High throughput assay system

• US 20040185464A1
• Filed: 10/09/2003
• Published: 09/23/2004
• Est. Priority Date: 09/15/2000
• Status: Abandoned Application

• Current Assignee: Richard M. Kris, Stephen Felder
• Original Assignee: Richard M. Kris, Stephen Felder
• Inventors: Felder, Stephen, Kris, Richard M.
• Application Number:
  US10/681,208
• Publication Number:
  US 20040185464A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2539/105 Involving introns, exons, or splice junctions

Abstract

The present invention relates to compositions, apparatus and methods useful for concurrently performing multiple, high throughput, biological or chemical assays, using repeated arrays of probes. A combination of the invention comprises a surface, which comprises a plurality of test regions, at least two of which, and in a preferred embodiment, at least twenty of which, are substantially identical, wherein each of the test regions comprises an array of generic anchor molecules. The anchors are associated with bifunctional linker molecules, each containing a portion which is specific for at least one of the anchors and a portion which is a probe specific for a target of interest. The resulting array of probes is used to analyze the presence or test the activity of one or more target molecules which specifically interact with the probes. In one embodiment of the invention, the test regions (which can be wells) are further subdivided into smaller subregions (indentations, or dimples).

In one embodiment of the invention, ESTs are mapped. In another embodiment, the presence of a target nucleic acid is detected by protecting the target against nuclease digestion with a polynucleotide fragment, and analyzing the protected polynucleotide by mass spectrometry.

Claims

1. A method to detect one or more nucleic acids of interest, comprising subjecting a sample comprising said nucleic acid(s) to nuclease protection with one or more protection fragments, and detecting the hybridized duplex molecules, or the single strand protected nucleic acid(s), or the protection fragment(s), with mass spectrometry.

2. The method of claim 1, wherein the method is a high throughput method.

3. The method of claim 2, wherein the nucleic acid(s) which is detected is a protection fragment(s).

4. The method of claim 3, wherein at least two different protection fragments are detected.

5. The method of claim 3, wherein at least 16 different protection fragments are detected.

6. The method of claim 2, wherein the nucleic acid(s) which is detected is a hybridized duplex molecule.

7. The method of claim 2, wherein the nucleic acid(s) which is detected is the protected nucleic acid.

8. The method of claim 2, wherein said nucleic acid(s) of interest is measured.

9. The method of claim 8, wherein the nucleic acid(s) which is measured is a protection fragment(s).

10. The method of claim 2, wherein said protection fragment is modified chemically, and said chemical modification, with or without the nucleic acid portion of the protection fragment, is detected.

11. A combination useful for the detection of one or more target(s) in a sample, which comprises, before the addition of said sample,

a) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising

b) at least eight different oligonucleotide anchors, each in association with

c) a bifunctional linker which has a first portion that is specific for the oligonucleotide anchor, and a second portion that comprises a probe which is specific for said target(s).

12. The combination of claim 11, having about 96 substantially identical regions, wherein each region comprises about 16, 36, 46 or 100 different oligonucleotide anchors.

13. The combination of claim 11, having about 384 substantially identical regions, wherein each region comprises about 9, 16, or 25 different oligonucleotide anchors.

14. The combination of claim 11, having about 1536 substantially identical regions, wherein each region comprises about 4 or 9 different oligonucleotide anchors.

15. A method of detecting at least one target, comprising

a) contacting a sample which may comprise said target(s) with the combination of claim 11, under conditions effective for said target to bind to said combination,

b) contacting said combination and any bound targets with a labeled detection probe, and

c) detecting said detection probe.

16. The method of claim 15, wherein said labeled detection probe produces a chemiluminescent signal.

17. The method of claim 15, wherein the target is measured.

18. A method of detecting at least one target, comprising

a) contacting a sample which may comprise said target(s) with a bifunctional linker which has a first portion that is specific for an oligonucleotide anchor and a second portion that comprises a probe which is specific for said target(s), under conditions effective to obtain a first hybridization product between said target(s) and said linker,

b) contacting said first hybridization product with a combination under conditions effective to obtain a second hybridization product between said first hybridization product and said combination, wherein said combination comprises, before the addition of said first hybridization product, a surface comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising

at least 8 different oligonucleotide anchors,

c) contacting said first hybridization product or said second hybridization product with a labeled detector probe, and

d) detecting said detection probe.