Methods and systems for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-11326203-B2
Application number	US-202016822121-A
Country	US
Kind code	B2
Filing date	Mar 18, 2020
Priority date	Sep 27, 2017
Publication date	May 10, 2022
Grant date	May 10, 2022

Abstract

The disclosure provides methods for sequencing nucleic acids using, including with nucleotide analogs and subsequently appended labels.

First claim

What is claimed is:

1. A method for determining a sequence of a template nucleic acid molecule, comprising:
(a) providing a reaction mixture comprising said template nucleic acid molecule and a plurality of nucleotide analogs, wherein a nucleotide analog of said plurality of nucleotide analogs comprises a functional group;
(b) subjecting said reaction mixture to conditions sufficient to incorporate said nucleotide analog comprising said functional group into a growing nucleic acid strand having sequence complementarity with said template nucleic acid molecule;
(c) subsequent to (b), subjecting said growing nucleic acid strand and a labeling reagent comprising a label to conditions sufficient for said functional group and said labeling reagent to react to provide a labeled functional group comprising said label;
(d) detecting one or more signals indicative of said labeled functional group; and
(e) subjecting said labeled functional group to conditions sufficient to convert said labeled functional group to a moiety that is substantially unreactive with said labeling reagent,.
wherein, subsequent to (e), said label is removed from said nucleotide analog, and
wherein, subsequent to (e), said moiety is attached to said growing nucleic acid strand.

2. The method of claim 1 , wherein said template nucleic acid molecule is immobilized to a support.

3. The method of claim 2 , wherein said support is a bead or a substantially planar surface.

4. The method of claim 1 , wherein said functional group comprises sulfur or selenium.

5. The method of claim 4 , wherein said plurality of nucleotide analogs comprises a plurality of alpha-thio-deoxynucleotide triphosphates (α-S-dNTPs).

6. The method of claim 1 , wherein the functional group comprises an azido group.

7. The method of claim 1 , wherein said labeling reagent comprises a luminescent moiety, an optically-active moiety, a self-quenching dye, or a proximity quenching dye.

8. The method of claim 1 , wherein (c) comprises covalently coupling at least a portion of said labeling reagent with at least a portion of said functional group.

9. The method of claim 1 , further comprising, subsequent to (c), subjecting said growing nucleic acid strand to one or more washing cycles.

10. The method of claim 1 , wherein (c) comprises an alkylation reaction between said labeling reagent and said functional group.

11. The method of claim 1 , wherein said label is derived from Atto-647N-iodoacetamide, an S-pyridyl-containing reagent, monobromobimane, Cy5, Cy5-azide, Atto-633-iodoacetamide, Bodipy FL iodoacetamide, tetramethylrhodamine iodoacetamide, or Atto-488 iodoacetamide.

12. The method of claim 1 , wherein (c) comprises a click reaction.

13. The method of claim 12 , wherein said click reaction is a copper-free click reaction.

14. The method of claim 1 , wherein, in (d), said one or more signals are optical signals, electrical signals or mechanical signals.

15. The method of claim 1 , wherein (e) comprises contacting said labeled functional group with a metal ion, an oxidant, or an oxime.

16. The method of claim 15 , wherein said metal ion comprises a silver-, mercury- or lead-containing ion.

17. The method of claim 15 , wherein said oxidant comprises iodine, iodosobenzoate, or potassium peroxymonosulfate (oxone).

18. The method of claim 15 , wherein said oxime comprises 2-pyridine aldoxime, 4-pyridine aldoxime, obidoxime, HI-6, HLö-7, E-2-nitrobenzaldoxime, or E-4-nitrobenzldoxime.

19. The method of claim 18 , wherein said labeled functional group comprises a disulfide bond, and wherein (e) comprises subjecting said disulfide bond to conditions sufficient to reduce said disulfide bond, thereby generating a modified labeled functional group.

20. The method of claim 18 , wherein said conditions comprise a reducing agent selected from the group consisting of Tris(2-carboxyethyl phosphine (TCEP), tris(hydroxypropyl)phosphine (THP), and dithiothreitol (DTT).

21. The method of claim 1 , further comprising repeating (a)-(e) using an additional plurality of nucleotide analogs.

22. The method of claim 1 , further comprising determining a nucleic acid sequence of said sequence of said template nucleic acid molecule based at least in part on said one or more signals.

23. The method of claim 1 , further comprising performing (a)-(e) for a plurality of template nucleic acid molecules.

24. The method of claim 1 , wherein (e) comprises cleaving said label from said labeled functional group.

25. The method of claim 1 , wherein nucleotide analogs of said plurality of nucleotide analogs are of a same type.

26. The method of claim 1 , wherein said reaction mixture comprises magnesium ions or manganese ions.

27. The method of claim 1 , wherein (i) said moiety that is substantially unreactive with said labeling reagent is optically undetectable, and (ii) said labeled functional group is optically detectable.

28. The method of claim 21 , wherein nucleotide analogs of said plurality of nucleotide analogs are of a same type, and wherein said additional plurality of nucleotide analogs does not comprise nucleotide analogs of said same type.

29. The method of claim 1 , wherein (b) comprises a primer extension reaction using a primer molecule comprising a sequence complementarity with said template nucleic acid molecule.

30. The method of claim 23 , wherein said plurality of template nucleic acid molecules has identical nucleic acid sequences.

Assignees

• Ultima Genomics Inc

Inventors

• Lee Linda
• Menchen Steven
• Nikiforov Theo
• Almogy Gilad
• Oberstrass Florian

Classifications

• C12Q1/6874

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

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• B01J31/00

  Catalysts comprising hydrides, coordination complexes or organic compounds (catalyst compositions used only in polymerisation reactions C08 {; catalytic antibodies C12N9/0002}) · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title