Multiplex Q-PCR arrays

• US 9,458,497 B2
• Filed: 09/22/2011
• Issued: 10/04/2016
• Est. Priority Date: 07/28/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hassibi, Arjang, Hassibi, Babak, Vikalo, Haris
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/240,603
• Publication Number:
  US 20120077692A1
• Time in Patent Office: 1,839 Days
• Field of Search: None
• US Class Current: 1/1
• 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) providing an array comprising a solid surface with a plurality of nucleic acid probes at independently addressable locations, which plurality of nucleic acid probes comprises 5 or more different nucleotide sequences;

(b) using the array to measure an amount of 5 or more amplicons corresponding to the 5 or more different nucleotide sequences in a single fluid volume during or after multiple amplification cycles to determine amplicon amount-amplification cycle values; and

(c) using the amplicon amount-amplification cycle values to determine a presence or amount of the 5 or more nucleotide sequences in a sample,

wherein the amount of the 5 or more amplicons is measured by measuring kinetics of binding of the 5 or more amplicons to nucleic acid probes.

2. The method of claim 1, wherein 20 or more amplicons corresponding to 20 or more different nucleotide sequences are used to determine an amount of 20 or more nucleotide sequences.

3. The method of claim 1, wherein 50 or more amplicons corresponding to 50 or more different nucleotide sequences are used to determine an amount of 50 or more nucleotide sequences.

4. The method of claim 1, wherein the multiple amplification cycles comprise about 10-40 amplification cycles.

5. The method of claim 1, wherein the amount of the 5 or more amplicons is measured in real-time.

6. The method of claim 1, wherein the amount of the 5 or more amplicons is measured by measuring quenching of fluorescence.

7. The method of claim 1, wherein each of the plurality of nucleic acid probes comprises a donor and each of the 5 or more amplicons comprises an acceptor.

8. The method of claim 7, wherein the donor is a fluorophore and the acceptor is a quencher.

9. A method comprising:

(a) providing an array comprising a solid surface with a plurality of nucleic acid probes at independently addressable locations, which plurality of nucleic acid probes comprises 5 or more different nucleotide sequences;

(b) using the array to measure an amount of 5 or more amplicons corresponding to the 5 or more different nucleotide sequences in a single fluid volume during or after multiple amplification cycles to determine amplicon amount-amplification cycle values; and

(c) using the amplicon amount-amplification cycle values to determine a presence or amount of the 5 or more nucleotide sequences in a sample,

wherein the amount of the 5 or more amplicons is measured by measuring quenching of a detectable optical signal.

10. The method of claim 9, wherein 20 or more amplicons corresponding to 20 or more different nucleotide sequences are used to determine an amount of 20 or more nucleotide sequences.

11. The method of claim 9, wherein 50 or more amplicons corresponding to 50 or more different nucleotide sequences are used to determine an amount of 50 or more nucleotide sequences.

12. The method of claim 9, wherein the multiple amplification cycles comprise about 10-40 amplification cycles.

13. The method of claim 9, wherein the amount of the 5 or more amplicons is measured in real-time.

14. The method of claim 9, wherein the detectable optical signal is fluorescence.

15. The method of claim 9, wherein each of the plurality of nucleic acid probes comprises a donor and each of the 5 or more amplicons comprises an acceptor.

16. The method of claim 15, wherein the donor is a fluorophore and the acceptor is a quencher.