Method of sequencing

• US 9,447,465 B2
• Filed: 09/30/2011
• Issued: 09/20/2016
• Est. Priority Date: 09/30/2010
• Status: Active Grant

• Current Assignee: Geneseque AS
• Original Assignee: Geneseque AS
• Inventors: Kotsbak, Jarle
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US13/876,175
• Publication Number:
  US 20140031239A1
• Time in Patent Office: 1,817 Days
• Field of Search: 435/6.12, 435/91.2, 435/91.52
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

Abstract

The present invention provides a method of sequencing a single polynucleotide in which the nucleotide base and/or probe which is incorporated during sequencing is labelled with a bead which is detected to identity the base and/or probe which has bound to the single polynucleotide. Particularly, the detection of the bead may be carried out using an apparatus comprising a surface provided with a means for detecting a bead e.g. with one or more light sensitive elements.

Claims

1. A method for determining a nucleotide sequence of a single polynucleotide immobilised on a solid support comprising the steps of:

(i) contacting said polynucleotide with at least one test complementary base and/or at least one probe comprising a portion which may be complementary to one or more bases in said polynucleotide to which a bead is attached or to which a first binding partner of a binding pair is attached and covalently binding said test complementary base and/or probe to said polynucleotide when said test complementary base and/or portion of said test complementary probe is complementary to said one or more bases in said polynucleotide;

(ii) when a binding partner is attached to said test complementary base and/or probe, binding a bead attached to a second binding partner of said binding pair to said test complementary base and/or probe to which said first binding partner is attached through said binding pair;

(iii) optionally repeating steps (i), or (i) and (ii), with at least one different test complementary base and/or probe until a test base which is complementary and/or a test probe which has a portion which is complementary to said one or more bases has bound to said polynucleotide;

(iv) determining which test complementary base and/or complementary portion of the test probe bound to said one or more bases of the polynucleotide by determining whether said bead attached to said test complementary base and/or probe bound to said polynucleotide during steps (i) to (iii) to identify said one or more bases of the polynucleotide; and

(v) removing said bead if not removed during step (iv);

wherein each cycle of steps (i) to (v) is performed one or more times and in each cycle one or more bases of said sequence are identified, and wherein binding of said bead to said polynucleotide during steps (i) to (iii) is detected using an apparatus comprising a surface provided with one or more light-sensitive elements, and said detection is by detecting light changes resulting from the bead blocking light from reaching the one or more light-sensitive elements.

2. A method as claimed in claim 1 wherein at least two cycles are performed.

3. The method as claimed in claim 1 wherein the covalent binding of the complementary base and/or the probe is by ligation or polymerisation.

4. The method as claimed in claim 1 wherein the method is one of sequencing by synthesis, sequencing by ligation or sequencing by stepwise ligation and cleavage.

5. The method as claimed in claim 1 wherein said method comprises the steps of:

(i) contacting said polynucleotide with at least one test probe comprising a portion which may be complementary to one or more bases in said polynucleotide, and optionally at least one test complementary base which may be complementary to a base in said polynucleotide, to either of which a bead is attached or to which a first binding partner of a binding pair is attached, and covalently binding said test complementary base and/or probe to said polynucleotide when said test complementary base and/or portion of said test complementary probe is complementary to said one or more bases in said polynucleotide by ligation of said probe to said polynucleotide;

(ii) when a binding partner is attached to said test complementary base and/or probe, binding a bead attached to a second binding partner of said binding pair to said test complementary base and/or probe to which said first binding partner is attached through said binding pair;

(iii) optionally repeating steps (i), or (i) and (ii), with at least one different test complementary base and/or probe until a test base which is complementary and/or a test probe which has a portion which is complementary to said one or more bases has bound to said polynucleotide;

(iv) after each step (i), or (i) and (ii), or after step (iii) adding an enzyme capable of removing said bead by a cleavage reaction which removes at least part of the test probe and at least one base of the polynucleotide being sequenced if said test probe bound to said polynucleotide and;

(v) determining which test complementary base and/or complementary portion of the test probe bound to said one or more bases of the polynucleotide by determining whether said bead attached to said test complementary base and/or probe bound to said polynucleotide during steps (i) to (iii) to identify said one or more bases of the polynucleotide;

wherein each cycle of steps (i) to (v) is performed one or more times and in each cycle one or more bases of said sequence are identified.

6. The method of claim 5 wherein said enzyme is a nuclease which has a cleavage site separate from its recognition site and said test probe contains a recognition site for said nuclease.

7. The method as claimed in claim 6 wherein said enzyme is restriction enzyme.

8. The method as claimed in claim 1 wherein in step (iv) multiple enzymes each capable of removing a bead from a different test complementary probe are used sequentially.

9. The method as claimed in claim 1 wherein determining step (iv) or (v) comprises determining the presence, absence or level of signal associated with said bead, wherein the presence of signal is indicative of binding of said test base and/or probe.

10. The method as claimed in claim 9 wherein the level of signal associated with said bead before and after said cleavage step is determined and a decrease of signal after cleavage is indicative of said test base and/or probe.

11. The method as claimed in claim 1 wherein the radius of said bead is larger than the length of the polynucleotide.

12. The method as claimed in claim 1 wherein said probes comprise a recognition site for a restriction enzyme, or said beads and said test complementary probe and/or base are attached through a linkage and said linkage comprises a recognition site for a restriction enzyme.

13. The method as claimed in claim 1 wherein the bead is magnetic.

14. The method as claimed in claim 1 wherein the complementary base or probe is terminating on the binding of further complementary bases or probes.

15. The method as claimed in claim 1 wherein said bead is attached to said complementary base and/or said probe before or after binding of the complementary base and/or probe to the polynucleotide.

16. The method as claimed in claim 1 wherein said bead is removed and/or detected by its magnetism.

17. The method as claimed in claim 1 wherein said solid support is a chip.

18. The method of claim 12, wherein said probes comprise a recognition site for a restriction enzyme, or said beads and said test complementary probe and/or base are attached through a linkage and said linkage comprises a recognition site for a restriction enzyme, and said recognition site is different for the different test complementary probes and/or bases used in steps (i) and (iii) and wherein in step (iv) multiple restriction enzymes, each of which recognizes one of said different recognition sites and is capable of removing a bead from a different test complementary probe and/or base, are used sequentially.

19. The method as claimed in claim 7 wherein said restriction enzyme is a type IIb restriction enzyme.