Methods for polymer synthesis

• US 6,747,143 B2
• Filed: 05/20/2002
• Issued: 06/08/2004
• 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., Stryer, Lubert, Read, J. Leighton, Fodor, Stephen P. A.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/152,440
• Publication Number:
  US 20030064391A1
• Time in Patent Office: 750 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 436/501, 436/518, 536/23.1, 536/25.32, 536/26.6
• US Class Current: 536/23.1
• 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 2219/00639 : the compounds being trapped in or bound to a porous medium
• 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: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• 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 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• 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 1/062 : for alpha- or omega-carboxy functions
• 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: C08G 69/00 : Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule products obtained from isocyanates or isothiocyanates C08G18/00; polyamide-imides C08G73/14
• 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 1/6837 : 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: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• 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: 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 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• 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
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/961 : including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• 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 435/973 : 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 428/31971 : Of carbohydrate
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

Claims

1. A method for synthesizing polymers on a substrate comprising the steps of:

(a) providing a substrate comprising polymers immobilized on a surface thereof, the polymers each coupled to a photoremovable protecting group;

(b) directing light using a mirror onto the surface of the substrate so as to reach only a first location to remove the protecting groups from the polymers in the first locations; and

(c) contacting the substrate with a first monomer to couple the first monomer to the polymers in the first location, the first monomer having a photoremovable protecting group thereon and wherein the polymers in the first location are different from polymers in a second location.

2. The method of claim 1, wherein the polymers are nucleic acids and the monomers are nucleotides.

3. The method of claim 1, wherein the polymers are peptides and the monomers are amino acids.

4. The method of claim 1, wherein the substrate is a glass substrate.

5. The method of claim 1, wherein the first location is smaller than 0.01 cm².

6. The method of claim 2, wherein the nucleic acids are attached to the substrate via a linker.

7. The method of claim 1, wherein the protecting group is selected from the group consisting of orthonitrobenzyl derivatives, 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, α

,α

-dimethyl-dimethoxybenzyloxycarbonyl, o-hydroxy-α

-methyl cinnamoyl derivatives and mixtures thereof.

8. A method for synthesizing polymers on a substrate comprising the steps of:

(a) providing a substrate comprising photoremovable protecting groups immobilized on a surface thereof;

(b) directing light using a mirror onto the surface of the substrate so as to reach a selected location to remove the protecting group in the selected location;

(c) contacting the substrate with a first monomer to immobilize the first monomer to the selected location, the first monomer having a photoremovable protecting group thereon; and

(d) repeating steps (b) and (c) until the desired polymers have been synthesized.

9. The method of claim 8, wherein the polymers are nucleic acids and the monomers are nucleotides.

10. The method of claim 8, wherein the polymers are peptides and the monomers are amino acids.

11. The method of claim 8, wherein the substrate is a glass substrate.

12. The method of claim 8, wherein the selected location is smaller than 0.01 cm².

13. The method of claim 9, wherein the protecting groups are attached to the substrate via a linker.

14. The method of claim 8, wherein the protecting group is selected from the group consisting of orthonitrobenzyl derivatives, 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, α

,α

-dimethyl-dimethoxybenzyloxycarbonyl, o-hydroxy-α

-methyl cinnamoyl derivatives and mixtures thereof.

15. A method for synthesizing polymers on a solid phase comprising the steps of:

(a) providing a substrate as the solid phase, wherein the substrate comprises polymers immobilized on a surface thereof, the polymers each coupled to a photoremovable protecting group;

(b) irradiating a first location of the substrate by directing light using a mirror to remove the protecting group from the polymers in the first location without irradiating a second location of the substrate; and

(c) contacting the substrate with a first monomer to couple the first monomer to the polymers in the first location, the first monomer having a protecting group thereon, thereby forming a first polymer on the substrate in the first location that is different from a polymer in the second location.

16. The method of claim 15, wherein the polymers are nucleic acids and the monomers are nucleotides.

17. The method of claim 15, wherein the polymers are peptides and the monomers are amino acids.

18. The method of claim 15, wherein the substrate is a glass substrate.

19. The method of claim 15, wherein the first location is smaller than 0.01 cm².

20. The method of claim 16, wherein the nucleic acids are attached to the substrate via a linker.

21. The method of claim 18, wherein the protecting group is selected from the group consisting of orthonitrobenzyl derivatives, 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, α

,α

-dimethyl-dimethoxybenzyloxycarbonyl, o-hydroxy-α

-methyl cinnamoyl derivatives and mixtures thereof.

22. A method of synthesizing polymers comprising the sequential steps of:

(a) generating a pattern of light and dark regions by selectively irradiating at least a first region of a surface of a substrate by directing light using a mirror, the surface comprising monomers immobilized on the surface, the monomers each coupled to a photoremovable protecting group, with irradiating at least a second region of the surface, to remove the protecting group from the monomers in the first region;

(b) simultaneously contacting the first region and the second region of the surface with a first monomer to couple the first monomer to the immobilized monomers in the first region, and not in the second region, the first monomer having the photoremovable protecting group;

(c) generating another pattern of light and dark regions by selectively irradiating with light using a mirror at least a part of the first region of the surface and at least a part of the second region to remove the protecting groups in said part of the first region and said part of the second region;

(d) simultaneously contacting the first region and the second region of the surface with a second monomer to couple the second monomer to the immobilized monomers on which the protecting groups have been removed in said part of the first region and said part of the second region; and

(e) performing additional irradiating with light using a mirror and performing additional monomer contacting and coupling steps so that a matrix array of more than 100 different polymers is formed on the surface, each different polymer synthesized in a region of less than 0.1 cm², whereby the different polymers have sequences and locations on the surface defined by patterns of light and dark regions formed during the irradiating steps and the monomers coupled in the contacting steps.

23. The method of claim 22, wherein the polymers are nucleic acids and the monomers are nucleotides.

24. The method of claim 22, wherein the polymers are peptides and the monomers are amino acids.

25. The method of claim 22, wherein the substrate is a glass substrate.

26. The method of claim 22, wherein each polymer is synthesized in a region smaller than 0.01 cm².

27. The method of claim 23, wherein the nucleic acids are attached to the substrate via a linker.

28. The method of claim 22, wherein the protecting group is selected from the group consisting of orthonitrobenzyl derivatives, 6-nitroveratryloxycarbonyl, 2-nitrobenzyloxycarbonyl, α

,α

-dimethyl-dimethoxybenzyloxycarbonyl, o-hydroxy-α

-methyl cinnamoyl derivatives and mixtures thereof.