# US 10,767,221 B2 · Enzyme-linked nucleotides

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- Number: US 10,767,221 B2
- Document ID: US10767221B2
- Status: Granted
- Kind: B2
- Application number: 14/776,551
- Filed: 2013-03-15
- Published: 2016-02-04
- Granted: 2020-09-08
- Earliest claimed: 2013-03-15
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Christian Gloeckner; Matthew William Kellinger; Lea Pickering
- Web page: https://patentgrep.com/patent/US10767221B2

## 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 modified DNA polymerase comprising:
a) a point mutation wherein a surface-exposed amino acid residue is replaced with a cysteine residue;
b) a flexible linker having a first end and second end, the first end covalently attached to the cysteine residue;
c) a nucleotide attached to the second end of the flexible linker, wherein the flexible linker attachment to the nucleotide allows, the nucleotide to interact at a catalytically active region of said modified DNA polymerase and incorporate the nucleotide to the 3′ end of a polynucleotide in the presence of a template nucleic acid sequence thereby producing a detectable increase in dwell time at the site of incorporation; and
c) an optically detectable label comprising a fluorophore attached to the modified DNA polymerase.

### Claim 2 (depends on 1)

The modified DNA polymerase of claim 1, wherein the linker attachment to the nucleotide occurs at the 5′ gamma phosphate of the nucleotide.

### Claim 3 (depends on 1)

The modified DNA polymerase of claim 1, wherein said nucleotide comprise one of the group of nucleotides consisting of: dCTP, dATP, dGTP, dTTP, and dUTP.

### Claim 4 (depends on 1)

The modified DNA polymerase of claim 1, wherein said nucleotide comprises polymeric phosphate comprising 3, 4, 5, 6 or greater than 6 phosphates.

### Claim 5 (depends on 4)

The modified DNA polymerase of claim 4, wherein said polymeric phosphate comprises hexaphosphate.

### Claim 6 (depends on 1)

The modified DNA polymerase of claim 1, wherein said flexible linker is of a length so as to create a relative concentration of said nucleotide in solution of greater than 1 μM, 10 μM, 100 μM, or greater than 1 mM.

### Claim 7 (depends on 1)

The modified DNA polymerase of claim 1, said flexible linker having a length of greater than 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or greater than 200 Angstrom.

### Claim 8 (depends on 1)

The modified DNA polymerase of claim 1, said flexible linker having a length of less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 or less than 50 Angstrom.