Combined polynucleotide sequence as discrete assay endpoints

• US 6,316,608 B1
• Filed: 03/20/2000
• Issued: 11/13/2001
• Est. Priority Date: 03/20/2000
• Status: Expired due to Fees

• Current Assignee: Incyte Corporation
• Original Assignee: Incyte Corporation
• Inventors: Reynolds, Mark A., Ruvolo, Michael, Arnold, Lyle J. Jr.
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/532,656
• Time in Patent Office: 603 Days
• Field of Search: 435/6, 435/395, 536/22.1, 536/25.32, 536/25.4, 536/25.5
• US Class Current: 536/22.1
• 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

Abstract

Methods are provide for determining the relative amounts of individual polynucleotides in a complex mixture. The polynucleotides, after fluorescent labeling, are contacted under hybridization conditions with an array having element disposed at discrete locations on a substrate. The elements comprise two or more distinct polynucleotides that are combined prior to arraying. The level of fluorescence associated with each element provides a measure of its relative amount in the mixture.

Claims

1. A microarray of non-contiguous polynucleotides, the array comprising:

a solid support having a surface; and

a plurality of elements arranged in a defined manner and stably associated with the surface, wherein each element comprises two or more combined, distinct polynucleotides having distinct sequences.

2. The array of claim 1, wherein the solid support is selected from the group consisting of glass, silica, plastic, ceramic, beads, and nylon, and combinations thereof.

3. The array of claim 1, wherein each element comprises about two to about ten combined, distinct polynucleotides having distinct sequences.

4. The array of claim 1, wherein each element comprises about two to about five combined, distinct polynucleotides having distinct sequences.

5. The array of claim 1, wherein the polynucleotide sequences are complementary to a single target polynucleotide.

6. The array of claim 1, wherein the polynucleotides independently comprise from about 5 to about 10000 nucleotides.

7. The array of claim 6, wherein the polynucleotides independently comprise from about 20 to about 300 nucleotides.

8. The array of claim 7, wherein the polynucleotides independently comprise from about 50 to about 65 nucleotides.

9. The array of claim 1, wherein the polynucleotides are double stranded.

10. The array of claim 1, wherein the elements are associated with the surface by non-covalent means.

11. The array of claim 1, wherein the elements are associated with the surface by covalent means.

12. The array of claim 11, wherein the covalent means comprises a linker.

13. The array of claim 1, wherein the elements are associated with the surface by non-covalent and covalent means.

14. A hybridization assay comprising the steps of:

contacting the array of claim 1, 3, 5, 6, or 10 with detectable nucleic acid probes under hybridization conditions to produce a hybridization pattern; and

detecting the hybridization pattern.

15. A kit for use in measuring hybridization, comprising:

an array according to claim 1, 3, 5, 6 or 10; and

instructional material for measuring hybridization with the array.