# US 2020/0392575 A1 · Enzyme-linked nucleotides

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2020/0392575 A1
- Document ID: US20200392575A1
- Status: Published application
- Kind: A1
- Application number: 17/005,130
- Filed: 2020-08-27
- Published: 2020-12-17
- Earliest claimed: 2015-09-14
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Christian Gloeckner; Matthew William Kellinger; Lea Pickering
- Web page: https://patentgrep.com/patent/US20200392575A1

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- A61K 47/59: obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K 47/64: 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)
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12Q 1/6869: Methods for sequencing
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 2)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 4)

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

### Claim 7 (depends on 4)

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

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

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.

### Claim 10 (depends on 9)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 12)

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

### Claim 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.

### Claim 14 (depends on 13)

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.

### Claim 15 (depends on 13)

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.

### Claim 16 (depends on 13)

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.

### Claim 17 (depends on 16)

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

### Claim 18 (depends on 16)

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

### Claim 19 (depends on 18)

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

### Claim 20 (depends on 13)

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.