Multiplex Q-PCR arrays

• US 8,048,626 B2
• Filed: 07/27/2007
• Issued: 11/01/2011
• 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
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US11/829,861
• Publication Number:
  US 20080176757A1
• Time in Patent Office: 1,558 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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;

(b) providing an array comprising a solid surface with a plurality of nucleic acid probes at independently addressable locations, said array configured to contact said fluid; and

(c) 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 wherein the amplicons comprise a quencher.

2. The method of claim 1 wherein primers are used to create the amplicons and the primers comprise a quencher.

3. The method of claim 2 wherein one of the primers in a primer pair comprises a quencher.

4. The method of claim 2 wherein both of the primers in a primer pair comprise a quencher.

5. The method of claim 1 wherein quenchers are incorporated into the amplicons as they are formed.

6. The method of claim 5 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.

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

8. The method of claim 7, wherein the fluorescent moieties are covalently attached to the nucleic acid probes.

9. The method of claim 7, wherein the fluorescent moieties are attached to the substrate and are not covalently attached to the nucleic acid probes.

10. The method of claim 7 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.

11. 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.

12. The method of claim 11 wherein the determining of the amount of amplified molecules is performed during or after multiple temperature cycles of the PCR amplification.

13. The method of claim 11 wherein more than one PCR primer for each nucleic acid sequence comprises a quencher.

14. The method of claim 11 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.

15. The method of claim 11 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.

16. The method of claim 11 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.

17. The method of claim 11 wherein two or more PCR primers corresponding to two or more different nucleotide sequences have different quenchers.

18. The method of claim 11 wherein two or more different addressable locations comprise different fluorescent moieties.

19. The method of claim 17 or 18 wherein the different quenchers and/or different fluorescent moieties are used to determine cross-hybridization.

20. A diagnostic test for determining the state of health of an individual comprising performing the method of claim 11 on a sample from such individual.