Preparation of templates for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-9498763-B2
Application number	US-201514738591-A
Country	US
Kind code	B2
Filing date	Jun 12, 2015
Priority date	Jul 20, 2005
Publication date	Nov 22, 2016
Grant date	Nov 22, 2016

Abstract

The invention relates to methods of generating templates for a nucleic acid sequencing reaction which comprise: providing at least one double-stranded nucleic acid molecule, wherein both strands of the double-stranded nucleic acid molecule are attached to a solid support at the 5′ end, cleaving one or both strands of the double-stranded nucleic acid molecule, and subjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the solid support, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction.

First claim

The invention claimed is:

1. A method of preparing single-stranded templates for a nucleic acid sequencing reaction comprising,
(i) providing a solid support comprising a plurality of amplification primers, wherein a subset of said plurality of amplification primers comprises a cleavage site;
(ii) amplifying a template using subset of the primers on the support to produce a plurality of double-stranded nucleic acid molecules, wherein both strands of each double-stranded nucleic acid molecule are attached to the solid support at their 5′ ends, whereby the cleavage site is positioned in a double-stranded region of each double-stranded molecule;
(iii) cleaving only one strand of the double stranded molecules at the cleavage site;
(iv) subjecting the cleaved strand to denaturing conditions to remove the portion of the cleaved strand not attached to the solid support, followed by re-annealing the cleaved strand to the opposite strand, thereby generating immobilized partially or substantially single-stranded templates; and
(v) hybridizing a sequencing primer to the immobilized partially or substantially single-stranded templates, thereby preparing single-stranded templates for a nucleic acid sequencing reaction.

2. The method of claim 1 , further comprising performing a sequencing reaction to determine the sequence of at least one region of the immobilized single-stranded templates.

3. The method of claim 1 , further comprising treating the cleaved strand with a capping agent prior to step (v).

4. The method of claim 3 , wherein said cleaving and treating with a capping agent occur concurrently.

5. The method of claim 3 , wherein said cleaving and treating with a capping agent occur non-concurrently.

6. The method of claim 1 , wherein cleavage comprises cleaving via a chemical cleavage reaction.

7. The method of claim 1 , wherein cleavage comprises generating an abasic site at the cleavage site.

8. The method of claim 1 , wherein the cleavage site comprises a uracil.

9. The method of claim 1 , wherein the cleavage site comprises 8-oxo-guanine.

10. The method of claim 1 , wherein the cleavage site comprises deoxyinosine.

11. The method of claim 1 , wherein the cleavage site comprises one or more ribonucleotides.

12. The method of claim 1 , wherein cleavage occurs by exposure to a metal ion.

13. The method of claim 1 , wherein the cleavage site comprises one or more methylated nucleotides.

14. The method of claim 1 , wherein cleavage occurs by a photochemical mechanism.

15. The method of claim 1 , wherein amplification of the template forms a cluster comprising the plurality of double-stranded molecules.

16. The method of claim 1 , wherein the sequencing reaction comprises sequencing-by-synthesis.

17. The method of claim 1 , wherein the sequencing reaction comprises pyrosequencing or sequencing-by-ligation.

18. The method of claim 1 , wherein the solid support comprises a hydrogel.

19. The method of claim 18 , wherein the hydrogel is a polyacrylamide hydrogel.

20. The method of claim 18 , wherein the solid support is prepared by a method comprising polymerizing on the solid support a mixture of:
(i) a first comonomer which is acrylamide, methacrylamide, hydroxyethyl methacrylate or N-vinyl pyrrolidinone; and
(ii) a second comonomer which is a functionalized comonomer selected from acrylamide or acrylate of formula (I):
H2C═C(H)—C(═O)-A-B—C  (I);

or a methacrylate or methacrylamide of formula (II):
H2C═C(CH3)—C(═O)-A-B—C  (II);

wherein
A is NR or O, wherein R is hydrogen or an optionally substituted saturated hydrocarbyl group comprising 1 to 5 carbon atoms;
B is an optionally substituted alkylene biradical of formula —(CHn)- wherein n is an integer from 1 to 50; and wherein n=2 or more, one or more optionally substituted ethylene biradicals —CH2CH2-of said alkylene biradical may be independently replaced by ethenylene and ethynylene moieties; and wherein n=1 or more, one or more methylene biradicals—CH2-may be replaced independently with an optionally substituted mono- or polycyclic hydrocarbon biradical comprising from 4 to 50 carbon atoms, or a corresponding heteromonocyclic or heteropolycyclic biradical wherein at least 1 CH2 or CH2 is substituted by an oxygen sulfur or nitrogen atom or an NH group; and
C of A-B—C of formulas (I) or (II) is a group for reaction with a compound to bind said compound covalently to said hydrogel to form a polymerized product, characterized in that polymerization is conducted on and immobilized the polymerized product to a solid support that is not covalently surface-modified.

21. The method of claim 1 , wherein the plurality of amplification primers comprises a set of forward amplification primers and a set of reverse amplification primers, and wherein the subset comprising the cleavage site is the set of forward primers.

22. The method of claim 1 , wherein the plurality of amplification primers comprises a set of forward amplification primers and a set of reverse amplification primers, and wherein the subset comprising the cleavage site is the set of reverse primers.

Assignees

• Illumina Cambridge Ltd

Inventors

• Liu Xiaohai
• Milton John
• Smith Geoffrey Paul
• Barnes Colin Lloyd
• Rasolonjatovo Isabelle
• Rigatti Roberto
• Wu Xiaolin
• Ost Tobias William Barr
• Worsley Graham John
• Earnshaw David James
• Turcatti Gerardo
• Romieu Anthony

Classifications

• B01J2219/00608

  DNA chips · CPC title

• C12Q2521/331

  Methylation site specific nuclease · CPC title

• B01J2219/00596

  Solid-phase processes · CPC title

• B01J2219/00675

  In-situ synthesis on the substrate · CPC title

• C12Q2523/113

  Denaturating agents · CPC title