Dna sequence analysis

• US 20040175716A1
• Filed: 02/13/2004
• Published: 09/09/2004
• Est. Priority Date: 08/13/2001
• Status: Abandoned Application

• Current Assignee: Solexa Ltd. (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Williamson, Alan Rowe, Balasubramanian, Shankar, Klenerman, David, Barnes, Colin
• Application Number:
  US10/486,951
• Publication Number:
  US 20040175716A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention concerns a method for determining the identity of one or more single nucleotide polymorphisms (SNP) in a genome, comprising: (i) fragmenting a sample genome; (ii) contacting the fragments with an excess of a plurality of different oligonucleotide primers under conditions that permit a primer to form a duplex with a complementary region on a fragment, each primer having a predetermined sequence complementary to a sequence on the genome that is proximal to a putative SNP site, and the resulting duplexes being immobilised on a solid support; (iii) carrying out the sequencing reaction(s) and detecting the incorporation of bases onto the oligonucleotide primers to extend the primers to at least the SNP site; and (iv) comparing the resulting sequences to those of the reference one or more SNPs.

Claims

1. A method for determining the identity of one or more single nucleotide polymorphisms (SNP) in a genome, comprising:

(i) fragmenting a sample genome;

(ii) contacting the fragments with an excess of a plurality of different oligonucleotide primers under conditions that permit a primer to form a duplex with a complementary region on a fragment, the primers having a predetermined sequence complementary to a sequence on the genome that is proximal to a SNP site, and the resulting duplexes being immobilised on a solid support;

(iii) carrying out the sequencing reaction(s) and detecting the incorporation of bases onto the oligonucleotide primers to extend the primers to at least the SNP site; and

(iv) comparing the resulting bases to those of the reference one or more SNPs.

2. A method according to claim 1, wherein the duplex is immobilised to the solid support via a covalent linkage to the fragment.

3. A method according to claim 1 or claim 2, wherein prior to step (ii), a nucleotide is incorporated onto one end of the fragments, the nucleotide comprising a linker molecule for immobilisation of the fragments with the solid support.

4. A method according to any of claims 1 to 3 wherein immobilisation is at a density that allows each immobilised duplex to be individually resolved by optical microscopy.

5. A method according to any preceding claim, wherein step (ii) comprises between 300 to 10⁶ different oligonucleotide primers.

6. A method according to any preceding claim, wherein step (ii) comprises from 10³ to 10⁵ different oligonucleotide primers.

7. A method according to any preceding claim, wherein step (ii) comprises from 10³ to 10⁴ different oligonucleotide primers.

8. A method according to any preceding claim, wherein the oligonucleotide primers comprise from 10 to 70 bases.

9. A method according to any preceding claim, wherein the oligonucleotide primers comprise from 30 to 50 bases.

10. A method according to any preceding claim, wherein the oligonucleotide primers comprise about 40 bases.

11. A method according to any preceding claim, wherein the primers are complementary to a sequence less than 20 bases from the SNP site.

12. A method according to any preceding claim, wherein the primers are complementary to a sequence less than 10 bases from the SNP site.

13. A method according to any preceding claim, wherein the primers are complementary to a sequence from 1 to 6 bases from the SNP site.

14. A method according to any preceding claim, wherein the primers are complementary to a sequence adjacent to the SNP site.

15. A method according to any preceding claim, wherein step (iii) comprises the sequential addition of fluorescently-labelled bases.