Expression monitoring by hybridization to high density oligonucleotide arrays

• US 6,927,032 B2
• Filed: 01/28/2003
• Issued: 08/09/2005
• Est. Priority Date: 09/15/1995
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Lockhart, David J., Gingeras, Thomas R., Brown, Eugene L., Chee, Mark, Wong, Gordon G.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/353,792
• Publication Number:
  US 20030215841A1
• Time in Patent Office: 924 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.51, 435/183, 435/283.1, 435/287.1, 435/287.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: G01N 15/1433 using image recognition

Abstract

This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm², and the olignucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Claims

1. A method for monitoring hybridization of an eukaryotic nucleic acid sample to a nucleic acid array, said method comprising the steps of:

(a) providing a nucleic acid array comprising a plurality of nucleic acid test probes that specifically bind to eukaryotic nucleic acid targets and at least one control probe that specifically binds to a bacterial gene;

(b) providing a spiked sample comprising an eukaryotic nucleic acid sample with a nucleic acid control, said control comprising a nucleic acid that specifically binds to the control probe;

(c) contacting said spiked sample with said array; and

(d) determining whether hybridization occurred between the control probe and the nucleic acid control.

2. The method according to claim 1, wherein said nucleic acid array is an oligonucleotide array.

3. The method according to claim 2, wherein said eukaryotic nucleic acid sample comprises cRNA.

4. The method according to claim 3, wherein said cRNA is labeled with a label to allow detection of hybridization of said cRNA to a nucleic acid array.

5. The method according to claim 3, wherein said cRNA is fragmented before hybridization to said oligonucleotide array.

6. The method according to claim 3, wherein said cRNA is labeled with biotin.

7. The method according to claim 1, wherein said at least one control probe specifically binds to the bacterial gene Bio B.

8. The method according to claim 5, wherein said nucleic acid control is an oligonucleotide probe.

9. The method according to claim 2, wherein said nucleic acid control is a cRNA.

10. The method according to claim 4, wherein said cRNA is fragmented before hybridization to said oligonucleotide array.

11. The method according to claim 7, wherein said nucleic acid control is cRNA.

12. The method according to claim 11, wherein said cRNA is labeled with a label to allow detection of hybridization of said cRNA to an array.

13. The method according to claim 12, wherein said cRNA is fragmented prior to hybridization of an array.

14. The method according to claim 13, wherein said cRNA is labeled with biotin.

15. The method of claim 1, wherein the array comprises greater than 100 probes per cm².

16. The method of claim 1, wherein the array comprises greater than 1000 probes per cm².

17. The method of claim 14, further comprising staining with a streptavidin conjugate to detect the biotin.

18. The method of claim 14, further comprising detecting the biotin with an avidin-conjugated fluorophore.

19. The method of claim 4, wherein the label is a fluorescent label.

20. The method of claim 1, wherein the determining is performed using confocal microscopy.

21. The method of claim 20, wherein the microscope is equipped with a phototransducer attached to an automated data acquisition system to record the fluorescent signal produced by hybridization to each probe in the array.

22. The method of claim 1, further comprising amplifying the eurkaryotic nucleic acid sample.

23. The method of claim 22, wherein the amplification is performed with a labeled primer or labeled nucleotide.

24. The method of claim 1, further comprising reverse transcribing the eukaryotic nucleic acid samle using a primer consisting of oligo dT and a sequence encoding the phage T7 premtoter.

25. A method for performing control hybridization reactions for hybridization of an eukaryotic nucleic acid sample to a nucleic acid array, the method comprising the steps of:

(a) providing a nucleic acid array comprising a plurality of probes, wherein two or more of said probes comprise control probes that specifically bind to different bacterial genes not normally found in said eukaryotic nucleic acid sample;

(b) providing a spike sample comprising said eukaryotic nucleic acid sample with nucleic acid controls, each of the controls comprising a nucleic acid that specifically binds to one of the control probes;

(c) contacting said spiked sample to the array; and

(d) determining whether hybridization occurred to said control probes.