Reagents, methods, and libraries for bead-based sequencing

Patent metadata

Field	Value
Publication number	US-10323277-B2
Application number	US-201615291982-A
Country	US
Kind code	B2
Filing date	Oct 12, 2016
Priority date	Feb 1, 2005
Publication date	Jun 18, 2019
Grant date	Jun 18, 2019

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.

First claim

What is claimed:

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.

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.

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

Assignees

• Applied Biosystems Llc

Inventors

• Mckernan Kevin
• Blanchard Alan
• Kotler Lev
• Costa Gina

Classifications

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• C12Q2565/137

  Chromatographic separation · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• B82Y30/00

  Nanotechnology for materials or surface science, e.g. nanocomposites · CPC title