# US 2022/0033894 A1 · Methods for single molecule sequencing

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- Number: US 2022/0033894 A1
- Document ID: US20220033894A1
- Status: Published application
- Kind: A1
- Application number: 17/499,718
- Filed: 2021-10-12
- Published: 2022-02-03
- Earliest claimed: 2016-12-16
- Assignees: Innovasion Labs, Inc.
- Applicants: Innovasion Labs, Inc.
- Inventors: Inanc ORTAC
- Web page: https://patentgrep.com/patent/US20220033894A1

## CPC

- C12Q 1/6869: Methods for sequencing
- 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

## Abstract

Provided herein are methods and systems for sequencing a single nucleic acid molecule utilizing a polymerase enzyme, a template nucleic acid, and a polymerase reagent solution.

## Claims

### Claim 1

A method for sequencing a nucleic acid template comprising:
providing a sequencing mixture comprising (i) a polymerase enzyme, (ii) a template nucleic acid, and (iii) a polymerase reagent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, wherein said polymerase reagent solution includes a component for a requenching reaction and a plurality of types of nucleotide analogs; wherein each type of nucleotide analog has a labeled leaving group that is cleavable by the polymerase, and each type of nucleotide analog has a different label, wherein the labeled leaving group is cleaved upon polymerase-dependent binding of a respective nucleotide analog to the template strand;
carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially to the template whereby: a) a nucleotide analog associates with the polymerase, b) the nucleotide analog is incorporated on the template strand by the polymerase when the labeled leaving group on that nucleotide analog is cleaved by the polymerase, wherein the labeled leaving group emits light upon cleavage, then c) the labeled leaving group on the nucleotide analog is quenched by the requenching reaction; and
detecting light from the labels while nucleic acid synthesis is occurring, and using light detected in the time between step b) when the labelled leaving group is cleaved, and step c) in which the labeled leaving group is quenched, to determine a sequence of the template nucleic acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleotide analog has been modified by a fluorophore attached to a terminal phosphate.

### Claim 3 (depends on 1, 2)

The method of claims 1-2, wherein the leaving group is a labelled pyrophosphate.

### Claim 4 (depends on 1, 2, 3)

The method of claims 1-3, wherein the pyrophosphate is labeled with a fluorophore.

### Claim 5 (depends on 1, 2, 3, 4)

The method of claims 1-4, wherein each base of a nucleotide is labeled with a unique fluorophore relative to other bases.

### Claim 6 (depends on 1, 2, 3, 4, 5, 6)

The method of claims 1-5, wherein the requenching reaction uses a quenching enzyme or Plasmonics. The method of claims 1-6, wherein the quenching enzyme is ATP sulfurylase.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The method of claims 1-7, wherein requenching is achieved by an adenosine nucleobase on APS.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of claims 1-8, wherein the polymerase enzyme is DNA polymerase.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The method of claims 1-9, wherein types of nucleotide analogs comprise dATP, dTTP, dGTP, dCTP and dUTP.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of claims 1-10, wherein the requenching reaction uses plasmonics.

### Claim 12 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method of claims 2-11, wherein the fluorophore is quenched by the nucleobase on the nucleotide.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The method of claims 1-12, wherein a removable quencher molecule is attached to the nucleotide analog through electrostatic interaction or hydrogen bonding.

### Claim 14 (depends on 13)

The method of claim 13, wherein the removable quencher molecule is attached to the nucleotide analog at the nucleobase or sugar.