Preparation of templates for nucleic acid sequencing

• US 20090118128A1
• Filed: 07/20/2006
• Published: 05/07/2009
• Est. Priority Date: 07/20/2005
• Status: Abandoned Application

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Rasolonjatovo, Isabelle Marie Julia, Earnshaw, David James, Smith, Geoffrey Paul, Worsley, Graham John, Rigatti, Roberto, Milton, John, Liu, Xiaohai, Ost, Tobias William Barr, Barnes, Colin, Romieu, Anthony, Turcatti, Gerardo, Wu, Xiaolin
• Application Number:
  US11/989,169
• Publication Number:
  US 20090118128A1
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 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);

H₂C═

C(H)—

C(═

O)-A-B—

C 

 

(I);

or a methacrylate or methacrylamide of formula (II);

or 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 —

(CH₂)_n—

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 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.

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.

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.

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.

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.