein the measuring of the kinetics of hybridization comprises measuring a light signal at multiple time points.

19. The method of claim 1 wherein the amplicons comprise a quencher.

20. The method of claim 19 wherein primers are used to create the amplicons and the primers comprise a quencher.

21. The method of claim 20 wherein one of the primers in a primer pair comprises a quencher.

22. The method of claim 20 wherein both of the primers in a primer pair comprise a quencher.

23. The method of claim 19 wherein quenchers are incorporated into the amplicons as they are formed.

24. The method of claim 23 wherein d-NTP'"'"'s are used to make the amplicons, and one or more of the d-NTP'"'"'s used to make the amplicon comprises a quencher.

25. The method of claim 1 wherein the amplicon hybridization measurement is performed by measuring fluorescence from fluorescent moieties attached to the solid surface.

26. The method of claim 25, wherein the fluorescent moieties are covalently attached to the nucleic acid probes.

27. The method of claim 25, wherein the fluorescent moieties are attached to the substrate and are not covalently attached to the nucleic acid probes.

28. The method of claim 25 wherein the amplicons comprise quenchers, and the measuring of hybridization is performed by measuring a decrease in fluorescence due to hybridization of amplicons to the nucleic acid probes.

29. The method of claim 1 wherein the array comprises at least about 3, 4, 5 10, 50, 100, 100, 1000, 10,000, 100,000, or 1,000,000 nucleic acid probes.

30. The method of claim 1 wherein the nucleic acid probe comprises a PNA or LNA probe.

31. A method comprising:

(a) providing an array comprising a solid support having a surface and a plurality of different probes, the different probes immobilized to the surface at different addressable locations, each addressable location comprising a fluorescent moiety;

(b) performing PCR amplification on a sample comprising a plurality of nucleotide sequences;

the PCR amplification carried out in a fluid, wherein;

(i) a PCR primer for each nucleic acid sequence comprises a quencher; and

(ii) the fluid is in contact with the probes, whereby amplified molecules can hybridize with probes, thereby quenching signal from the fluorescent moiety;

(c) detecting the signals from the fluorescent moieties at the addressable locations over time;

(d) using the signals detected over time to determine the amount of amplified molecules in the fluid; and

(e) using the amount of amplified molecules in the fluid to determine the amount of the nucleotide sequences in the sample.

32. The method of claim 31 wherein the determining of the amount of amplified molecules is performed during or after multiple temperature cycles of the PCR amplification.

33. The method of claim 31 wherein more than one PCR primer for each nucleic acid sequence comprises a quencher.

34. The method of claim 31 wherein the detecting of the signals from the fluorescent moieties at the addressable locations over time comprises measuring the rate of hybridization of the amplified molecules with the probes.

35. The method of claim 31 wherein the sample comprises messenger RNA or nucleotide sequences derived from messenger RNA, and the determination of the amount of nucleic acid sequence in the sample is used to determine the level of gene expression in a cell or group of cells from which the sample was derived.

36. The method of claim 31 wherein the sample comprises genomic DNA or nucleotide sequences derived from genomic DNA, and the determination of the amount of nucleic acid sequence in the sample is used to determine the genetic makeup of a cell or group of cells from which the sample was derived

37. The method of claim 31 wherein two or more PCR primers corresponding to two or more different nucleotide sequences have different quenchers.

38. The method of claim 31 wherein two or more different addressable locations comprise different fluorescent moieties.

39. The method of claims 37 or 38 wherein the different quenchers and/or different fluorescent moieties are used to determine cross-hybridization.

40. A diagnostic test for determining the state of health of an individual comprising performing the method of claim 31 on a sample from such individual.

41. A method comprising

(a) measuring the amount of 10 or more amplicons corresponding to 10 or more different nucleotide sequences in a single fluid volume during or after multiple amplification cycles to determine amplicon amount-amplification cycle values, and

(b) using the amplicon amount-amplification cycle values to determine the presence or amount of the 10 or more nucleotide sequences in a sample.

42. The method of claim 41 wherein 20 or more amplicons corresponding to 20 or more different nucleotide sequences are used to determine the amount of 20 or more nucleotide sequences.

43. The method of claim 41 wherein 50 or more amplicons corresponding to 50 or more different nucleotide sequences are used to determine the amount of 50 or more nucleotide sequences.

44. The method of claim 41 wherein the multiple amplification cycles comprise about 10-40 amplification cycles.

45. The method of claim 41 wherein the amount of the 10 or more amplicons is measured in real-time.

46. The method of claim 41 wherein the amount of the 10 or more amplicons is measured by measuring the kinetics of binding of the amplicons to nucleic acid probes.

47. The method of claim 41 wherein the amount of the 10 or more amplicons is measured by measuring the quenching of fluorescence.

48. A system comprising:

(a) a PCR amplification reaction chamber capable of receiving;

(i) a substrate comprising a surface with an array of nucleic acid probes at independently addressable locations, and

(ii) a fluid to be held contact with the substrate, the fluid comprising a nucleic acid sample comprising multiple nucleotide sequences, primers, and enzymes;

(b) a temperature controller capable of carrying out multiple PCR temperature phases and temperature cycles comprising;

(i) a heating and cooling module for raising and lowering the temperature of the fluid and/or the substrate; and

(ii) a temperature sensor; and

(c) a detector capable of detecting light signals as a function of time from the independently addressable locations on the substrate within the chamber at a specific phase or phases during or after a plurality of temperature cycles while the fluid is in contact with the substrate.

49. The system of claim 48 further comprising:

(d) an analysis block comprising a computer and software capable of determining the amounts of amplified products hybridized to the array of probes using the detected light as a function of time, and of determining the amounts of multiple nucleotide sequences in a sample using the amounts of amplified products determined during or after a plurality of temperature cycles.

50. The system of claim 48 wherein the detector comprises a photodiode, a CCD array, or a CMOS array.

51. The system of claim 48 wherein the detector is in contact with the substrate, and different areas of the detector correspond to different detectable locations.

52. The system of claim 48 wherein the detector is optically coupled to the substrate with lenses and/or waveguides.

53. The system of claim 48 wherein the heating and cooling module is capable of raising or lowering the temperature at a rate of greater than 5°

C. per sec.

54. The system of claim 48 wherein the heating and cooling module is capable of raising or lowering the temperature at a rate of greater than 10°

C. per sec.

55. The system of claim 48 wherein the heating and cooling module is capable of raising or lowering the temperature at a rate of greater than 20°

C. per sec.

56. The system of claim 48 wherein the temperature sensor is capable of measuring temperature at an accuracy of 0.1°

C.

57. The system of claim 48 wherein system comprises a microfluidic device having multiple arrays, each of the multiple arrays is in a chamber which can hold the fluid to be held in contact with the substrate.

58. The system of claim 57 wherein at least some of the multiple arrays are addressed with different temperature profiles.

59. The system of claim 57 wherein the microfluidic device has about 4, 5, 6, 7, 8, 9, or 10 arrays.