Preparation of templates for nucleic acid sequencing
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.
1. A method comprising,
(i) amplifying a template using amplification primers on a solid support to produce a plurality of double-stranded nucleic acid molecules such that both strands of each double-stranded nucleic acid molecule are attached to the solid support at their 5′ ends, wherein a subset of the amplification primers comprise a cleavage site and wherein the cleavage site is positioned in a double-stranded region of each double-stranded molecule; and
(ii) cleaving only one strand of the double stranded molecules at the cleavage site to produce a cleaved strand and an un-cleaved strand.
2. The method of claim 1 , further comprising hybridizing a sequencing primer to the un-cleaved strand.
3. The method of claim 2 , further comprising performing a sequencing reaction to determine the sequence of at least one region of the un-cleaved strand.
4. The method of claim 3 , wherein the sequencing reaction comprises sequencing-by-synthesis.
5. The method of claim 3 , wherein the sequencing reaction comprises pyrosequencing or sequencing-by-ligation.
6. The method of claim 1 , wherein the cleaving occurs by a chemical cleavage reaction.
7. The method of claim 1 , wherein the cleaving 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 the cleaving 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 the cleaving occurs by a photochemical mechanism.
15. The method of claim 1 , further comprising subjecting the cleaved strand to denaturing conditions to remove a portion of the cleaved strand not attached to the solid support.
16. The method of claim 1 , wherein the amplification primers comprise 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 either the set of forward amplification primers or the set of reverse amplification primers.
17. The method of claim 1 , wherein the amplification of the template forms a cluster comprising the plurality of double-stranded molecules.
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 19 , 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 —CH 2 CH 2 — of said alkylene biradical may be independently replaced by ethenylene and ethynylene moieties; and wherein n=1 or more, one or more methylene biradicals —CH 2 — 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 CH 2 or CH 2 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.