Preparation of templates for nucleic acid sequencing

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.

1 . A method of generating a template for a nucleic acid sequencing reaction comprising,
 (i) providing a solid supported polyacrylamide hydrogel having attached thereto one or more double-stranded nucleic acid molecules, wherein both strands of the double-stranded nucleic acid molecule are attached to the polyacrylamide hydrogel at the 5′ end,   (ii) cleaving one or both strands of the double-stranded nucleic acid molecule(s), and   (iii) subjecting the cleaved strand(s) to denaturing conditions to remove the portion of the cleaved strand(s) not attached to the polyacrylamide hydrogel, thereby generating a partially or substantially single-stranded template for a nucleic acid sequencing reaction.   
     
     
         2 . The method according to  claim 1  wherein the double-stranded stranded nucleic acid molecule is cleaved at a pre-determined cleavage site. 
     
     
         3 . The method according to  claim 1  wherein the double-stranded nucleic acid molecule is cleaved in one or both strands via a chemical cleavage reaction. 
     
     
         4 . The method according to  claim 3  wherein one strand of the double-stranded nucleic acid molecule comprises a diol linker and this strand is cleaved by treatment with periodate. 
     
     
         5 . The method according to  claim 1  wherein one strand of the double-stranded nucleic acid molecule is treated to generate an abasic site and then cleaving at the abasic site. 
     
     
         6 . The method according to  claim 5  wherein one strand of the double-stranded nucleic acid molecule includes a uracil base and the abasic site is generated by treatment with uracil glycosylase. 
     
     
         7 . The method according to  claim 1  wherein one strand of the double-stranded nucleic acid comprises one or more ribonucleotides and step (ii) comprises cleaving this strand at a phosphodiester bond between a deoxyribonucleotide and a ribonucleotide. 
     
     
         8 . The method according to  claim 7  wherein step (ii) comprises exposure to a metal ion, preferably a rare earth metal ion. 
     
     
         9 . The method according to  claim 1  wherein one strand of the double-stranded nucleic acid comprises one or more methylated nucleotides and step (ii) comprises cleaving this strand using an enzyme specific for a recognition sequence including said methylated nucleotide(s). 
     
     
         10 . The method according to  claim 1  wherein step (ii) comprises cleaving one or both strands of the double-stranded nucleic acid by a photochemical mechanism. 
     
     
         11 . The method according to  claim 1  wherein step (ii) comprises cleaving one or both strands of the double-stranded nucleic acid molecule with an enzyme which is not a restriction endonuclease or a nicking endonuclease. 
     
     
         12 . The method according to  claim 11  wherein one strand of the double-stranded nucleic acid comprises two or more consecutive ribonucleotides and step (ii) comprises cleavage with a ribonuclease. 
     
     
         13 . The method according to  claim 12  wherein the ribonuclease is RNaseA. 
     
     
         14 . The method according to  claim 1  wherein one strand of the double-stranded nucleic acid molecule has a peptide covalently linked at the 5′ end and step (ii) comprises cleaving the peptide. 
     
     
         15 . The method according to  claim 1  wherein the polyacrylamide hydrogel is substantially free of binder silane. 
     
     
         16 . The method according to  claim 15  wherein the solid supported hydrogel is prepared by a method comprising polymerising on a 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 functionalised acrylamide or acrylate of formula (I):
   H 2 C═C(H)—C(═O)-A-B—C  (I); 
   or a methacrylate or methacrylamide of formula (II):
   or H 2 C═C(CH 3 )—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 —(CH 2 ) n — 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 is a group for reaction with a compound to bind said compound covalently to said hydrogel) to form a polymerised product,   characterised in that polymerisation is conducted on, and immobilises the polymerised product to, a solid support which is not covalently surface-modified.   
     
     
         17 . A method of generating a template for a nucleic acid sequencing reaction comprising,
 (i) 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,   (ii) cleaving one or both strands of the double-stranded nucleic acid molecule, and   (iii) 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,   characterised in that step (ii) does not comprise cleavage with a restriction endonuclease or a nicking endonuclease.   
     
     
         18 . The method according to  claim 17  wherein the double-stranded stranded nucleic acid molecule is cleaved at a pre-determined cleavage site. 
     
     
         19 . The method according to  claim 17  wherein the double-stranded nucleic acid molecule is cleaved in one or both strands via a chemical cleavage reaction. 
     
     
         20 . The method according to  claim 19  wherein one strand of the double-stranded nucleic acid molecule comprises a diol linkage and step (ii) comprises cleaving this strand at the diol linkage by treatment with periodate. 
     
     
         21 . The method according to  claim 17  wherein step (ii) comprises treating one strand of the double-stranded nucleic acid molecule to generate an abasic site and then cleaving at the abasic site. 
     
     
         22 . The method according to  claim 21  wherein one strand of the double-stranded nucleic acid molecule includes a uracil base and the abasic site is generated by treatment with uracil glycosylase. 
     
     
         23 . The method according to  claim 17  wherein one strand of the double-stranded nucleic acid comprises one or more ribonucleotides and step (ii) comprises cleaving this strand at a phosphodiester bond between a deoxyribonucleotide and a ribonucleotide. 
     
     
         24 . The method according to  claim 23  wherein step (ii) comprises exposure to a metal ion, preferably a rare earth metal ion. 
     
     
         25 . The method according to  claim 17  wherein one strand of the double-stranded nucleic acid comprises one or more methylated nucleotides and step (ii) comprises cleaving this strand using an enzyme specific for a recognition sequence including said methylated nucleotide(s). 
     
     
         26 . The method according to  claim 17  wherein step (ii) comprises cleaving one or both strands of the double-stranded nucleic acid by a photochemical mechanism. 
     
     
         27 . The method according to  claim 17  wherein one strand of the double-stranded nucleic acid molecule has a peptide covalently linked at the 5′ end and step (ii) comprises cleaving the peptide. 
     
     
         28 . The method according to  claim 17  wherein one strand of the double-stranded nucleic acid comprises two or more consecutive ribonucleotides and step (ii) comprises cleavage with a ribonuclease. 
     
     
         29 . The method according to  claim 28  wherein the ribonuclease is RNaseA. 
     
     
         30 . The method according to  claim 1  or  17  wherein the double-stranded nucleic acid molecule forms part of a clustered array of nucleic acid molecules. 
     
     
         31 . The method according to  claim 30  wherein the clustered array is formed by solid phase nucleic acid amplification. 
     
     
         32 . The method according to  claim 6  or  claim 22  wherein the double-stranded nucleic acid molecule forms part of a clustered array of double-stranded nucleic acid molecules and one strand of each of said double-stranded nucleic acid molecules includes a uracil base. 
     
     
         33 . The method according to  claim 32  wherein said clustered array is prepared by solid-phase nucleic acid amplification using forward and reverse amplification primers attached to said solid support at the 5′ end and said uracil base is present in either said forward or said reverse amplification primers. 
     
     
         34 . The method according to  claim 33  wherein said uracil base is present as the 3′ terminal base in either said forward or said reverse amplification primers. 
     
     
         35 . The method according to  claim 1 , or  17  wherein the double-stranded nucleic acid molecule forms part of a clustered array of double-stranded nucleic acid molecules prepared by solid-phase nucleic acid amplification using forward and reverse amplification primers attached to said solid support at the 5′ end and either said forward or said reverse primers include a PCR stopper. 
     
     
         36 . The method of nucleic acid sequencing comprising forming a template for nucleic acid sequencing using the method of  claim 1  and performing a nucleic acid sequencing reaction to determine the sequence of at least one region of the template. 
     
     
         37 . The method according to  claim 36  wherein the nucleic acid sequencing reaction comprises hybridising a sequencing primer to a single-stranded region of the template generated in step (iii), sequentially incorporating one or more nucleotides into a polynucleotide strand complementary to the region of the template to be sequenced, identifying the base present in one or more of the incorporated nucleotide(s) and thereby determining the sequence of a region of the template. 
     
     
         38 . The method of nucleic acid sequencing comprising forming a template for nucleic acid sequencing using a method comprising steps (i) and (ii) only of the method of  claim 1  followed by carrying out a strand displacement sequencing reaction by sequentially adding one or more nucleotides to a free 3′ hydroxyl group generated on the strand cleaved in step (ii), identifying the base present in one or more of the incorporated nucleotide(s) and thereby determining the sequence of a region of the template.