Quantitative microarray hybridizaton assays

• US 6,004,755 A
• Filed: 04/07/1998
• Issued: 12/21/1999
• Est. Priority Date: 04/07/1998
• Status: Expired due to Fees

• Current Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Original Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Inventors: Wang, Bruce
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US09/056,338
• Time in Patent Office: 623 Days
• Field of Search: 435/6, 536/26.6, 536/24.3, 536/24.31, 536/24.32
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

Methods are provided for quantitative gene expression analysis. In the subject methods, end-labeled target nucleic acid is contacted with an array of probe molecules stably associated with the surface of a solid support under hybridization conditions sufficient to produce a hybridization pattern. The resultant hybridization pattern can be used to obtain a quantitative information about the genetic profile of the end-labeled target nucleic acid sample, as well as the physiological source from which it is derived. As such, the subject methods find use in a variety of applications.

Claims

1. A hybridization assay comprising the steps of:

contacting an array of probe molecules stably associated with the surface of a solid support with an end labeled target nucleic acid sample under hybridization conditions sufficient to produce a hybridization pattern, wherein each of said end labeled target nucleic acids is capable of generating a signal of substantially the same specific activity; and

detecting said hybridization pattern.

2. The assay according to claim 1 further comprising the steps of removing unhybridized target nucleic acid prior to said detecting step.

3. The assay according to claim 1, wherein said method further comprises preparing said end labeled target nucleic acid sample by contacting an mRNA source with an end-labeled oligo(dT) primer, a reverse transcriptase and nucleotides under conditions sufficient for reverse transcription of said mRNA into said end-labeled target nucleic acid sample, wherein said-labeled oligo(dT) primer comprises a known number of labeled nucloetides.

4. The assay according to claim 1, wherein said end-labeled nucleic acid sample further comprises end-labeled standard DNA.

5. The assay according to claim 4, wherein said assay further comprises:

(a) preparing said end-labeled standard DNA by contacting a standard RNA sample with said end-labeled oligo(dT) primer, a reverse transcriptase and nucleotides under conditions sufficient for reverse transcription of said standard RNA into said end labeled standard DNA; and

(b) combining said end labeled standard DNA with said end-labeled target nucleic acid sample.

6. The assay according to claim 1, wherein said array is contacted with a nuclease prior to said detecting step.

7. An assay to determine the genetic profile of a physiological source, said assay comprising the steps of:

(a) preparing an end labeled target nucleic acid sample by contacting mRNA from a physiological source with an end-labeled oligo(dT) primer, a reverse transcriptase and nucleotides under conditions sufficient for reverse transcription of said mRNA into said end-labeled target nucleic acid sample, wherein said end-labeled oligo (dT) primer is comprises a known number of labeled nucleotides;

(b) preparing end labeled standard DNA by contacting standard RNA with said end-labeled oligo(dT) primer, a reverse transcriptase and nucleotides under conditions sufficient for reverse transcription of said standard RNA into said end labeled standard DNA;

(c) combining said end labeled standard DNA with said end-labeled target nucleic acid sample;

(d) contacting said end labeled target nucleic acid sample with an array of probe molecules stably associated with the surface of a solid support under hybridization conditions to produce a hybridization pattern;

(e) separating unhybridized target from said array;

(f) contacting said array with a nuclease; and

(g) detecting said hybridization pattern.

8. The assay according to claim 7, wherein said end-labeled target nucleic acids are not directly detectable and said method further comprises contacting said hybridization pattern with at least one additional member of a signal producing system to provide a detectable hybridization pattern.

9. The assay according to claim 8, wherein said contacting with at least one additional member of a signal producing system is prior to said step (f).

10. The assay according to claim 8, wherein said end-labeled target nucleic acids are labeled with biotin.

11. The assay according to claim 9, wherein said at least one additional member of a signal producing system is fluorescently labeled streptavidin.

12. A kit for use in the preparation of end-labeled target nucleic acids to be used in an array based assay, said kit comprising:

end labeled oligo(dT) primers capable of generating a signal of known value; and

a polymerase.

13. The kit according to claim 12, wherein said kit further comprises nucleotides.

14. The kit according to claim 12, wherein said kit further comprises a buffer.

15. The kit according to claim 12, wherein said kit further comprises an array of probe molecules and said kit is for use in a hybridization assay.

16. The kit according to claim 12, wherein said kit further comprises standard RNA.

17. The kit according to claim 12, wherein said kit further comprises a nuclease.