Methods and systems for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-12071653-B2
Application number	US-202217712765-A
Country	US
Kind code	B2
Filing date	Apr 4, 2022
Priority date	Sep 27, 2017
Publication date	Aug 27, 2024
Grant date	Aug 27, 2024

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) contacting two or more nucleotide analogs with a growing nucleic acid strand having sequence complementarity with said template nucleic acid molecule, under conditions sufficient to incorporate said two or more nucleotide analogs into said growing nucleic acid strand, wherein said nucleotide analogs comprise a modified phosphate group comprising sulfur or selenium;
(b) reacting at least one of said modified phosphate group of said nucleotide analogs with a labeling reagent comprising a label to generate a label-S-P, label-S-S-P, or label-Se-P group on said growing nucleic acid strand;
(c) detecting one or more signals indicative of said label; and
(d) converting said label-S-P, label-S-S-P, or label-Se-P group to an O═P group on said growing nucleic acid strand.

2. The method of claim 1 , further comprising sequencing said template nucleic acid molecule based at least in part on said one or more signals detected in (c).

3. The method of claim 1 , wherein (a) comprises: contacting a plurality of nucleotides of a same canonical base, including said two or more nucleotide analogs, to said growing nucleic acid strand, under conditions sufficient to incorporate at least two nucleotides of said plurality of nucleotides, including said two or more nucleotide analogs, into said growing nucleic acid strand.

4. The method of claim 3 , wherein said plurality of nucleotides of said same canonical base each comprises a modified phosphate group comprising sulfur or selenium.

5. The method of claim 3 ,
wherein (a) comprises: contacting said plurality of nucleotides of said same canonical base, including said two or more nucleotide analogs each comprising a modified phosphate group comprising sulfur or selenium, to a plurality of growing nucleic acid strands, including said growing nucleic acid strand, under conditions sufficient to incorporate at least two nucleotides of said plurality of nucleotides into each of said plurality of growing nucleic acid strands, wherein said plurality of growing nucleic acid strands has sequence identity;
wherein (b) comprises: reacting a plurality of labeling reagents, at least a subset each comprising said label, with at least a subset of modified phosphate groups incorporated in said plurality of growing nucleic acid strands, to generate label-S-P, label-S-S-P, or label-Se-P groups across said plurality of growing nucleic acid strands;
wherein in (c): said one or more signals are indicative of said labels; and
further comprising determining a sequence of a homopolymer region on said template nucleic acid molecule based at least in part on said one or more signals.

6. The method of claim 5 , wherein in (b) at least two label-S-P, label-S-S-P, or label-Se-P groups of said label-S-P, label-S-S-P, or label-Se-P groups are generated on a same growing nucleic acid strand.

7. The method of claim 1 , wherein said two or more nucleotide analogs each comprises alpha-thio-deoxynucleotide triphosphate (α-S-dNTP).

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

9. The method of claim 8 , wherein said support is a bead.

10. The method of claim 8 , wherein said support is a substantially planar surface.

11. 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.

12. The method of claim 11 , wherein said labeling reagent comprises a dye that is functionalized with an iodoacetamide moiety.

13. The method of claim 11 , wherein said labeling reagent comprises a bimane derivative.

14. The method of claim 13 , wherein said bimane derivative is monobromobimane.

15. The method of claim 1 , wherein said converting in (d) comprises providing to said growing nucleic acid strand one or more of a metal ion, an oxidant, an oxime, or a reducing agent.

16. The method of claim 15 , wherein said converting in (d) comprises providing to said growing nucleic acid strand said metal ion selected from the group consisting of a silver-, mercury- and lead-containing ion.

17. A method for determining a sequence of a template nucleic acid molecule, comprising:
(a) contacting a nucleotide analog with a growing nucleic acid strand having sequence complementarity with said template nucleic acid molecule, under conditions sufficient to incorporate said nucleotide analog into said growing nucleic acid strand, wherein said nucleotide analog comprises a modified phosphate group comprising sulfur or selenium;
(b) reacting said modified phosphate group of said nucleotide analog with a labeling reagent comprising a label to generate a label-S-P, label-S-S-P, or label-Se-P group on said growing nucleic acid strand;
(c) detecting one or more signals indicative of said label; and
(d) converting said label-S-P, label-S-S-P, or label-Se-P group to an O═P group on said growing nucleic acid strand; wherein said converting in (d) comprises providing to said growing nucleic acid strand one or more of a metal ion, an oxidant, an oxime, or a reducing agent; wherein said oxidant selected from the group consisting of iodine, iodosobenzoate, and potassium peroxymonosulfate (oxone).

18. A method for determining a sequence of a template nucleic acid molecule, comprising:
(a) contacting a nucleotide analog with a growing nucleic acid strand having sequence complementarity with said template nucleic acid molecule, under conditions sufficient to incorporate said nucleotide analog into said growing nucleic acid strand, wherein said nucleotide analog comprises a modified phosphate group comprising sulfur or selenium;
(b) reacting said modified phosphate group of said nucleotide analog with a labeling reagent comprising a label to generate a label-S-P, label-S-S-P, or label-Se-P group on said growing nucleic acid strand;
(c) detecting one or more signals indicative of said label; and
(d) converting said label-S-P, label-S-S-P, or label-Se-P group to an O═P group on said growing nucleic acid strand; wherein said converting in (d) comprises providing to said growing nucleic acid strand one or more of a metal ion, an oxidant, an oxime, or a reducing agent; wherein said oxime selected from the group consisting of 2-pyridine aldoxime, 4-pyridine aldoxime, obidoxime, HI-6, HLö-7, E-2-nitrobenzaldoxime, and E-4-nitrobenzldoxime.

19. The method of claim 15 , wherein said converting in (d) comprises providing to said growing nucleic acid strand said reducing agent selected from the group consisting of Tris(2-carboxyethyl phosphine (TCEP), tris(hydroxypropyl)phosphine (THP), and dithiothreitol (DTT).

20. The method of claim 1 , wherein said reacting in (b) comprises an alkylation reaction between said labeling reagent and said modified phosphate group.

21. The method of claim 1 , wherein the label comprises a dye and a functional group configured to react to said modified phosphate group.

22. The method of claim 21 , wherein the functional group configured to react to said modified phosphate group comprises iodoacetamide.

23. The method of claim 21 , wherein the functional group configured to react to said modified phosphate group comprises an alkylating agent.

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

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · 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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6806

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