Reagents, methods, and libraries for bead-based sequencing

• US 10,323,277 B2
• Filed: 10/12/2016
• Issued: 06/18/2019
• Est. Priority Date: 02/01/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: McKernan, Kevin, Blanchard, Alan, Kotler, Lev, Costa, Gina
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/291,982
• Publication Number:
  US 20170081717A1
• Time in Patent Office: 979 Days
• Field of Search: None
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to immobilized beads. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides.

Claims

1. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:

(I) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a phosphorothiolate linkage and is from one of at least two distinguishably labeled probe families to which the template polynucleotide is exposed prior to the ligating, and wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise;

(1) a constrained portion of least two constrained nucleosides, wherein the constrained nucleosides;

(i) are at least 2-fold degenerate among the probes in the probe family, (ii) need not be located contiguously, and (iii) are not selected independently of each other, such that

(2) knowing the label of a probe eliminates one or more combinations of nucleosides as possibilities for the sequence of the constrained portion, and

(3) knowing the identity of a constrained nucleoside in that probe further eliminates at least one possible identity for another constrained nucleoside in the probe;

(II) identifying one or more nucleotides of the polynucleotide; and

(III) repeating steps I and II until the sequence of nucleotides is determined;

wherein the template polynucleotide is attached to a solid support and/or immobilized in or on a semi-solid support during the extending step.

2. The method of claim 1, comprising the steps of:

(a) providing a probe-template duplex comprising an oligonucleotide probe from one of the at least two distinguishably labeled probe families hybridized to a template polynucleotide, the probe covalently attached to the extended duplex and having an extendable terminus;

(b) ligating an extension oligonucleotide probe from one of the at least two distinguishably labeled probe families to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe, wherein the extension probe comprises a phosphorothiolate linkage;

(c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe;

(d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined.

3. The method of claim 2, wherein each extension probe has a non-extendable moiety at one terminus.

4. The method of claim 2, wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe.

5. The method of claim 2, wherein the step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

6. The method of claim 2, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

7. The method of claim 2, wherein the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

8. The method of claim 2, wherein the ligating and generating steps are performed while the template polynucleotide is attached to a solid support and/or immobilized in or on a semi-solid support.

9. The method of claim 2, wherein step (a) includes providing, in separate aliquots, a plurality of distinct probe-template duplexes, each distinct duplex comprising an initializing oligonucleotide probe hybridized to a template polynucleotide, wherein the template polynucleotide in each duplex is the same, but the initializing oligonucleotide probe in each duplex is bound to a different sequence of the template polynucleotide;

and steps (b) to (e) are carried out independently on each aliquot.

10. The method of claim 9, wherein for each aliquot, said extension oligonucleotide probe has a non-extendable moiety at one terminus and, optionally, wherein for each aliquot, said step of identifying includes (a) detecting a label attached to the most recently ligated extension probe or (b) removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

11. The method of claim 10, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

12. The method of claim 2, further comprising the steps of:

(f) removing the ligated probes and the initializing oligonucleotide from the template;

(g) repeating step (a) using a second oligonucleotide that is bound to a different sequence of the template polynucleotide; and

(h) repeating steps (b) to (e).

13. The method of claim 12, wherein the method is repeated a plurality of times using initializing oligonucleotides bound to different sequences of the template polynucleotide.

20. The method of claim 1 comprising the steps of:

(a) providing a probe-template duplex comprising an oligonucleotide probe from one of the at least two distinguishably labeled probe families hybridized to a template polynucleotide, the probe having an extendable terminus, the probe-template duplex being attached to a microparticle that is embedded in or on a semi-solid support;

(b) ligating an extension oligonucleotide probe from one of the at least two distinguishably labeled probe families to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe, wherein the extension probe comprises a phosphorothiolate linkage;

(c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe;

(d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined.

25. The method of claim 1, wherein the method is performed in parallel on a plurality of templates attached to one or more solid supports and/or immobilized in or on a semi-solid support during the extending step.

14. A method for identifying a sequence of nucleotides in a template polynucleotide attached to a support at a point of attachment, the method comprising the steps of:

(a) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein extension proceeds along the template towards its point of attachment to the support;

(b) identifying one or more nucleotides of the polynucleotide; and

(c) repeating steps (a) and (b) until the sequence of nucleotides is determined.

15. The method of claim 14, wherein each extension probe has a non-extendable moiety at one terminus.

16. The method of claim 14, wherein the step of identifying includes detecting a label attached to the most recently ligated extension probe.

23. The method of claim 14, wherein the oligonucleotide probe is from one of at least two distinguishably labeled probe families to which the template polynucleotide is exposed prior to the ligating.

24. The method of claim 14, wherein the oligonucleotide probe is fluorescently labeled and the identifying comprises detecting the fluorescently labeled probe.

26. The method of claim 23, comprising the steps of:

(a) providing a probe-template duplex comprising an oligonucleotide probe from one of the at least two distinguishably labeled probe families hybridized to a template polynucleotide, the primer having an extendable terminus;

(b) ligating an extension oligonucleotide probe from one of the at least two distinguishably labeled probe families to said extendable terminus, to form an extended duplex containing an extended oligonucleotide probe;

(c) identifying, in the extended duplex, at least one nucleotide in the template polynucleotide that is either (1) complementary to the just-ligated extension probe or (2) a nucleotide residue in the template polynucleotide which is immediately downstream of the extended oligonucleotide probe;

(d) generating an extendable terminus on the extended oligonucleotide probe, if an extendable terminus is not already present, such that the terminus generated is different from the terminus to which the last extension probe was ligated; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides in the template polynucleotide is determined.

27. The method of claim 26, wherein the step of identifying includes removing said non-extendable moiety and extending said extended oligonucleotide probe with a nucleic acid polymerase in the presence of one or more labeled chain-terminating nucleoside triphosphates.

28. The method of claim 26, further including a step of capping an extended oligonucleotide probe whenever no extension probe has ligated to the extendable terminus in the ligation step.

29. The method of claim 26, wherein the oligonucleotide probe contains a phosphorothiolate linkage, and wherein the generating step comprises cleaving the phosphorothiolate linkage with a cleavage agent comprising an atom selected from the group consisting of:

Ag, Hg, Cu, Mn, Zn and Cd.

30. The method of claim 26, further comprising the steps of:

(f) removing the ligated probes and the initializing oligonucleotide from the template;

(g) repeating step (a) using a second oligonucleotide that is bound to a different sequence of the template polynucleotide; and

(h) repeating steps (b) to (e).

31. The method of claim 26, wherein the ligating and generating steps are performed in or on a semi-solid support.

17. A method for identifying a sequence of nucleotides in a template polynucleotide, the method comprising the steps of:

(a) providing a template polynucleotide attached to a microparticle that is immobilized in or on a semi-solid support;

(b) extending an initializing oligonucleotide along the template polynucleotide by ligating an oligonucleotide probe thereto to form an extended duplex, wherein the oligonucleotide probe comprises a scissile linkage and is from one of at least two distinguishably labeled probe families to which the template polynucleotide is exposed prior to the ligating, and wherein each probe family comprises a plurality of oligonucleotide probes having identical labels but different sequences that comprise;

(1) a constrained portion of least two constrained nucleosides, wherein the constrained nucleosides;

(i) are at least 2-fold degenerate among the probes in the probe family, (ii) need not be located contiguously, and (iii) are not selected independently of each other, such that

(2) knowing the label of a probe eliminates one or more combinations of nucleotides nucleosides as possibilities for the sequence of the constrained portion, and

(3) knowing the identity of a constrained residue nucleoside in that probe further eliminates at least one possible identity for another constrained residue nucleoside in the probe;

(c) identifying one or more nucleotides of the polynucleotide; and

(d) repeating steps (b) and (c) until the sequence of nucleotides is determined.

18. The method of claim 17, wherein the extending step is performed in the semi-solid support.

19. The method of claim 17, wherein the template is attached to a microparticle that is attached to a substantially planar, rigid substrate.

21. The method of claim 17 wherein step (a) comprises amplifying a template polynucleotide molecule in a compartment of an emulsion in the presence of microparticle so that a microparticle having a clonal population of template polynucleotides attached thereto is produced, recovering the microparticle from the emulsion, and embedding the microparticle in or on a semi-solid support.

22. The method of claim 21, wherein

(i) a plurality of template polynucleotide molecules comprising different sequences are amplified in individual compartments of the emulsion;

(ii) a plurality of microparticles, each having attached thereto a clonal populations of template polynucleotides, wherein the clonal populations have different sequences, is recovered from the emulsion and embedded in or on the support, and

(iii) steps (b), (c), and (d) are performed in parallel on the clonal populations attached to the embedded microparticles so as to determine a plurality of sequences in parallel.