Normalization control for hybridization reactions

• US 6,423,535 B1
• Filed: 11/24/1999
• Issued: 07/23/2002
• Est. Priority Date: 11/24/1999
• Status: Expired due to Fees

• Current Assignee: Incyte Pharmaceuticals, Inc. (Incyte Corporation)
• Original Assignee: Incyte Genomics, Inc. (Incyte Corporation)
• Inventors: Theriault, Thomas P., Lei, Warren, Bjeldanes, Erik, Bedilion, Tod, Arnold, Lyle
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/449,059
• Time in Patent Office: 972 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 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
• US Class Current: 435/287.2
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods are provide for quantitatively determining the amount of polynucleotides in a sample. In one method, the distinct polynucleotide targets and standard polynucleotide targets are contacted with detectable probes and independently detectable single or double stranded complements to the standard targets under hybridization conditions. The hybridization pattern from the probe is compared to the hybridization pattern from the standard sequences to obtain quantitative information about the genetic profile of the labeled nucleic acid sample. Also provided are kits for the use of the methods.

Claims

1. A hybridization assay using an array comprising samples separately arrayed on and stably associated with a surface of a solid support, each sample comprising a distinct sample polynucleotide target and a standard polynucleotide target, wherein the sample target and the standard target arm co-arrayed to the same location on the array, the method comprising the steps of:

contacting each sample of the anay with a detectable sample probe specific for the sample target, and an independently detectable standard probe specific for the standard target, under hybridization conditions to produce a hybridization pattern comprising a sample probe signal and a standard probe signal at each sample, wherein the standard probe signal provides a normalization control for the sample signal; and

detecting the hybridization pattern.

2. The assay of claim 1, wherein the sample targets and the standard targets are covalently linked.

3. The assay of claim 1, wherein the sample targets and the standard targets are not covalently linked.

4. The assay of claim 1, wherein the standard targets are about 5 to about 10000 nucleotides in length.

5. The assay of claim 1, wherein the standard targets are about 20 to about 80 nucleotides in length.

6. The assay of claim 1 wherein the sample probes comprise a detectable first fluorescent label and the standard probes comprise an independently detectable second fluorescent label.

7. The assay of claim 1, wherein the standard probes are single-stranded probes.

8. The assay of claim 1, wherein the standard probes are anti-sense.

9. The assay of claim 1, wherein the standard probes are double-stranded probes.

10. The assay of claim 1, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the corresponding standard targets.

11. The assay of claim 1, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the corresponding standard targets, and wherein the sense and anti-sense complements are present in approximately equimolar concentration.

12. The assay of claim 1, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the corresponding standard targets, and wherein the sense and anti-sense complements are covalently attached.

13. The assay of claim 1, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the corresponding standard targets, and wherein the sense and anti-sense complements are covalently attached to form a hairpin.

14. The assay of claim 1, wherein each sample has a common standard target.

15. The assay of claim 1, wherein each sample has a different standard target.

16. The assay of claim 1, wherein preceding the contacting step the method comprises the step of amplifying the sample targets from cDNA.

17. The assay of claim 1, 2, 3 or 9, wherein the sample targets and standard targets are in predetermined and known stoichiometry.

18. The assay of claim 1, wherein the sample targets and standard targets are in equimolar concentration.

19. A hybridization assay using a microarray comprising samples separately arrayed on and stably associated with a surface of a solid support, each sample comprising a distinct sample polynucleotide target and a standard polynucleotide target, wherein the sample target and the standard target are co-arrayed to the same location on the array, the method comprising the steps of:

contacting each sample of the microarray with a detectable sample probe specific for the sample target, and an independently detectable standard probe specific for the standard target, under hybridization conditions to produce a hybridization pattern comprising a sample probe signal and a standard probe signal at each sample, wherein the standard probe signal provides a normalization control for the sample signal, and wherein the sample probes comprise a detectable first fluorescent label and the standard probes comprise an independently detectable second fluorescent label, and detecting the hybridization pattern.

20. The assay of claim 19, wherein the sample targets and the standard targets are covalently linked.

21. The assay of claim 19, wherein the sample targets and the standard targets are not covalently linked.

22. The assay of claim 19, wherein the standard targets comprise from about 5 to about 10000 nucleotides in length.

23. The assay of claim 19, wherein the standard targets comprise from about 20 to about 80 nucleotides in length.

24. The assay of claim 19, wherein the standard probes are single-stranded probes.

25. The assay of claim 19, wherein the standard probes are anti-sense.

26. The assay of claim 19, wherein the standard probes are double-stranded probes.

27. The assay of claim 19, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the standard probes.

28. The assay of claim 19, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the standard targets, and wherein the sense and anti-sense complements are present in approximately equimolar concentration.

29. The assay of claim 19, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the standard targets, and wherein the sense and anti-sense complements are covalently attached.

30. The assay of claim 19, wherein the standard probes are double-stranded probes which comprise sense and anti-sense complements to the standard probes, and wherein the sense and anti-sense complements are covalently attached to form a hairpin.

31. The assay of claim 19, wherein each sample has a common standard target.

32. The assay of claim 19, wherein each sample has a different standard target.

33. The assay of claim 19, wherein preceding the contacting step the method comprises the step of amplifying the sample targets from cDNA.

34. The assay of claim 19, 20, 21, or 26 wherein the sample targets and standard targets are in predetermined and known stoichiometry.

35. the assay of claim 19, wherein the sample targets and standard targets are in equimolar concentration.

36. The assay of claim 19 wherein preceding the contacting step the method comprises the step of making the microarray by microdeposition.

37. The assay of claim 19 wherein preceding the contacting step the method comprises the step of making the microarray by microdeposition effected by an ink jet application apparatus.

38. The assay of claim 19 wherein preceding the contacting step the method comprises the step of making the microarray by in situ synthesis by photolithography.

39. A hybridization assay using a microarray comprising samples separately arrayed on and stably associated with a surface of a solid support, each sample comprising a distinct sample polynucleotide target and a standard polynucleotide target, wherein the sample target and the standard target are co-arrayed to the same location on the array, the method comprising the steps of:

contacting each sample of the microarray with a detectable sample probe specific for the sample target, and an independently detectable standard probe specific for the standard target, under hybridization conditions to produce a hybridization pattern comprising a sample probe signal and a standard probe signal at each sample, wherein the standard probe signal provides a normalization control for the sample signal, and wherein the sample probes comprise a detectable first fluorescent label and the standard probes comprise an independently detectable second fluorescent label, and wherein each sample has a common standard target, and wherein the sample targets and standard targets are in predetermined and known stoichiometry, and

detecting the hybridization pattern.

40. A hybridization assay using a microarray comprising samples separately arrayed on and stably associated with a surface of a solid support, each sample comprising a distinct sample polynucleotide target and a standard polynucleotide target, wherein the sample target and the standard target are co-arrayed to the same location on the array, the method comprising the steps of:

contacting each sample of the microarray with a detectable sample probe specific for the sample target, and an independently detectable standard probe specific for the standard target, under hybridization conditions to produce a hybridization pattern comprising a sample probe signal and a standard probe signal at each sample, wherein the standard probe signal provides a normalization control for the sample signal, and wherein the sample probes comprise a detectable first fluorescent label and the standard probes comprise an independently detectable second fluorescent label, and wherein the standard targets comprise from about 20 to about 80 nucleotides in length, and wherein each sample has a common standard target, and wherein the sample targets and standard targets are in predetermined and known stoichiometry, and

detecting the hybridization pattern.