Photolithographic and other means for manufacturing arrays

• US 6,379,895 B1
• Filed: 09/01/2000
• Issued: 04/30/2002
• 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: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): SIEW, JEFFREY
• Application Number:
  US09/654,435
• Time in Patent Office: 606 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 506/18
• 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 2219/00621 : 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: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• 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 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• 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: C07K 7/06 : having 5 to 11 amino acids
• 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: 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 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 : Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• 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. A binary synthesis method for synthesizing a plurality of different polypeptides at known locations on a substrate, said method comprising:

placing a first reactant in a first localized area of said substrate and not in a second localized area, said substrate having at least first and second localized areas each of less than 0.01 cm² and said first localized area comprising at least first and second fractions at known locations in the first localized area, and said second localized area comprising at least first and second fractions at known locations in said second localized area;

placing a second reactant in said first fraction of said first localized area and not in said second fraction of the first localized area;

placing a third reactant in said first fraction of said second localized area and not in the second fraction of said second localized area.

2. The method of claim 1, wherein said localized area is less than about 1×

10<sup>−

2</sup> cm² to about 1×

10<sup>−

5</sup> cm².

3. The method of claim 2, wherein said localized area is less than about 1×

10<sup>−

2</sup> cm² to about 1×

10<sup>−

4</sup> cm².

4. The method of claim 3, wherein said localized area is less than about 1×

10<sup>−

2</sup> cm² to about 1×

10<sup>−

3</sup> cm².

5. The method of claim 1, wherein said polypeptides occupy a density of about 400 different polypeptides/cm².

6. The method of claim 1, wherein said polypeptides occupy a density of about 1000 different polypeptides/cm².

7. The method of claim 1, wherein said substrate comprises 10³ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

8. The method of claim 1, wherein said substrate comprises 10⁴ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

9. A binary synthesis method for synthesizing a plurality of different polypeptides at known locations on a substrate at a density exceeding 400 different polypeptides occupying a total area of less than 1 cm² on said substrate, said groups of polypeptides having different polypeptide sequences, said method comprising:

placing a first reactant in a first localized area of said substrate and not in a second localized area, said substrate having at least first and second localized areas and said first localized area comprising at least first and second fractions at known locations in the first localized area, and said second localized area comprising at least first and second fractions at known locations in said second localized area;

placing a second reactant in said first fraction of said first localized area and not in said second fraction of the first localized area;

placing a third reactant in said first fraction of said second localized area and not in the second fraction of said second localized area.

10. The method of claim 9, wherein said at least first and second localized areas are each of less than 0.01 cm² in size.

11. The method of claim 9, wherein said at least first and second localized areas are each of less than 0.001 cm² in size.

12. The method of claim 9, wherein said substrate comprises 10³ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

13. The method of claim 9, wherein said substrate comprises 10⁴ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

14. A binary synthesis method for synthesizing a plurality of different polypeptides at known locations on a substrate at a density exceeding 1000 different polypeptides occupying a total area of less than 1 cm² on said substrate, said groups of polypeptides having different polypeptide sequences, said method comprising:

placing a first reactant in a first localized area of said substrate and not in a second localized area, said substrate having at least first and second localized areas and said first localized area comprising at least first and second fractions at known locations in the first localized area, and said second localized area comprising at least first and second fractions at known locations in said second localized area;

placing a second reactant in said first fraction of said first localized area and not in said second fraction of the first localized area;

placing a third reactant in said first fraction of said second localized area and not in the second fraction of said second localized area.

15. The method of claim 14, wherein said at least first and second localized areas are each of less than 0.01 cm² in size.

16. The method of claim 14, wherein said at least first and second localized areas are each of less than 0.001 cm² in size.

17. The method of claim 14, wherein said substrate comprises 10³ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

18. The method of claim 14, wherein said substrate comprises 10⁴ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

22. The method of claim 14, wherein said at least first and second localized areas are each of less than 0.01 cm² is size.

23. The method of claim 14, wherein said at least first and second localized areas are each of less than 0.001 cm² is size.

19. A binary synthesis method for synthesizing a plurality of different polypeptides at known locations on a substrate, said method comprising:

placing a first reactant in a first localized area of said substrate and not in a second localized area, said substrate having at least first and second localized areas and said first localized area comprising at least first and second fractions at known locations in the first localized area, and said second localized area comprising at least first and second fractions at known locations in said second localized area;

placing a second reactant in said first fraction of said first localized area and not in said second fraction of the first localized area;

placing a third reactant in said first fraction of said second localized area and not in the second fraction of said second localized area, wherein said substrate comprises 10³ or more different groups of polypeptides with known sequences bound to known regions of said substrate.

20. The method of claim 19, wherein said polypeptides occupy a density of about 400 different polypeptides/cm².

21. The method of claim 19, wherein said polypeptides occupy a density of about 1000 different polypeptides/cm².

24. An ordered method for forming a plurality of different polypeptides occupying known locations on a substrate by sequential addition of reactants to said substrate, said method comprising:

(a) providing a substrate having a surface with a localized area occupied by a protected amino acid;

(b) deprotecting the protected amino acid in a fraction of the localized area to produce a deprotected amino acid;

(c) immersing the surface of said substrate in a solution comprising a protected amino acid whereby the protected amino acid attaches to the deprotected amino acid to produce a further protected amino acid in the fraction of said localized area;

(d) repeating (b) and (c) at least once wherein the fraction in step (b) is a fraction of a fraction in a previous deprotecting step (b) to produce an array of different polypeptides occupying different fractions at known locations within the localized area.

25. The method of claim 24, wherein the localized area is less than 0.01 cm².

26. The method of claim 24, wherein the localized area is less than 0.001 cm².

27. The method of claim 24, wherein said polypeptides occupy a density of about 400 different polypeptides/cm².

28. The method of claim 24, wherein said polypeptides occupy a density of about 1000 different polypeptides/cm².

29. The method of claim 24, wherein said substrate comprises 10³ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

30. The method of claim 24, wherein said substrate comprises 10⁴ or more different groups of polypeptides with known sequences bound to known locations of said substrate.

31. A method of forming an array of diverse polypeptides on a substrate, the surface of said substrate comprising at least first and second regions having polypeptide molecules thereon, said polypeptide molecules coupled to a protective group at a first functional group, said first functional group capable of binding to a second functional group of selected amino acids which also have a first functional group coupled to a photoremovable protective group, said method comprising:

removing said protective group from said polypeptide molecules in said first region of said substrate to expose said first functional group, but not removing said protective group from said polypeptide molecules in said second region;

contacting said first and second regions of said surface with first selected amino acid molecules to covalently bond said second functional group of said first amino acid molecules to said first functional group of said polypeptides by means of a peptide bond in said first region, but not said second region;

removing the protective group from at least a portion of said first selected amino acid molecules in said first region to expose said first functional group on said at least a portion of said first selected amino acid molecules; and

contacting said first and said second regions with second selected amino acid molecules to covalently bond a second functional group of said second selected amino acid molecules to said first functional group of said first selected amino acid molecules by means of a peptide bond, forming polypeptides in said first region having a different amino acid sequence than polypeptides in said second region.

32. The method of claim 31, wherein aid protective group is a photoremovable protective group.

33. The method of claim 31, further comprising removing and coupling to form at least 100 different polypeptides on said substrate.

34. The method of claim 31, further comprising removing and coupling to form at least 1000 different polypeptides on said substrate.

35. The method of claim 31, wherein said substrate is glass.

36. The method of claim 32, wherein said removing step is accomplished with light.

37. The method of claim 31, wherein said first said second regions individually have an area of less than 1 mm².

38. The method of claim 31, wherein said first and said second regions individually have an area of less than 250,000 square microns.

39. The method of claim 36, wherein the step of removing protective groups with light comprises illuminating said substrate through a mask.

40. The method of claim 39, further comprising illuminating said second region, wherein illuminating said second region illuminates a portion of the substrate that was illuminated when said photoremovable protective groups on said polypeptide molecules in said first region were removed, and does not illuminate a portion of the substrate that was illuminated when said photoremovable protective groups on said polypeptide molecules in said first region were removed.

41. The method of claim 40, wherein said step of illuminating said second region illuminates a portion of the substrate that was not illuminated when said photoremovable protective groups on said polypeptide molecules in said first region were removed, and does not illuminate a portion of the substrate that was not illuminated when said photoremovable protective groups on said polypeptide molecules in said first region were removed.

42. The method of claim 32, wherein said photoremovable protective groups are ortho-nitrobenzyl protecting groups.

43. A method of making an array comprising at least two different polypeptides immobilized on a substrate, wherein said polypeptides differ from one another with respect to monomer sequence, by synthesizing said polypeptides on a surface of said substrate, said surface having functional groups capable of coupling with an amino acid, said functional groups protected by protecting groups, said method comprising:

removing said protecting groups from said functional groups with light so that a first pattern of activated and unactivated regions is formed on said surface;

contacting said surface with a first amino acid, said first amino acid having at least a first functional group capable of coupling with said functional groups in said activated regions of said surface and a second functional group capable of coupling with another amino acid, said second functional group protected by a protecting group, under conditions such that said first amino acid is coupled to said functional groups in said activated regions by means of a peptide bond;

selectively activating said surface by removing said protecting groups from said functional groups with light so that a second pattern of activated and unactivated regions is formed on said surface;

contacting said surface with a second amino acid, said second amino acid having at least a first functional group capable of coupling with said functional groups in said activated regions of said surface and a second functional group capable of coupling with another amino acid, said second functional group protected by a protecting group, under conditions such that said second amino acid is coupled to said functional groups in said activated regions by means of a peptide bond; and

repeating said activating and coupling until said two different polypeptides are formed on said surface, so that each different peptide is present at a defined location on said surface and has a defined amino acid sequence, whereby the amino acid sequence and location of each different polymer is defined by the patterns of activated and unactivated regions formed during each activating step and the monomers coupled in each coupling.

44. The method of claim 43, wherein said protecting group is a photoremovable protecting group.

45. The method of claim 44, further comprising placing a mask between a light source and said substrate, and exposing said mask to light, said mask comprising first selected regions that transmit at least one selected wavelength of light and second selected regions that do not transmit said selected wavelength of light.

46. The method of claim 45, wherein said mask is placed in direct contact with said substrate.

47. The method of claim 44, wherein said photoremovable protecting groups are ortho-nitrobenzyl protecting groups.

48. The method of claim 47, wherein said ortho-nitrobenzyl protecting groups are selected from the group consisting of nitroveratryloxycarbonyl and nitrobenzyloxycarbonyl.

49. The method of claim 43, wherein said steps are repeated to synthesize an array of more than 100 different polymers.

50. The method of claim 43, wherein said steps are repeated to synthesize an array of more than 1000 different polymers.

51. The method of claim 43, wherein said at least two different polymers each occupy an area on said substrate of less than 1 mm².

52. The method of claim 43, wherein said at least two different polymers each occupy an area on said substrate of less than 1×

10<sup>−

1</sup> cm².

53. The method of claim 43, wherein said at least two different polymers each occupy an area on said substrate of less than 1×

10<sup>−

2</sup> cm².

54. The method of claim 43, wherein said at least two different polymers each occupy an area on said substrate of less than 1×

10<sup>−

4</sup> cm².