Method of sequencing

• US 10,167,504 B2
• Filed: 09/30/2011
• Issued: 01/01/2019
• Est. Priority Date: 09/30/2010
• Status: Active Grant

• Current Assignee: Geneseque AS
• Original Assignee: Geneseque AS
• Inventors: Kotsbak, Jarle
• Primary Examiner(s): Priest, Aaron A
• Application Number:
  US13/876,176
• Publication Number:
  US 20140045703A1
• Time in Patent Office: 2,650 Days
• Field of Search: 435 612, 435 912
• 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 polynucleotide using a stepwise ligation and cleavage method in which two or more cleavage enzymes which are specific to two or more test probes are used sequentially to identify which test probe bound and hence the sequence of the target polynucleotide. In a preferred aspect the probes and/or bases used in the reaction are labelled, e.g. with a bead.

Claims

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

(i) contacting said polynucleotide with a) at least a first and second test probe each comprising a portion which may be complementary to one or more bases in said polynucleotide, and optionally, in addition, b) at least one test complementary base each of which may be complementary to a base in said polynucleotide, and covalently binding said first or second test probe to said polynucleotide when said portion of said first or second test probe is complementary to said one or more bases in said polynucleotide by ligation of said first or second test probe to said polynucleotide;

(ii) adding a first enzyme capable of removing at least part of said first test probe and at least one base of the polynucleotide being sequenced by a cleavage reaction if said first test probe bound to said polynucleotide and then sequentially adding at least a second enzyme capable of removing at least part of said second test probe if said second test probe bound to said polynucleotide, wherein each of said enzymes is different and specific for said first or second test probe;

(iii) optionally repeating steps (i) and (ii) with at least two different test probes and at least two enzymes wherein said enzymes may be the same or different to the enzymes used in step (ii) until a test probe which has a portion which is complementary to said one or more bases has bound to said polynucleotide and optionally, in addition, a test complementary base has bound to said polynucleotide, wherein only one test probe used in step (i) or step (iii) has a portion which is complementary to said one or more bases in said polynucleotide and optionally, in addition, only one test complementary base used in step (i)or step (iii) is complementary to a base in said polynucleotide, and said only one test probe binds to said polynucleotide in step (i) or step (iii) and optionally, in addition, said only one test complementary base binds to said polynucleotide in step (i) or (iii);

(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 test complementary base and/or test probe bound to said polynucleotide during steps (i), (ii) or (iii) to thereby identify said one or more bases of the polynucleotide;

wherein each cycle of steps (i) to (iv) is performed one or more times, and in each cycle one or more bases of said sequence are identified, and wherein determining step (iv) comprises determining the presence, absence or level of a signal associated with said test probe and/or test complementary base, wherein the presence of signal is indicative of binding of said test complementary base and/or test probe and said signal is provided by a label associated with said test probe and/or test complementary base, and wherein said label is a bead and wherein the same bead is used for each test probe and/or test complementary base and said label is used to identify the one or more bases which are identified by determining which test probe is cleaved during step (ii).

2. The method as claimed in claim 1 wherein in step (i) at least a first, second, third and fourth test probe each comprising a portion which may be complementary to one or more bases in said polynucleotide is used and in step (ii) at least a first, second, third and fourth enzyme is used.

3. The method as claimed in claim 2 wherein in each cycle one base is identified.

4. The method as claimed in claim 1 wherein in step (i) at least a first and second test probe each comprising a portion which may be complementary to one or more bases in said polynucleotide is used and in step (ii) at least a first and second enzyme is used and in each cycle more than one base of said sequence is identified.

5. The method as claimed in claim 1 wherein at least two cycles are performed.

6. The method as claimed in claim 1 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 and each enzyme used in said method has a different recognition site.

7. The method as claimed in claim 6 wherein said enzyme is a restriction enzyme, preferably a type IIb restriction enzyme and each enzyme used in said method is a different restriction enzyme.

8. The method as claimed in claim 1 wherein the level of signal associated with said test probe and/or test complementary base before and after said cleavage step is determined and a decrease of signal after cleavage is indicative that said test complementary base and/or test probe bound to said polynucleotide.

9. The method as claimed in claim 1 wherein said label is attached to said test probe and/or test complementary base.

10. The method as claimed in claim 9 wherein a first binding partner of a binding pair is attached to said test probe and/or test complementary base and said label is attached to a second binding partner of said binding pair and said first binding partner is attached to said second binding partner during said method.

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 test probes comprise a recognition site for a restriction enzyme, or wherein said label and said test probe and/or test complementary base are attached through a linkage and said linkage comprises a recognition site for a restriction enzyme, and said enzyme is an enzyme used in said method.

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

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

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

16. The method as claimed in claim 1 wherein said first and second test probes comprise a first and second set of test probes and different labels are attached to at least two test probes of each set of test probes and/or test complementary bases and the signal associated with said different labels is detected to determine which test complementary base and/or test probe bound to said polynucleotide to determine the sequence of one or more bases of said polynucleotide, wherein the one or more bases which are identified are different to the one or more bases which are identified by determining which test probe is cleaved during step (ii).

17. The method as claimed in claim 1 wherein said label is detected using an apparatus comprising a surface provided with a means for detecting said label.

18. The method as claimed in claim 17 wherein said surface is provided with one or more sensory elements, preferably light sensitive elements.

19. The method as claimed in claim 1 wherein said determining the presence, absence or level of a signal is by detecting light changes resulting from the presence of the label on a light sensitive surface.

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

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

22. The method as claimed in claim 1, wherein said bead is tethered.

23. The method as claimed in claim 22, wherein said bead is tethered to said solid support.