Synthesis of spatially addressed molecular arrays

• US 7,384,737 B2
• Filed: 08/01/2002
• Issued: 06/10/2008
• Est. Priority Date: 02/02/2000
• Status: Expired due to Term

• Current Assignee: Solexa Ltd. (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Barnes, Colin
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US10/210,211
• Publication Number:
  US 20030064398A1
• Time in Patent Office: 2,140 Days
• Field of Search: 435/6, 435/7.1, 435/174, 435/283.1, 435/91.1, 435/287.2, 536/23.1, 536/25.3
• US Class Current: 435/6.11
• 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 2219/00596 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 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

Methods are disclosed for forming spatially addressable arrays of polynucleotides of known squence, by using blocking groups that prevent the incorporation of multiple nucleotides during each incorporation step.

Claims

1. A method for forming a spatially addressed array of a plurality of single polymers, wherein the polymers are polynucleotides immobilized on a single solid support, and wherein the solid support is a planar surface comprising the steps of:

(i) contacting the array of single molecules with one or more detectably labeled monomers, under conditions that permit template independent incorporation of a monomer onto a molecule of the array, wherein the labelled monomer comprises a removable blocking group that prevents further monomer incorporation occurring;

(ii) removing non-incorporated monomers from the array of single molecules and detecting the label on the incorporated monomer;

(iii) removing the blocking group and any separate label; and

(iv) repeating steps (i)-(iii) to form single polymers of defined sequence;

wherein the array has a surface density which allows each polymer to be individually resolved by optical microscopy and each of the polymers is synthesized by template independent random incorporation of said labeled monomers, and wherein a spatially addressed array of single polymers is formed by determining the sequence of each single polymer on the array via detection of incorporated monomers.

2. A method according to claim 1, wherein the detectably labeled monomers are any of the bases A, C, T and G.

3. A method according to claim 2, wherein each of the bases A, C, T and G comprises a different label, and step (i) is carried out in the presence of all four bases.

4. A method according to claim 1, wherein the label is a fluorophore.

5. A method according to claim 4, wherein the label is detected using a 2-D fluorescent imaging device, a confocal fluorescence microscope or a CCD camera.

6. A method according to claim 5, wherein the label is removed by chemical or enzymatic cleavage.

7. A method according to claim 6, wherein the array has a density of from 10⁵ to 10⁹ polymers per cm².

8. A method according to claim 7, wherein the density is 10⁷ to 10⁸ polymers per cm².

9. A method according to claim 8, wherein the polymers are separated by a distance of at least 100 nm.

10. A method according to claim 9, wherein the polymers are separated by a distance of at least 250 nm.