METHODS FOR SEQUENCE A POLYNUCLEOTIDE TEMPLATE

• US 20100311597A1
• Filed: 07/20/2006
• Published: 12/09/2010
• Est. Priority Date: 07/20/2005
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Swerdlow, Harold Philip
• Granted Patent: US 9,765,391 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The invention relates to methods for pairwise sequencing of a polynucleotide template which result in the sequential determination of nucleotide sequence in two distinct and separate regions of the polynucleotide template.

Claims

1. A method for pairwise sequencing of a polynucleotide template, the method comprising:

(a) providing a polynucleotide template having a first free 3′

hydroxyl group which is positioned to initiate sequencing of a first region for sequencing determination on a first template strand,

(b) carrying out a first sequencing reaction by sequential addition of nucleotides to the first free 3′

hydroxyl group to determine the sequence of the first template strand in the first region,

(c) generating a second free 3′

hydroxyl group within the polynucleotide template at a nucleotide position which is spaced apart from the last nucleotide added in the sequencing reaction of step (b), wherein the second free 3′

hydroxyl group is positioned to initiate sequencing of a second region for sequencing determination on a second template strand, and wherein the second free hydroxyl group is in continuous covalent linkage with the last nucleotide added in the sequencing reaction of step (b), and

(d) carrying out a second sequencing reaction by sequential addition of nucleotides to the second free 3′

hydroxyl group to determine the sequence of the second template strand in the second region.

2. The method of claim 1 wherein the 3′

end of the first template strand is linked to the 5′

end of a self-complementary hairpin linker polynucleotide and the 3′

end of the linker polynucleotide forms the first free 3′

hydroxyl group of part (a).

3. The method of claim 1 or claim 2 wherein the polynucleotide template or linker polynucleotide is linked to a solid support.

4. The method of claim 3 wherein the polynucleotide template forms part of an array.

5. The method according to claim 4 wherein the addressable array is a single molecule array.

6. The method according to claim 4 wherein the addressable array is a clustered array.

7. The method of any one of claims 1 to 6 wherein the first and second regions for sequence determination are in the same strand of the polynucleotide template.

8. The method of claim 7 wherein the second free 3′

hydroxyl group is generated by sequential addition of one or more nucleotides to the 3′

end of the last nucleotide added in the sequencing reaction of part (b) without determining the identity of the nucleotides to produce an extended complementary strand, wherein the last such nucleotide added provides the second free 3′

hydroxyl group which is spaced apart from but in continuous covalent linkage with the last nucleotide added in the sequencing reaction of part (b).

9. The method of claim 8 wherein at least 50, preferably at least 100, and more preferably at least 500 nucleotides are added to the last nucleotide added in the sequencing reaction of part (b).

10. The method of claim 7 wherein the second free 3′

hydroxyl group is generated by sequential addition of nucleotides including dUTP to the 3′

end of the last nucleotide added in the sequencing reaction of part (b) to produce an extended complementary strand incorporating one or more uracil bases, followed by treatment with an enzyme which hydrolyses uracil glycosidic bonds, thereby generating a second free 3′

hydroxyl group.

11. The method of any one of claims 1 to 6 wherein the first and second regions for sequence determination are in complementary strands of the polynucleotide template.

12. The method of claim 11 wherein the second free 3′

hydroxyl group is generated by (i) removing or blocking any 5′

phosphate group present on the first template strand such that it is not capable of being ligated to a free 3′

hydroxyl group, (ii) sequentially adding nucleotides to the 3′

end of the last nucleotide added in the sequencing reaction of part (b) to produce a second template strand complementary to the full length of the first template strand, thereby generating a double-stranded blunt ended polynucleotide, (iii) ligating a self-complementary hairpin polynucleotide having a free 3′

hydroxyl group to the free 3′

hydroxyl group of the second template strand, whereby the free 3′

hydroxyl group of the linker polynucleotide provides a second free 3′

hydroxyl group.

13. The method of claim 12 wherein the second sequencing reaction of part (d) is carried out using a strand-displacing polymerase enzyme.

14. The method of claim 12 wherein prior to step (d) a portion of the first template strand is removed.

15. The method of claim 14 wherein a portion of the first template strand is removed by cleavage of the first template strand with a nicking endonuclease followed by denaturation of the resulting product to remove the portion of the first template strand upstream of the cleavage site.

16. The method of claim 15 wherein the 3′

end of the first template strand is joined to the 5′

end of one strand of a self-complementary hairpin linker polynucleotide, which linker polynucleotide comprises a recognition site for a nicking endonuclease which directs cleavage by the nicking endonuclease at a site before, at or beyond the 5′

end of the linker polynucleotide.

17. The method of claim 16 wherein the template is linked to a solid support via the self-complementary hairpin linker polynucleotide.

18. The method of claim 11 wherein the polynucleotide template of step (a) is generated by:

(i) providing a fully double-stranded polynucleotide consisting of a first template strand having a 5′

phosphate group and a complementary strand lacking a 5′

phosphate group;

(ii) ligating a second self-complementary hairpin linker polynucleotide having hydroxyl groups at both the 3′ and

5′

ends to a first end of the double-stranded polynucleotide such that the 3′

end of the second linker is joined to the 5′

end of the first template strand but the 5′

end of the second linker remains free;

(iii) ligating a first self-complementary hairpin linker polynucleotide having a 5′

phosphate group and a 3′

hydroxyl group to the second end of the double-stranded polynucleotide such that the 5′

end of the first linker is joined to the 3′

end of the first template strand but the 3′

end of the first linker remains free;

(iv) denaturing the product of step (iii) to remove the complementary strand of the double-stranded polynucleotide, thereby forming a polynucleotide template having a first free 3′

hydroxyl group; and

(v) optionally restoring a 5′

phosphate group to the 5′

end of the second linker polynucleotide.

19. The method of claim 18 wherein following the first sequencing reaction of part (b) a second free 3′

hydroxyl group is generated by;

(i) sequentially adding unmodified nucleotides to the 3′

end of the last nucleotide added in the first sequencing reaction, thereby forming a second template strand complementary to the entire length of the first template strand;

(ii) ligating the 3′

end of the second template strand to the 5′

end of the second hairpin polynucleotide linker, which linker has been treated to restore the 5′

phosphate group, thereby forming a self-complementary closed circle;

(iii) generating a second free 3′

hydroxyl group by cleavage of the first template strand or the second hairpin linker polynucleotide ligated to the 5′

end of the first template strand.

20. The method of claim 19 wherein step (iii) comprises cleaving the first template strand at two cleavage sites spaced apart from each other and then denaturing the product to remove the portion of the first template strand located between the two cleavage sites.

21. The method of claim 20 wherein the second linker polynucleotide includes a recognition site for a nicking endonuclease which directs cleavage by the nicking endonuclease at a cleavage site before, at or beyond the 3′

end of the second linker.

22. The method of claim 20 or claim 21 wherein the first hairpin polynucleotide linker includes a recognition site for a nicking endonuclease, which directs cleavage by the nicking endonuclease at a site before, at or beyond the 5′

end of the first linker.

23. The method of claim 19 wherein step (iii) comprises cleaving the first template strand at a site before, at or beyond the 3′

end of the second linker and the second sequencing reaction is carried out using a strand displacement polymerase.

24. The method of any one of claims 18 to 23 wherein the first hairpin polynucleotide linker is linked to a solid support.

25. The method of claim 11 wherein the polynucleotide template of step (a) is generated by:

(i) providing a fully double-stranded polynucleotide consisting of a first template strand and a complementary second template strand;

(ii) ligating a first self-complementary linker polynucleotide to a first end of the double-stranded polynucleotide, the first linker polynucleotide having a recognition site for a first nicking endonuclease which directs cleavage by the nicking endonuclease at a site before, at or beyond the 3′

end of the first linker;

(iii) ligating a second self-complementary linker polynucleotide to a second end of the double-stranded polynucleotide, the second linker polynucleotide having a recognition site for a second nicking endonuclease which directs cleavage by the nicking endonuclease at a site before, at or beyond the 3′

end of the second linker, thereby producing a self-complementary closed circle;

(iv) cleaving one strand of the self-complementary closed circle produced in step (iii) with a first nicking endonuclease at a site before, at or beyond the 3′

end of the first linker, thereby generating a first free 3′

hydroxyl group.

26. The method of claim 25 wherein the first sequencing reaction of step (b) is carried out using a strand displacement polymerase enzyme.

27. The method of claim 26 wherein the second free 3′

hydroxyl group is generated by nicking one strand of the self-complementary closed circle produced in step (iii) with a second nicking endonuclease at a site before, at or beyond the 3′

end of the second linker, thereby generating a second free 3′

hydroxyl group.

28. The method of claim 27 wherein the second sequencing reaction of step (d) is carried out using a strand displacement enzyme.

29. The method of claim 11 wherein the polynucleotide template of step (a) is generated by:

(i) providing a fully double-stranded polynucleotide consisting of a first template strand and a complementary second template strand;

(ii) ligating a first self-complementary linker polynucleotide to a first end of the double-stranded polynucleotide, the first linker polynucleotide having a recognition site for a first nicking endonuclease which directs cleavage by the nicking endonuclease at a site before, at or beyond the 3′

end of the first linker;

(iii) ligating a second self-complementary linker polynucleotide to a second end of the double-stranded polynucleotide, the second linker polynucleotide having a recognition site for a second nicking endonuclease which directs cleavage by the nicking endonuclease at a site before, at or beyond the 5′

end of the second linker;

(iv) cleaving one template strand of the ligated product of step (iii) with a first nicking endonuclease at a site before, at or beyond the 3′

end of the first linker and cleaving the same strand with a second nicking endonuclease at a second site before, at or beyond the 5′

end of the second linker;

(v) denaturing the product of step (iv) to remove the portion of the template strand cleaved in step (iv) located between the two cleavage sites, thereby generating a polynucleotide template having a first free 3′

hydroxyl group.

30. The method of claim 29 wherein following the first sequencing reaction a second free 3′

hydroxyl group is generated by;

(vi) sequentially adding unmodified nucleotides to the 3′

end of the last nucleotide added in the first sequencing reaction of step (b), thereby forming a second template strand complementary to the entire length of the first template strand;

(vii) ligating the 3′

end of the second template strand to the 5′

end of the second polynucleotide linker;

(viii) generating a second free 3′

hydroxyl group by cleavage of the first template strand or the second linker polynucleotide ligated to the 5′

end of the first template strand.

31. The method of claim 30 wherein step (viii) comprises cleaving the first template strand at two cleavage sites spaced apart from each other and then denaturing the product to remove the portion of the first template strand located between the two cleavage sites.

32. The method of claim 30 or claim 31 wherein the second polynucleotide linker includes a recognition site for a nicking endonuclease which directs cleavage by the nicking endonuclease at a cleavage site before, at or beyond the 3′

end of the second linker.

33. The method of claim 31 or claim 32 wherein the first polynucleotide linker includes a recognition site for a nicking endonuclease which directs cleavage by the nicking endonuclease at a site before, at or beyond the 5′

end of the first linker.

34. The method of claim 32 wherein the second sequencing reaction is carried out using a strand displacement polymerase.