Enzyme-linked nucleotides

Patent metadata

Field	Value
Publication number	US-2025257395-A1
Application number	US-202519026055-A
Country	US
Kind code	A1
Filing date	Jan 16, 2025
Priority date	Mar 15, 2013
Publication date	Aug 14, 2025
Grant date	—

Abstract

Presented herein are polymerase-linked nucleotides for improved distinguishing nucleotide sequences for different nucleic acid molecules. Also presented are methods and systems using the polymerase-linked nucleotides for improved distinguishing nucleotide sequences for different nucleic acid molecules.

First claim

What is claimed is:

1 . A method of distinguishing nucleotide sequences for different nucleic acid molecules, comprising
(a) providing a plurality of different nucleic acid molecules, wherein the different nucleic acid molecules are attached to a surface in the form of an array of nucleic acid features; (b) adding a plurality of polymerase-linked nucleotides to said nucleic acid features, wherein the polymerase-linked nucleotide comprises a polymerase having a point mutation wherein a surface-exposed amino acid residue is replaced with a cysteine residue, a flexible linker having a first end and a second end, wherein the first end is attached to the cysteine residue and a nucleotide is attached to the second end of the flexible linker, (b) monitoring binding of the polymerase molecules to the nucleic acid features, thereby determining dwell time of the polymerase molecules at the nucleic acid features; and (c) identifying nucleic acid features of the array that correctly incorporate the nucleotide molecules based on the dwell time of the polymerase molecules at the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

2 . The method of claim 1 , wherein (b) comprises monitoring a detectable signal indicative of the binding of the polymerase molecules to the nucleic acid features and catalysis of nucleotide incorporation into the polynucleotide feature.

3 . The method of claim 2 , wherein (c) comprises identifying the nucleic acid features of the array that correctly incorporate the nucleotide molecules based on detectable signal of the polymerase molecules at the nucleic acid features, whereby the dwell time determines nucleotide molecules that are correctly incorporated into the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

4 . The method of claim 1 , wherein (b) comprises monitoring a detectable signal indicative of the binding of the polymerase molecules to the nucleic acid features and catalysis of nucleotide incorporation into the polynucleotide feature.

5 . The method of claim 4 , wherein said detectable label comprises a detectable label attached to the polymerase.

6 . The method of claim 4 , wherein said detectable label comprises an optically detectable label.

7 . The method of claim 4 , wherein said optically detectable label comprises a fluorophore.

8 . The method of claim 1 , wherein (b) comprises simultaneously adding a plurality nucleotide species that base-pair with four different nucleotide species in the polynucleotide features.

9 . The method of claim 8 , wherein each of said nucleotide species is distinguished from the other nucleotide species in the plurality of species by a distinct detectable label.

10 . The method of claim 9 , wherein said detectable label comprises a fluorophore.

11 . The method of claim 1 , wherein successive incorporation events are identified as discrete binding events.

12 . The method of claim 12 , wherein detection discrete binding events permit detection of a homopolymer sequence.

13 . A system for distinguishing nucleotide sequences for different nucleic acid molecules, the system comprising
(a) an array comprising nucleic acid features having different nucleotide sequences; (b) a fluidic apparatus configured to deliver sequencing reagents to the array, wherein the sequencing reagents comprise polymerase-linked nucleotide comprising a nucleotide covalently attached to a catalytically active polymerase enzyme by a flexible linker; (c) a detection apparatus configured to measure binding events from the array at a resolution that distinguishes individual nucleic acid features of the array; (d) a control module comprising instructions for
(i) adding the sequencing reagents to said nucleic acid features,
(ii) obtaining measurements of binding of the polymerase molecules to the nucleic acid features; and
(e) an analysis module comprising instructions for
(i) processing the measurements of binding of the polymerase molecules to the nucleic acid features, thereby determining dwell time of the polymerase molecules at the nucleic acid features; and
(ii) identifying nucleic acid features of the array that correctly incorporate the nucleotide molecules based on the dwell time of the polymerase molecules at the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

14 . The system of claim 13 , wherein (d)(ii) comprises monitoring a detectable signal indicative of the binding of the polymerase molecules to the nucleic acid features and catalysis of nucleotide incorporation into the polynucleotide feature.

15 . The system of claim 13 , wherein (e)(ii) comprises identifying the nucleic acid features of the array that correctly incorporate the nucleotide molecules based on detectable signal of the polymerase molecules at the nucleic acid features, whereby the dwell time determines nucleotide molecules that are correctly incorporated into the nucleic acid features, thereby distinguishing the nucleotide sequences for the different nucleic acid molecules.

16 . The system of claim 13 , wherein (d)(ii) comprises monitoring a detectable signal indicative of the binding of the polymerase molecules to the nucleic acid features and catalysis of nucleotide incorporation into the polynucleotide feature.

17 . The system of claim 16 , wherein said detectable label comprises a detectable label attached to the polymerase.

18 . The system of claim 16 , wherein said detectable label comprises an optically detectable label.

19 . The system of claim 18 , wherein said optically detectable label comprises a fluorophore.

20 . The system of claim 13 , wherein (d)(ii) comprises simultaneously adding a plurality nucleotide species that base-pair with four different nucleotide species in the polynucleotide features.

Assignees

• Illumina Inc

Inventors

• Gloeckner Christian
• Kellinger Matthew William
• Pickering Lea

Classifications

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• A61K47/59

  obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes · CPC title

• A61K47/64

  Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65) · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title