Arrays and methods for detecting nucleic acids

• US 6,576,424 B2
• Filed: 01/25/2001
• Issued: 06/10/2003
• Est. Priority Date: 06/07/1989
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Solas, Dennis W., Dower, William J., Fodor, Stephen P. A.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US09/768,626
• Publication Number:
  US 20020164590A1
• Time in Patent Office: 866 Days
• Field of Search: 435/6, 435/287.2, 435/288.3, 536/23.1, 536/24.3, 536/24.31, 536/24.32
• 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 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/0063 : Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00641 : the porous medium being continuous, e.g. porous oxide substrates
• 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 1/6804 : Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 2525/151 : repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/179 : incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/185 : incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/204 : specific length of the oligonucleotides
• CPC Class Codes: C12Q 2545/101 : with an internal standard/control
• CPC Class Codes: C12Q 2563/119 : the label being proteinic
• CPC Class Codes: C12Q 2563/149 : Particles, e.g. beads
• CPC Class Codes: C12Q 2563/173 : staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 : characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• 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: 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 7/265 : 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 and apparatus for sequencing, fingerprinting and mapping biological macromolecules, typically biological polymers. The methods make use of a plurality of sequence specific recognition reagents which can also be used for classification of biological samples, and to characterize their sources.

Claims

1. An array comprising a solid substrate and a plurality of positionally distinguishable sequence specific polynucleotides attached to the solid substrate of at least 100 polynucleotides per cm², at least a plurality of said polynticleotides comprising at least 25 nucleotides.

2. The array of claim 1, comprising at least 300 of said positionally distinguishable polynucleotides per cm² attached to the solid substrate.

3. The array of claim 1, comprising at least 1000 of said positionally distinguishable polynucleotides per cm² attached to the solid substrate.

4. The array of claim 1, comprising at least 3000 of said positionally distinguishable polynucleotides per cm² attached to the solid substrate.

5. The array of claim 1, comprising at least 10,000 of said positionally distinguishable polynucleotides per cm² attached to the solid substrate.

6. The array or claim 1, comprising at least 30,000 of said positionally distinguishable polynucleotides per cm² attached to the solid substrate.

7. The array of claim 1, comprising at least 100,000 of said positionally distinguishable polynucleotides per cm² attached to the solid substrate.

8. The array of claim 1, wherein said plurality of attached polynucleotides comprises at least 3000 different polynucleotides.

9. The array of claim 1, wherein the solid substrate has a surface area of less than 4 square centimeters.

10. The array of claim 1, wherein the solid substrate has a surface area of less than 11 cm².

11. The array of claim 1, wherein the solid substrate is glass.

12. The array of claim 1, wherein the solid substrate comprises a polymeric substrate.

13. The array of claim 1, wherein the solid substrate is plastic.

14. A method of producing the array of claim 1 comprising:

attaching the plurality of sequence specific polynucleotides at positions which are positionally distinguishable on the solid substrate.

15. A method of detecting hybridization of a target nucleic acid to the array of claim 1 comprising:

contacting the target nucleic acid with the polynucleotides of the array under conditions permitting hybridization and detecting hybridization.

16. A method of comparing a target nucleic acid with a reference nucleic acid comprising:

(a) contacting the target nucleic acid and the reference nucleic acid with the array of claim 1 under hybridization conditions,

(b) determining a first pattern of hybridization with the target nucleic acid and a second pattern of hybridization with the reference nucleic acid, and

(c) comparing the first and second patterns of hybridization to determine similarities or differences between the target nucleic acid and the reference nucleic acid.

17. A method for detecting nucleotide sequences in two or more collections of nucleic acids comprising:

(a) providing the array of claim 1 including said positionally distinguishable sequence specific polynucleotides;

(b) contacting said array of positionally distinguishable sequence specific polynucleotides with

(i) a first collection of labeled nucleic acids comprised of a nucleotide sequence that specifically binds to at least one known position of the array, and

(ii) at least a second collection of labeled nucleic acids comprised of a nucleotide sequence that specifically binds to at least one distinguishable position of the array;

wherein the labels of the nucleic acids of the first and second collections are distinguishable from each other; and

(c) detecting hybridization of the nucleic acids of the first and second collections to the polynucleotides of the array.

18. The method of claim 17, wherein the labels of the first and second nucleic acid collections are distinguishable fluorescent labels.

19. A method of identifying a sample containing nucleic acids comprising:

(a) contacting the sample with the array of claim 1 under hybridization conditions to obtain a hybridization pattern, and

(b) comparing the hybridization pattern with a reference database of hybridization patterns from the same array to identify the sample.

20. An array comprising a solid substrate and a plurality of positionally distinguishable polynucleotides attached to the solid substrate at a density of at least 100 polynucleotides per cm²;

wherein each of the attached polynucleotides comprises at least 50 nucleotides and has a predetermined nucleotide sequence.

21. The array of claim 20, wherein the positionally distinguishable polynucleotides are attached to the solid substrate within a square of less than 500 microns in dimension.

22. The array of claim 20, wherein each of the positionally distinguishable polynucleotides is attached to the solid substrate within a square of less than 100 microns in dimension.

23. The array of claim 20, wherein each of the positionally distinguishable polynucleotides is attached to the solid substrate within a square of less than 50 microns in dimension.

24. The array of claim 20, wherein each of the positionally distinguishable polynticleotides is attached to the solid substrate within a square of less than 10 microns in dimension.

25. The array of claim 20, wherein the density of positionally distinguishable polynucleotides attached to the solid substrate is at least 300 polynucleotides per cm².

26. The array of claim 20, wherein the density of positionally distinguishable polynucleotides attached to the solid substrate is at least 1000 polynucleotides per cm².

27. The array of claim 20, wherein the density of positionally distinguishable polynucleotides attached to the solid substrate is at least 3000 polynucleotides per cm².

28. The array of claim 20, wherein the density of positionally distinguishable polynucleotides attached to the solid substrate is at least 10,000 polynucleotides per cm².

29. The array of claim 20, wherein the density of positionally distinguishable polynucleotides attached to the solid substrate is at least 30,000 polynucleotides per cm².

30. The array of claim 20, wherein the density of positionally distinguishable polynucleotides attached to the solid substrate is at least 100,000 polynucleotides per cm².

31. The array of claim 20, wherein the plurality of attached polynucleotides comprises at least 3000 different polynucleotides.

32. The array of claim 20, wherein the solid substrate has a surface area of less than 4 square centimeters.

33. The array of claim 20, wherein the solid substrate has a surface area of less than 11 cm².

34. The array of claim 20, wherein the solid substrate is glass.

35. The array of claim 20, wherein the solid substrate comprises a polymeric substrate.

36. The array of claim 20, wherein the solid substrate is plastic.

37. A method of producing the array of claim 20 comprising:

attaching said polynucleotides at positions which are positionally distinguishable on the solid substrate.

38. A method of detecting hybridization of a target nucleotide acid to the array of claim 20 comprising:

contacting the target nucleotide acid with the polynucleotides of the array under hybridization conditions and detecting hybridization.

39. A method of comparing a target nucleic acid with a reference nucleic acid comprising:

(a) contacting the target nucleic acid and the reference nucleic acid with the array of claim 28 under conditions permitting hybridization,

(b) determining a first pattern of hybridization with the target nucleic acid and a second pattern of hybridization with the reference nucleic acid, and

(c) comparing the first and second patterns of hybridization to determine similarities or differences between the target nucleic acid and the reference nucleic acid.

40. A method for detecting nucleotide sequences in two or more collections of nucleic acids comprising:

(a) providing the array of polynucleotides of claim 20;

(b) contacting the array of polynucleotides with

(i) a first collection of labeled nucleic acids comprised of a nucleotide sequence that specifically binds to at least one known position of the array, and

(ii) at least a second collection of labeled nucleic acids comprised of a nucleotide sequence that specifically binds to at least one known position of the array;

wherein the labels of the nucleic acids of the first and at least second collections are distinguishable from each other; and

(c) detecting hybridization of the nucleic acids of the first and at least second collections to the polynucleotides of the array thereby detecting nucleotide sequences in two or more collections of nucleic acids.

41. The method of claim 40, wherein the labels of the first and at least second nucleic acid collections are distinguishable fluorescent labels.

42. A method of identifying a sample containing nucleic acids comprising:

(a) contacting the sample with the array of claim 20 under hybridization conditions to obtain a hybridization pattern, and

(b) comparing the hybridization pattern with a reference database of hybridization patterns from the same array to identify the sample.

43. A method for detecting nucleic acid sequences in two or more collections of nucleic acid molecules, the method comprising:

(a) providing an array of nucleic acids bound to a solid surface, each of said nucleic acids comprising a determinable nucleic acid sequence;

(b) contacting the array of nucleic acids with;

(i) a first collection of labeled nucleic acids, and

(ii) at least a second collection of labeled nucleic acids;

wherein the labels on the first collection of nucleic acids and at least the second collection of nucleic acids are distinguishable from each other; and

(c) detecting hybridization of the first collection of labeled nucleic acids and at least the second collection of labeled nucleic acids to nucleic acids on said array to thereby detect nucleic acid sequences in two or more collections of nucleic acid molecules.

44. The method of claim 43, wherein the array comprises an array of beads.

45. The method of claim 43, wherein the first and at least second labels are fluorescent labels.

50. The method of claim 43 wherein the nucleic acids are individually attached to the array at specific positions.

51. The method of claim 43, wherein the array of nucleic acids has at least 10² distinct nucleic acids on a surface area of about 1 cm², each distinct nucleic acid being disposed at a separate, defined position in said array.

53. The method of claim 43 wherein the array comprises a support and a surface, the surface of the array comprising a different material than the support.

55. The method of claim 43 wherein the labels are selected from the group consisting of green and red fluorescent labels.

46. A method of detecting differential expression of a plurality of nucleic acids in a first cell or population of cells with respect to expression of nucleic acids in a second cell or population of cells, said method comprising:

adding nucleic acids from the two cells or populations of cells to an array of nucleic acids, under conditions that result in hybridization to nucleic acids on the array, the nucleic acids from the first and the second cell or cell populations having fluorescent labels that are distinguishable from one another; and

examining the array by fluorescence under fluorescence excitation conditions to detect differential expression of the nucleic acids from the two cells or cell populations.

47. The method of claim 46, wherein the array of nucleic acids has at least 10² distinct nucleic acids on a surface area of about 1 cm², each distinct nucleic acid being disposed at a separate, defined position in said array.

48. The method of claim 46, wherein the array of nucleic acids has at least 10³ distinct nucleic acids on a surface area of about 1 cm², each distinct nucleic acid being positionally distinguishable.

49. The method of claim 47 wherein the nucleic acids are individually attached to the array at specific positions.

52. The method of claim 46, wherein the array of nucleic acids has at least 10³ distinct nucleic acids on a surface area of about 1 cm², each distinct nucleic acid being disposed at a separate, defined position in said array.

54. The method of claim 46, wherein the array comprises a support and a surface, the surface of the array comprising a different material than the support.

56. The method of claim 46, wherein the fluorescent labels are selected from the group consisting of green and red fluorescent labels.

62. The method of claim 46, further comprising mixing the nucleic acids from the first and the second cell populations before they are added to the array.

63. The method of claim 46, further comprising simultaneously adding the nucleic acids from the first and the second cell populations to the array.

64. The method of claim 46, further comprising the sequential addition of the nucleic acids from the first and the second cell populations to the array.

57. A method for detecting nucleic acids in two or more collections of nuclcic acid molecules, the method comprising:

(a) providing an array of polynucleotides bound to a solid surface, each said polynucleotide comprising a determinable nucleic acid;

(b) contacting the array of polynucleotides with;

(i) a first collection of labeled nucleic acids, and

(ii) at least a second collection of labeled nucleic acids;

wherein the first and second labels are distinguishable from each other; and

(c) detecting hybridization of the first collection of labeled nucleic acids and at least the second collection of labeled nucleic acids to polynucleotides of said array to thereby detect nucleic acids in two or more collections of nucleic acid molecules.

58. The method of claim 57, wherein the solid substrate comprises an array of beads.

59. The method of claim 57, wherein the first and second labels are fluorescent labels.

60. A method of detecting differential expression of each of a plurality of genes in a first cell with respect to expression of the same genes in a second cell, said method comprising:

adding fluorescent labeled nucleic acids from the two cells to an array of polynucleotides complementary to a plurality of known genes of the two cells, under conditions that result in hybridization to polynucleotides on the array; and

examining the array by fluorescence under fluorescence excitation conditions in which labeled nucleic acids from one of the cells that are hybridized to polynucleotides in the array give a distinct fluorescence emission color and labeled nucleic acid from the other cell which are hybridized to polynucleotides of said array give a different fluorescence emission color thereby enabling the detection of the differential expression of the genes in said first and second cells.

61. The method of claim 60, wherein the array of polynucleotides has at least 10³ distinct polynucleotide in a surface area of about 1 cm², each distinct polynucleotide being disposed at a separate, defined position in said array.