Arrayed polynucleotides and their use in genome analysis

• US 20030022207A1
• Filed: 05/22/2002
• Published: 01/30/2003
• Est. Priority Date: 10/16/1998
• Status: Abandoned Application

• Current Assignee: Solexa Ltd. (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Klenerman, David, Barnes, Colin
• Application Number:
  US10/153,240
• Publication Number:
  US 20030022207A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0054 Means for coding or tagging the apparatus or the reagents
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel 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 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• 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 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 : Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

The invention encompasses a method for determining a single nucleotide polymorphism present in a genome, comprising: (a) immobilizing polynucleotide molecules onto the surface of a support to form an array comprising polynucleotides located at addresses capable of interrogation, wherein each address of at least a subset of addresses on the array corresponds to a single polynucleotide molecule, and the array permits the subset of addresses to be individually resolved by optical microscopy, and wherein each such single polynucleotide molecule comprises a portion that is immobilized by covalent bonding to the surface and a portion that is capable of interrogation; (b) interrogating an address that corresponds to a single polynucleotide molecule to identify nucleotide sequence in the single polynucleotide molecule; and (c) comparing the nucleotides identified in step (b) with a known consensus sequence, and thereby determining differences between the consensus sequence and the sequence of the single polynucleotide molecule.

Claims

1. A method for determining a single nucleotide polymorphism present in a genome, comprising

(a) immobilizing polynucleotide molecules onto the surface of a solid support to form an array comprising polynucleotides located at addresses capable of interrogation, wherein each address of at least a subset of addresses on the array corresponds to a single polynucleotide molecule, and the array permits said subset of addresses to be individually resolved by optical microscopy, and wherein each said single polynucleotide molecule comprises a first portion that is immobilized by bonding to the surface and a second portion that is capable of interrogation;

(b) interrogating a said address to identify nucleotides of a sequence in a said single polynucleotide molecule on said array; and

(c) comparing the nucleotides identified in step (b) with a known consensus sequence, and thereby determining differences between the consensus sequence and said sequence of said single polynucleotide molecule.

2. The method of claim 1 wherein said polynucleotide molecules comprise fragments of a genome.

3. The method of claim 1 wherein said interrogating comprises identifying nucleotides of a sequence in said second portion of said single polynucleotide molecule.

4. The method of claim 1, wherein step (b) comprises

(i) contacting the array with each of the nucleotides dATP, dTTP, dGTP and dCTP, under conditions that permit a nucleic acid polymerase reaction to proceed and thereby form sequences complementary to the polynucleotides immobilized on said array;

(ii) determining the incorporation of a nucleotide in the complementary sequences formed in step (i); and

(iii) optionally repeating said steps (i) and (ii).

5. The method of claim 4, wherein each nucleotide contains a removable fluorescent label.

6. The method of claim 4, wherein each nucleotide contains a removable blocking group that prevents further base incorporation, and wherein the blocking group is removed after each step of determining nucleotide incorporation.

7. The method of claim 4, wherein step (i) is carried out by first contacting the array with three of the four nucleotides dATP, dTTP, dCTP and dGTP under conditions that permit a nucleic acid polymerase reaction to proceed and thereby form sequences complementary to those in the array, then removing unincorporated nucleotides from the array, and then contacting the array with the remaining nucleotide under conditions that permit a nucleic acid polymerase reaction to proceed and thereby form sequences complementary to those in the array, so that step (ii) proceeds only after incorporation of said remaining nucleotide.

8. The method of any one of claims 1 to 5, wherein adjacent polynucleotides of the array are separated by a distance of at least 10 nm.

9. The method of claim 8, wherein the polynucleotides are separated by a distance of at least 100 nm.

10. The method of claim 8, wherein the polynucleotides are separated by a distance of at least 250 nm.

11. The method of claim 1, wherein the array has a density of from 10⁶ to 10⁹ single polynucleotides per cm².

12. The method of claim 11, wherein the density is from 10⁷ to 10⁹ single polynucleotides per cm².

13. The method of claim 1, wherein said polynucleotides are immobilised to said solid support via the 5′

terminus, the 3′

terminus or via an internal nucleotide.