Expression monitoring by hybridization to high density oligonucleotide arrays

• US 20080227653A1
• Filed: 04/19/2006
• Published: 09/18/2008
• Est. Priority Date: 06/07/1989
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Fodor, Stephen P.A., Solas, Dennis W., Dower, William J.
• Application Number:
  US11/406,531
• Publication Number:
  US 20080227653A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00619 : using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 : by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00644 : the porous medium being present in discrete locations, e.g. gel pads
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 : for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/325 : Single stranded exonuclease
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 15/1475 : using image analysis for extracting features of the particle
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/0045 : with organic non-macromolecular light-sensitive compounds not otherwise provided for, e.g. dissolution inhibitors
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules

Abstract

The present invention provides methods for comparing and identifying differences in nucleic acid sequences using a plurality of sequence specific recognition reagents (i.e., probes comprising a nucleic acid complementary to a nucleic acid sequence in collections to be compared) bound to a solid surface.

Claims

1. A method of monitoring RNA processing, said method comprising:

(a) providing a pool of target nucleic acids comprising RNA transcripts of one or more genes, or nucleic acids derived therefrom using said RNA transcripts as templates;

(b) hybridizing said pool of target nucleic acids to an array of oligonucleotide probes immobilized on a surface, said array comprising more than 100 different oligonucleotides, at least some of which comprise control probes, wherein each different oligonucleotide is localized in a predetermined region of said surface, the density of said different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm², and at least some of said oligonucleotide probes are complementary to said RNA transcripts or said nucleic acids derived therefrom using said RNA transcripts; and

(c) quantifying the hybridization of said nucleic acids to said array, wherein said quantification is proportional to the expression level of said genes.

2. The method of claim 1, wherein said pool of target nucleic acids comprises nascent RNA transcripts.

3. The method of claim 2, wherein said pool of target nucleic acids further comprises processing intermediates.

4. The method of claim 2, wherein said pool of target nucleic acids further comprises mature mRNA.

5. The method of claim 3, wherein said pool of target nucleic acids further comprises mature mRNA.

6. The method of claim 1, wherein said pool of target nucleic acids comprises alternatively spliced mRNA transcripts.

7. The method of claim 1, wherein said array of oligonucleotides further comprises mismatch control probes.

8. The method of claim 7, wherein said quantifying comprises calculating the difference in hybridization signal intensity between each of said oligonucleotide probes and its corresponding mismatch control probe.

9. The method of claim 8, wherein said quantifying comprises calculating the average difference in hybridization signal intensity between each of said oligonucleotide probes and its corresponding mismatch control probe for each gene.

10. The method of claim 1, wherein hybridization and quantification is accomplished in under 48 hours.

11. The method of claim 1, wherein said multiplicity of genes is 100 genes or more.

12. The method of claim 1, wherein for each gene, said array comprises at least 10 different oligonucleotide probes complementary to subsequences of that gene.

13. The method of claim 1, wherein said hybridization is performed with a fluid volume of about 250 μ

l or less.

14. The method of claim 1, wherein said quantifying comprises detecting a hybridization signal that is proportional to the concentration of said RNA in said nucleic acid sample.

15. The method of claim 1, wherein said oligonucleotides are from 5 to about 50 nucleotides in length.

16. The method of claim 1, wherein said oligonucleotides are synthesized by light-directed polymer synthesis.

17. The method of claim 1, wherein said control probes comprise sequences from constitutively expressed control genes.

18. The method of claim 12, wherein said control genes are selected from the group consisting of β

-actin, GAPDH, and the transferrin receptor.

19. The method of claim 1, wherein said hybridization comprises a hybridization at low stringency of 30°

C. to 50°

C. and 6×

SSPE-T or lower and a wash at higher stringency.

20. The method of claim 1, wherein said pool of target nucleic acids is a pool of mRNAs.

21. The method of claim 1, wherein said pool of target nucleic acids is a pool of RNAs in vitro transcribed from a pool of cDNAs.

22. The method of claim 1, wherein said pool of target nucleic acids is amplified from a biological sample by an in vivo or an in vitro method.

23. The method of claim 1, wherein said pool of target nucleic acids comprises fluorescently labeled nucleic acids.

24. The method of claim 1, wherein said detecting comprises quantifying fluorescence of a label on said hybridized nucleic acids at a spatial resolution of 100 μ

m or higher.

25. The method of claim 24, wherein said quantification is by means of a scanning confocal fluorescence microscope.

26. The method of claim 1, wherein said providing comprises:

(i) hybridizing a pool of RNAs with a pool of oligonucleotides having the same sequences as said oligonucleotide probes to form a pool of hybridized nucleic acids;

(ii) treating said pool of hybridized nucleic acids with RNase A, thereby digesting single stranded nucleic acid sequences and leaving intact the hybridized double stranded regions;

(iii) denaturing the hybridized double-stranded regions and removing said oligonucleotides thereby leaving a pool of RNAs enhanced for those RNAs complementary to the oligonucleotide probes in said high density array.

27. The method of claim 1, wherein said providing comprises:

(a) hybridizing a pool of RNAs with paired target specific oligonucleotides wherein said paired target specific oligonucleotides are complementary to regions on either side of a subsequence, said subsequence being complementary to at least one of said oligonucleotide probes in said array;

(b) treating said pool of nucleic acids with RNase H to digest the hybridized (double stranded) nucleic acid sequences; and

(c) isolating the remaining nucleic acid sequences having a length about equivalent to said subsequence.

28. The method of claim 1, wherein said providing comprises:

(a) hybridizing a pool of poly A⁺ mRNAs with oligonucleotides that hybridize specifically with preselected mRNA target messages;

(b) treating said pool of nucleic acids with RNase H to digest the hybridized (double stranded) nucleic acid sequences thereby separating the coding sequence from the polyA⁺ tail; and

(c) isolating or amplifying the polyA⁺ RNA in said pool.

29. The method of claim 1, wherein at least some of said oligonucleotides are up to about 500 nucleotides in length.

30. The method of claim 1, wherein at least some of said oligonucleotides are greater than 10 nucleotides in length.

31. The method of claim 1, wherein at least some of the oligonucleotides are chosen based on sequence information from at least one public database.

32. The method of claim 1, wherein said surface is glass.

33. The method of claim 1, wherein said surface is a chip.

34. The method as recited in claim 1 wherein said probes are provided by synthesis of RNA or DNA on the solid surface.

35. The method as recited in claim 1 wherein said probes are delivered to the surface through flow channels on the surface of the substrate.

36. The method as recited in claim 1 wherein said probes are delivered to the surface by spotting on predefined regions.

37. The method of claim 1 wherein at least some of said oligonucleotide probes are specific for splice variants associated with a disease state.

38. The method of claim 37 wherein at least some of said oligonucleotide probes are specific for splice variants associated with malignant transformation.

39. The method of claim 37 wherein at least some of said oligonucleotide probes are specific for splice variants associated with apoptosis.

40. The method of claim 1 wherein said method is used in toxicology.

41. The method of claim 1 wherein said method is used in drug discovery.

42. The method of claim 1 wherein said method is used in medical diagnostics.

43. A device for performing the method of claim 1 comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to nascent RNA transcripts.

44. A device for performing the method of claim 1 comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to RNA processing intermediates.

45. A device for performing the method of claim 1 comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to mature mRNA.

46. A device for performing the method of claim 1 comprising an array of oligonucleotide probes, wherein at least some of said oligonucleotide probes are complementary to alternatively spliced mRNA transcripts.