High throughput assay system

• US 6,232,066 B1
• Filed: 07/02/1998
• Issued: 05/15/2001
• Est. Priority Date: 12/19/1997
• Status: Expired due to Term

• Current Assignee: High Throughput Genomics, Inc.
• Original Assignee: Neogen Corporation
• Inventors: Felder, Stephen, Kris, Richard M.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/109,076
• Time in Patent Office: 1,048 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/22.1
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00662 Two-dimensional arrays within two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

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).

Claims

1. A method of detecting at least one nucleic acid target, comprising contacting a sample which may comprise said target(s) with a nuclease protection fragment(s) specific for and which binds to said target(s), exposing the s ple to a nuclease effective to digest remaining single strand nucleic acid, and then contacting the resultant sample with a combination which comprises, before the addition of said sample,

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

ii) at least two different anchors, each in association with

iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for said nuclease protection fragment(s),

under conditions effective for said nuclease protection fragment(s) to bind to said combination,

and detecting said bound protection fragment(s).

3. The method of claim 1, wherein each region comprises at least about four different oligonucleotide anchors.

4. The method of claim 1, wherein each region comprises at least about eight different oligonucleotide anchors.

5. The method of claim 1, wherein each region comprises at least about 64 different oligonucleotide anchors.

6. The method of claim 1, wherein each surface comprises at least about 96 to about 1536 substantially identical, spatially discrete regions.

7. The method of claim 1, wherein each surface comprises about 4-1536 substantially identical, spatially discrete regions, each region comprising about 8 to about 100 different oligonucleotide anchors.

8. The method of claim 1, wherein each surface comprises about 4, 8, 96 or 384 substantially identical, spatially discrete regions, each region comprising about 4 to about 100 different oligonucleotide anchors.

9. The method of claim 1, wherein each of said regions comprises about 30 to about 100 probes, each specific for a different target.

10. The method of claim 1, wherein said regions are further subdivided into smaller subregions.

11. The method of claim 1, wherein each region is a well of a microtiter plate.

12. The method of claim 1, wherein said anchors are oligonucleotides of about 0 to about 50 nucleotides in length.

13. The method of claim 1, wherein one or more regions further comprise controls for hybridization efficiency or specificity.

14. The method of claim 1, wherein one or more regions further comprise controls for the capacity of a locus to bind target.

15. The method of claim 1, further comprising contacting said combination and any bound nuclease protection fragments with a labeled detection probe, and detecting said detection probe.

16. The method of claim 1, further comprising

a) subjecting the sample comprising target(s) bound to nuclease protection fragment(s) to treatment with one or more nucleases effective for digesting nucleic acid other than the nuclease protection fragment(s) which have hybridized to the nucleic acid(s) of interest and, optionally, the portion(s) of said nucleic acid(s) which have been hybridized, and

b) removing nucleic acid material other than said nuclease protection fragment(s) which have hybridized to said nucleic acid(s) of interest, to provide a sample containing the nuclease protection fragment(s).

17. The method of claim 1, which identifies an RNA expression pattern, wherein at least two of said nuclease protection fragments are specific for different RNA molecules.

18. The method of claim 17, which identifies an agent which modulates an RNA expression pattern, further comprising comparing the RNA expression pattern produced in the presence of said agent to the RNA expression pattern produced under a different set of conditions.

19. The method of claim 1, for identifying an agent which modulates the interaction of said nuclease protection fragment(s) with said probe(s).

20. The method of claim 1, wherein said nuclease protection fragment is DNA.

21. The method of claim 20, wherein the DNA is amplified before said sample comprising said nuclease protection fragment(s) is contacted with said combination.

22. The method of claim 21, wherein said amplification is PCR amplification.

23. The method of claim 1, wherein said nucleic acid target comprises a polymorphism.

24. The method of claim 1, wherein said combination comprises a large number of said regions, and wherein the method is a high throughput method.

25. The method of claim 1, wherein a large number of samples are assayed rapidly.

26. The method of claim 1, fiwther comprising removing said bifunctional linkers from said oligonucleotide anchors and replacing them with substantially identical or with different bifunctional linkers, thereby reprogramming said surfaces, and repeating said method of detecting a target.

27. The method of claim 1, wherein said anchors have been dissociated from bifunctional linkers having a different target specificity.

28. The method of claim 1, wherein said sample is generated in vitro or in vivo.

29. The method of claim 1, wherein said sample is generated by incubating or growing cells which may contain said target(s) and extracting nucleic acid from the cells.

30. The method of claim 1 which is diagnostic for a disease, which measures a side effect of a drug, or which identifies an agent which has a therapeutic effect.

31. The method of claim 1, which is predictive of a disease, or which measures the efficacy, safety, or metabolism of a drug.

32. The method of claim 1, further comprising contacting said combination and any bound nuclease protection probes with a labeled detection probe which produces a chemiluminescent signal, and detecting said detection probe.

33. The method of claim 1, wherein at least of one said targets is a DNA molecule and at least one of said targets is an RNA molecule.

34. The method of claim 1, wherein at least of one said nuclease protection fragments is specific for a DNA molecule and at least one of said nuclease protection fragments is specific for an RNA molecule.

35. The method of claim 1, wherein said targets are DNA molecules.

36. The method of claim 1, wherein said anchors are oligonucleotide anchors.

2. A method of detecting at least one nucleic acid target, comprising contacting sample which may comprise said target(s) with a nuclease protection fragment(s) specific for and which binds to said target(s), thereby forming a nuclease protection fragment/target duplex, exposing the sample to a nuclease effective to digest remaining single strand nucleic acid, and then contacting the resultant sample with a combination which comprises, before the addition of said sample,

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

ii) at least two different anchors, each in association with

iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for a portion(s) of said duplex,

under conditions effective for hybridization,

and detecting any bound protected portion(s).

37. A method of detecting at least one nucleic acid target, comprising contacting a sample which may comprise said target(s) with a nuclease protection fragment(s) specific for and which binds to said target(s), exposing the sample to a nuclease effective to digest remaining single strand nucleic acid, and then contacting the resultant sample with a combination which comprises,

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

ii) at least two different anchors, each in association with

iii) a bifunctional linker which has a first portion that is specific for the anchor, and a second portion that comprises a probe which is specific for said nuclease protection fragment(s),

under conditions effective for said nuclease protection fragment(s) to bind to said combination,

and detecting said bound protection fragment(s).

38. A kit useful for the detection of at least one nucleic acid target in a sample, comprising

a) at least one nuclease protection fragment specific for said nucleic acid(s), but not for any of the oligonucleotide anchors in said kit,

b) a surface, comprising multiple spatially discrete regions, at least two of which are substantially identical, each region comprising at least two different oligonucleotide anchors, and

c) a container comprising at least one biflnctional linker molecule, which has a first portion specific for at least one of said oligonucleotide anchors and a second portion that comprises a probe which is specific for, and in said detection binds to, at least one of said nuclease protection fragments.

39. The kit of claim 38, further comprising

d) one or more nucleases effective for digesting single strand nucleic acid and/or the RNA strand of a DNA/RNA duplex.

40. A kit useful for the detection of at least one nucleic acid target in a sample, comprising

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

b) a container comprising at least one bifunctional linker molecule, which has a first portion specific for one of said oligonucleotide anchors and a second portion that comprises a probe which is specific for a nuclease protection fragment which is specific for one of said nucleic acids, and

c) one or more nucleases effective for digesting single strand nucleic acid and/or the RNA strand of a DNA/RNA duplex.

41. A kit useful for the detection of at least one nucleic acid target in a sample, comprising

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

b) a set of instructions for i) performing a nuclease protection procedure on said nucleic acid(s) of interest, using a nuclease protection fragment(s) specific for said nucleic acid(s), and ii) attaching to at least one of said oligonucleotide anchors a bifunctional linker molecule, which has a first portion specific for at least one of said oligonucleotide anchors and a second portion that comprises a probe which is specific for at least one of said nuclease protection fragments.