Preparation of templates for nucleic acid sequencing

• US 9,999,866 B2
• Filed: 10/06/2016
• Issued: 06/19/2018
• Est. Priority Date: 07/20/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• 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
• Primary Examiner(s): Garyu, Lianko G
• Application Number:
  US15/287,077
• Publication Number:
  US 20170021325A1
• Time in Patent Office: 621 Days
• 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 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

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.

Claims

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 the subset of the amplification primers on the solid 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, 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 re-annealing the cleaved strand to the opposite strand after removing the portion of the cleaved strand not attached to the solid support.

3. The method of claim 2, wherein the re-annealing occurs prior to the hybridizing of the sequencing primer to the immobilized partially or substantially single-stranded templates.

4. 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.

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

6. The method of claim 5, wherein the cleaving and treating with a capping agent occur concurrently.

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

8. The method of claim 1, wherein the cleaving occurs by a chemical cleavage reaction.

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

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

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

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

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

14. The method of claim 1, wherein the cleaving occurs by exposure to a metal ion.

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

16. The method of claim 1, wherein the cleaving occurs by a photochemical mechanism.

17. 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 of the amplification primers comprising the cleavage site is the set of forward amplification primers.

18. 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 of the amplification primers comprising the cleavage site is the set of reverse amplification primers.

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

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

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

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

23. The method of claim 22, wherein the hydrogel is a polyacrylamide hydrogel.

24. The method of claim 22, 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.

25. A method 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 the subset of the amplification primers on the solid 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 thereby leaving un-cleaved strands attached to the solid support; and

(iv) hybridizing a sequencing primer to the un-cleaved strands attached to the solid support.