Method for Sequencing a Polynucleotide Template

• US 20090181370A1
• Filed: 07/20/2006
• Published: 07/16/2009
• Est. Priority Date: 07/20/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Smith, Geoffrey Paul
• Granted Patent: US 8,017,335 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing

Abstract

The invention relates to methods for pairwise sequencing of a double-stranded polynucleotide template, which permit the sequential determination of nucleotide sequences in two distinct and separate regions on complementary strands of the double-stranded polynucleotide template. The two regions for sequence determination may or may not be complementary to each other.

Claims

1. A method for pairwise sequencing of first and second regions of a target double-stranded polynucleotide, wherein said first and second regions are in complementary strands of the target polynucleotide, the method comprising:

(a) providing a solid support having immobilised thereon a plurality of template polynucleotide duplexes each comprising the double-stranded target polynucleotide, wherein each template duplex is formed from complementary first and second template strands linked to the solid support at their 5′

ends;

(b) cleaving the second template strands of a sub-fraction of the template polynucleotide duplexes to remove all or a portion of said strands, thereby generating single-stranded regions on the complementary first template strands;

(c) hybridising first sequencing primers to the single-stranded regions of the first template strands generated in part (b);

(d) carrying out a first sequencing reaction by sequential addition of nucleotides to the first sequencing primer to determine the sequence of a first region of the target polynucleotide in the first template strand;

(e) cleaving the first template strands of substantially all intact template polynucleotide duplexes not cleaved in step (b) to remove all or a portion of said strands, thereby generating single-stranded regions on the second template strands that were not cleaved in step (b);

(f) hybridising a second sequencing primer to the single-stranded regions of the second template strands generated in part (e); and

(g) carrying out a second sequencing reaction by sequential addition of nucleotides to the second sequencing primer to determine the sequence of a second region of the target polynucleotide in the second template strand.

2. The method according to claim 1 wherein the sub-fraction of the template polynucleotide duplexes cleaved in step (b) is approximately half.

3. The method according to claim 1 wherein the plurality of template polynucleotide duplexes provided in step (a) comprises a mixture of first and second template polynucleotide duplexes each comprising the same double-stranded target polynucleotide, wherein a first template strand of both the first and second template polynucleotide duplexes includes a cleavage site Y and a second template strand of the first template polynucleotide duplexes but not the second template polynucleotide duplexes includes a cleavage site X, which is different to cleavage site Y;

step (b) comprises

cleaving the second template strand of the first template polynucleotide duplexes at cleavage site X;

and step (e) comprises

cleaving the first template strand of both the first and second template polynucleotide duplexes at cleavage site Y.

4. The method according to claim 3 wherein cleavage site X permits enzymatic, chemical or photochemical cleavage of the second template strand of the first template polynucleotide duplexes.

5. The method according to claim 3 wherein cleavage site Y permits enzymatic, chemical or photochemical cleavage of the first template strand of both the first and second template polynucleotide duplexes.

6. The method according to claim 3 wherein cleavage site X is a site for enzymatic cleavage and cleavage site Y is a site for chemical or photochemical cleavage.

7. The method according to claim 6 wherein cleavage site X is a site for cleavage with a restriction endonuclease.

8. The method according to claim 5 wherein cleavage site Y comprises a diol linkage which is cleaved by treatment with a chemical cleavage agent comprising periodate.

9. The method according to claim 1 wherein the plurality of template polynucleotide duplexes of step (a) are present on a clustered array.

10. The method according to claim 9 wherein the clustered array is formed by solid-phase nucleic acid amplification.

11. The method according to claim 9 wherein the plurality of template polynucleotide duplexes of step (a) are present within a single cluster on said clustered array.

12. The method according to claim 11 wherein each individual cluster on said clustered array comprises first and second polynucleotide duplexes derived from amplification of a single target polynucleotide molecule.

13. The method according to claim 11 wherein at least 95% of the clusters on the array comprise template polynucleotide duplexes derived from amplification of different single target polynucleotides.

14. The method according to claim 3 wherein the mixture of first and second template polynucleotide duplexes is formed by an amplification reaction using a combination of amplification primers immobilised on said solid support, wherein said combination includes first and second forward primers and reverse primers having the structures:

First forward primer;

A-L-X-S1

Second forward primer;

A-L-S1

Reverse primer;

A-L-Y-S2

Wherein A is a moiety which allows attachment to the solid support, L is an optional linker sequence, X is a cleavage site, Y is a cleavage site different to X, S1 and S2 are sequences which permit amplification of a nucleic acid molecule comprising the target double-stranded polynucleotide.