Very large scale immobilized polymer synthesis using mechanically directed flow paths

• US 5,885,837 A
• Filed: 10/23/1996
• Issued: 03/23/1999
• Est. Priority Date: 11/22/1991
• Status: Expired due to Fees

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Buchko, Christopher J., Winkler, James L., Ross, Debra A., Aldwin, Lois, Fodor, Stephen P. A.
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/740,170
• Time in Patent Office: 881 Days
• Field of Search: 435/6, 435/91.1, 435/287.2, 536/23.1, 536/25.3, 536/25.31, 536/25.6, 935/16, 935/76, 436/94
• 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 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00367 capillary
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00416 Vacuum
• CPC Class Codes: B01J 2219/0043 for direct application of reagents, e.g. through openings in a shutter
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• 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 2219/00536 in the shape of disks
• 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 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00686 : Automatic
• 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 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 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 1/045 : 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: 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 35/1072 : with provision for selective pipetting of individual channels
• CPC Class Codes: Y10S 435/968 : High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/97 : Test strip or test slide
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]

Abstract

A method and device for forming large arrays of polymers on a substrate (401). According to a preferred aspect of the invention, the substrate is contacted by a channel block (407) having channels (409) therein. Selected reagents are flowed through the channels, the substrate is rotated by a rotating stage (403), and the process is repeated to form arrays of polymers on the substrate. The method may be combined with light-directed methodolgies.

Claims

1. A method of forming an oligonucleotide arrays for hybridization comprising the steps of:

a) contacting a substrate with a channel block in a first orientation, said channel block comprising a substantially planar member with a plurality of recessed regions and raised walls on a surface thereof, said recessed regions forming channels, said raised wall effective to produce substantially fluid-tight seals between said raised walls and said substrate upon said contacting to define thereby a first plurality of flow channels between said channel block and said substrate, said first flow channels dividing said substrate surface into at least a first portion and a second portion;

b) flowing at least a first nucleotide through at least one of said first flow channels; and

covalently coupling said first nucleotide to said first portion of said substrate surface, said first nucleotide comprising a reactive group protected by a protecting group,

c) flowing at least a second nucleotide through at least one of said first channels, and covalently coupling said second nucleotide to said second portion of said substrate surface, said second nucleotide comprising a reactive group protected by a protecting group;

d) translating said channel block relative to said substrate and contacting said substrate with said channel block in a second orientation to produce substantially fluid-tight seals between said raised walls and said substrate upon said contacting to define thereby a second plurality of flow channels between said channel block and said substrate, said second flow channels dividing said substrate surface into at least a third portion and forth portion, said third and fourth portions each having at least one interaction with each of said first and second portions of said substrate surface;

e) removing said protective group from at least a portion of said first or second nucleotides to form a first set of deprotected first or second nucleotides and flowing a third nucleotide through at least one of said second flow channels, covalently coupling said third nucleotide to at least a portion of said fist set of deprotected fist or second nucleotides where said first and second portions of said substrate surface intersect with said third portion of said substrate surface to form first and second oligonucleotides; and

f) removing said protective group from at least a portion of said first and second nucleotides to form a second set of deprotected first or second nucleotides and flowing a fourth nucleotide through at least one of said second flow channels, covalently coupling said fourth nucleotide to at least a portion of said second set of deprotected first or second nucleotides where said fourth portion of said substrate surface intersects with said first and second portions of said substrate surface to form third and fourth oligonucleotides.

2. The method as recited in claim 1 wherein at least 10 different oligonucleotides are formed on said surface.

3. The method as recited in claim 1 wherein at least 100 different oligonucleotides are formed on said surface.

4. The method as recited in claim 1 wherein at least 1,000 different oligonucleotides are formed on said surface.

5. The method as recited in claim 1 wherein at least 10,000 different oligonucleotides are formed on said surface.

6. The method as recited in claim 1 wherein at least 100,000 different oligonucleotides are formed on said surface.

7. The method as recited in claim 1 wherein each different oligonucleotide is in a region having an area of less than about 1 cm².

8. The method as recited in claim 1 wherein each different oligonucleotide is in a region having an area of less than about 1 mm².

9. The method as recited in claim 8 wherein each different oligonucleotide is in a region having an area of less than about 10,000 microns².

10. The method as recited in claim 1 wherein the surface of said substrate comprises active groups capable of forming a covalent bond with nucleotides, said active groups protected with photoremovable groups, and wherein the step of forming a first plurality of flow channels is preceded or followed by a step of irradiating portions of said substrate with a light source shown through a mask whereby said photoremovable groups are removed from at least some of the active groups on said substrate surface in order for at least said first nucleotide to covalently bind thereto.

11. The method as recited in claim 10 wherein said irradiated portions are in the form of stripes, and wherein said step of forming said first plurality of flow channels comprises forming said first flow channels along a path of said stripes, and wherein different reagents are placed in at least a portion of said first flow channels, said reagents selected from the group consisting of nucleotides, wash solutions and protective group removal agents.

12. The method as recited in claim 1 wherein each said step of flowing said first, second, third or fourth nucleotide through its respective said first or second flow channels comprises:

placing a pipet in fluid communication with said first or second flow channel; and

injecting said first, second, third or fourth nucleotide from said pipet through its respective said first or second flow channel.

13. The method as recited in claim 12 wherein said step of placing the pipet in fluid communication with said first or second flow channel includes placing said pipet in contact with an orifice on a side of said substrate.

14. The method as recited in claim 12 wherein said step of placing the pipet in fluid communication with said first or second flow channel is a step of placing a plurality of pipettes in fluid communication with a plurality of said first or second flow channels and flowing reagents selected from the group consisting of said first, second, third and fourth nucleotides, wash solutions and protective group removal agents through at least two of said first or second flow channels.

15. A method of forming an array of diverse oligonucleotides at known locations on a single substrate, the substrate comprising a surface with a plurality of selected regions, the method comprising:

(1) placing a synthesis chamber adjacent the surface enclosing a first selected region of the surface;

(2) placing a first selected nucleotide in the synthesis chamber under conditions such that the first nucleotide attaches to the substrate within the first selected region;

(3) translating the synthesis chamber relative to the substrate such that the synthesis chamber encloses a second selected region of the surface, part of which is included in the first selected region and part of which is not;

(4) placing a second selected nucleotide in the synthesis chamber under conditions such that the second selected nucleotide attaches to the substrate or to the first nucleotide;

(5) further translating the synthesis chamber relative to the substrate such that the synthesis chamber encloses a further selected region of the surface, part of which is included in a previous selected region and part of which is not;

(6) placing a further selected nucleotide in the synthesis chamber under conditions such that the further selected nucleotide attaches to the substrate or to a previous nucleotide attached to the substrate; and

(7) repeating steps (5) and (6) until at least 10 diverse oligonucleotides are formed on the substrate.

16. The method of claim 15, wherein steps (5) and (6) are repeated until at least 100 diverse oligonucleotides are formed on the substrate.

17. The method of claim 16, wherein steps (5) and (6) are repeated until at least 1000 diverse oligonucleotides are formed on the substrate.

18. The method of claim 15, wherein the diverse oligonucleotides are at least 5 mers.

19. The method of claim 18, wherein the diverse oligonucleotides are at least 15 mers.

20. The method of claim 15, further comprising the step of screening the diverse oligonucleotides for complementarity to a polynucleotide.