Methods and reagents for nucleic acid analysis

Patent metadata

Field	Value
Publication number	US-2025333787-A1
Application number	US-202418968249-A
Country	US
Kind code	A1
Filing date	Dec 4, 2024
Priority date	Oct 30, 2020
Publication date	Oct 30, 2025
Grant date	—

Abstract

Provided herein are fluorescently-labeled nucleotide conjugates for nucleic acid analysis. Also provided are reagents used for forming binding complexes between a fluorescently-labeled nucleotide conjugate and a target nucleic acid sequence in the presence of one or more reagents disclosed herein. Binding complexes can be detected in the presence of the one or more reagents. For example, the one or more reagents may contain a photobleaching reducing agent configured to reduce photobleaching resulting from use of the fluorescently-labeled nucleotide conjugate to form the binding complex in a nucleic acid analysis. Such nucleic acid analysis may be used to identify sites of nucleobase binding or incorporation between the target nucleic acid sequence and one or more nucleotide moieties of the fluorescently-labeled nucleotide conjugate in a nucleic acid sequence reaction.

First claim

1 - 30 . (canceled)

31 . A method for sequencing a template nucleic acid, comprising:
a) providing a fluorescently-labeled multivalent molecule comprising a core attached to a plurality of nucleotide-arms wherein individual nucleotide-arms comprise (i) a core attachment moiety, and (ii) a nucleotide moiety; b) forming a binding complex by contacting the template nucleic acid with (i) a polymerizing enzyme, (ii) a sequencing primer comprising a 3′ extendible end, and (iii) the multivalent molecule, wherein one of the nucleotide moieties of the multivalent molecule is bound to the 3′ end of the sequencing primer at a position that is opposite a complementary nucleotide in the template nucleic acid; and c) contacting the binding complex with a trap reagent comprising a non-catalytic divalent cation that promotes formation of the binding complex and inhibits polymerase-catalyzed incorporation of the nucleotide moiety into the 3′ extendible end of the sequencing primer.

32 . The method of claim 31 , wherein the non-catalytic divalent cation is a strontium, barium, scandium, titanium, calcium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, rhodium, europium, tin or terbium cation.

33 . The method of claim 31 , wherein the trap reagent further comprises a pH buffering agent.

34 . The method of claim 33 , wherein the pH buffering agent comprises Tris, Tris-HCl, Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (MEA), a citrate compound, a citrate mixture, NaOH, or KOH, or a mixture thereof.

35 . The method of claim 31 , wherein the trap reagent further comprises a chelating agent.

36 . The method of claim 35 , wherein the chelating agent is selected from the group consisting of EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylene triamine pentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), citrate anhydrous, sodium citrate, calcium citrate, ammonium citrate, ammonium bicitrate, citric acid, potassium citrate, or magnesium citrate or a mixture thereof.

37 . The method of claim 31 , wherein the trap reagent further comprises a monovalent cation.

38 . The method of claim 37 , wherein the monovalent cation is sodium, potassium, or a mixture thereof.

39 . The method of claim 31 , wherein the trap reagent further comprises a detergent.

40 . The method of claim 39 , wherein the detergent comprises SDS (sodium dodecyl sulfate), Triton X-100, Tween 20, Tween 80, Nonidet P-40, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-Dodecyl-N,N-dimethyl-3-amonio-1-propanesulfate (DetX), LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, or sodium cholate, or a mixture thereof.

41 . The method of claim 31 , wherein the trap reagent further comprises a fluorescently-labeled nucleotide conjugate.

42 . The method of claim 41 , wherein the fluorescently-labeled nucleotide conjugate is dATP, dGTP, dCTP, dTTP and dUTP, or a mixture thereof, wherein one of more of the dATP, dGTP, dCTP, dTTP and dUTP is labeled with a detectable reporter moiety.

43 . The method of claim 31 , wherein the trap reagent further comprises a first sequencing polymerase enzyme.

44 . The method of claim 43 , wherein the first sequencing polymerase enzyme comprises the amino acid sequence of any one of SEQ ID NOs 1 to 12.

45 . The method of claim 31 , wherein the trap reagent further comprises a viscosity agent.

46 . The method of claim 45 , wherein the viscosity agent comprises trehalose, sucrose, cellulose, xylitol, mannitol, sorbitol or inositol, glycerol, ethylene glycol or propylene glycol, or a mixture thereof.

47 . The method of claim 31 , wherein the trap reagent comprises:
(i) HEPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), Tween-20 (0.02%), Gd—HCl (0.08 M), ethylene glycol (10%), dATP (0.04 μM), dGTP (0.04 μM), dCTP (0.04 μM), dUTP (0.04 μM), and a sequencing polymerase (200 nM); (ii) Bicine (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (50 mM), Ba-acetate (10 mM), Tween-20 (0.02%), Gd—HCl (0.06 M), ethylene glycol (10%), dATP (0.04 μM), dGTP (0.04 μM), dCTP (0.04 μM), dUTP (0.04 μM), and sequencing polymerase (100 nM); (iii) MOPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KCl (25 mM), Sr-acetate (5 mM), Tween-20 (0.02%), Gd—HCl (0.04 M), ethylene glycol (10%), dATP (0.02 μM), dGTP (0.02 μM), dCTP (0.02 μM), dUTP (0.02 μM), and sequencing polymerase (500 nM); (iv) BES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KCl (50 mM), Ba-acetate (10 mM), Tween-20 (0.02%), Gd—HCl (0.02 M), ethylene glycol (10%), dATP (0.02 μM), dGTP (0.02 μM), dCTP (0.02 μM), dUTP (0.02 μM), and sequencing polymerase (300 nM); (v) TES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (25 mM), Sr-acetate (5 mM), Tween-20 (0.02%), Gd—HCl (0.08 M), ethylene glycol (10%), dATP (0.04 μM), dGTP (0.04 μM), dCTP (0.04 μM), dUTP (0.04 μM), and sequencing polymerase (100 nM); (vi) CAPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KCl (50 mM), Ba-acetate (10 mM), Triton-X (0.02%), urea (0.08 M), ethylene glycol (10%), dATP (0.04 μM), dGTP (0.04 μM), dCTP (0.04 μM), dUTP (0.04 μM), and sequencing polymerase (200 nM); (vii) TAPS (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KCl (25 mM), Sr-acetate (5 mM), Triton-X (0.02%), urea (0.08 M), ethylene glycol (10%), dATP (0.04 μM), dGTP (0.04 μM), dCTP (0.04 μM), dUTP (0.04 μM), and sequencing polymerase (500 nM) (viii) ACES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), NaCl (50 mM), Ba-acetate (10 mM), Triton-X (0.02%), urea (0.06 M), ethylene glycol (10%), dATP (0.02 μM), dGTP (0.02 μM), dCTP (0.02 μM), dUTP (0.02 μM), and sequencing polymerase (400 nM); PIPES (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KCl (25 mM), Sr-acetate (5 mM), Triton-X (0.02%), urea (0.04 M), ethylene glycol (10%), dATP (0.02 μM), dGTP (0.02 μM), dCTP (0.02 μM), dUTP (0.02 μM), and sequencing polymerase (500 nM); or Tris (25 mM, pH 8), EDTA (0.5 mM, pH 7.5), KCl (50 mM), Ba-acetate (10 mM), Triton-X (0.02%), urea (0.02 M), ethylene glycol (10%), dATP (0.04 μM), dGTP (0.04 μM), dCTP (0.04 μM), dUTP (0.04 μM), and sequencing polymerase (200 nM).

48 . The method of claim 31 , wherein the trap reagent does not comprise manganese or magnesium ions.

49 . The method of claim 31 , further comprising: detecting a fluorescence signal emitted from the binding complex in response to an excitation illumination.

50 . The method of claim 31 , further comprising the step of contacting the binding complex with an imaging reagent, wherein the imaging reagent comprises a photobleaching reducing agent.

51 . The method of claim 49 , wherein the photobleaching reagent comprises ascorbate.

Assignees

• Element Biosciences Inc

Inventors

• Arslan Sinan
• He Molly
• Previte Michael
• Tippana Ramreddy
• Yu Hua
• Light William
• Zhao Junhua

Classifications

• C12Q1/6806

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

• C12Q2565/107

  Alteration in the property of hybridised versus free label oligonucleotides · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2525/197

  incorporating a spacer/coupling moiety · CPC title