# US 2019/0376133 A1 · Polymerase-template complexes

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- Number: US 2019/0376133 A1
- Document ID: US20190376133A1
- Status: Published application
- Kind: A1
- Application number: 16/505,496
- Filed: 2019-07-08
- Published: 2019-12-12
- Earliest claimed: 2016-02-29
- Assignees: Roche Sequencing Solutions, Inc.
- Applicants: Roche Sequencing Solutions, Inc.
- Inventors: Aruna Ayer; Preethi Sarvabhowman; Charles Schwab
- Web page: https://patentgrep.com/patent/US20190376133A1

## CPC

- C12Q 1/6869: Methods for sequencing
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- 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
- 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

The present disclosure provides methods and compositions for enhancing the processivity of a polymerase in catalyzing template-dependent DNA synthesis in high concentrations of salt. Also disclosed are methods and compositions for enhancing the assembly of polymerase-template complex compatible with active DNA synthesis in the presence of low levels of nucleotides and at a high temperature, such as temperatures at or near the melting temperature of the polymerase.

## Claims

### Claim 1

A method for preparing a polymerase-template complex, the method comprising incubating a primed DNA template with a molar excess of a DNA polymerase in a solution having up to 2.2 μM nucleotide polyphosphate for a period of time sufficient to allow the DNA polymerase and the DNA template to form the polymerase-template complex, wherein the incubation is performed at or above the melting temperature of the free polymerase and below the melting temperature of the DNA polymerase complexed with the DNA template.

### Claim 2 (depends on 1)

The method of claim 1, wherein the solution comprises at least 75 mM monovalent salt.

### Claim 3 (depends on 2)

The method of claim 2, wherein the salt is selected from the group consisting of potassium glutamate, potassium chloride (KCl), potassium sulfate (K2SO4), potassium nitrate (KNO3), cesium chloride (CsCl), and cesium nitrate (CsNO3), and mixtures thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the solution does not comprise a magnesium salt or a calcium salt.

### Claim 5 (depends on 1)

The method of claim 1, wherein the solution is essentially free of nucleotide polyphosphate.

### Claim 6 (depends on 1)

The method of claim 1, wherein the DNA polymerase comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 14.

### Claim 7 (depends on 1)

The method of claim 1, wherein said DNA polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 14.

### Claim 8 (depends on 1)

The method of claim 1, wherein the DNA polymerase comprises an amino acid sequence at least 75% identical to SEQ ID NO: 14, and wherein the solution comprises at least 75 mM a salt selected from the group consisting of potassium glutamate, potassium chloride (KCl), potassium sulfate (K2SO4), potassium nitrate (KNO3), cesium chloride (CsCl), and cesium nitrate (CsNO3), and mixtures thereof.

### Claim 9 (depends on 8)

The method of claim 8, wherein the solution is essentially free of nucleotide polyphosphate.

### Claim 10

A method of sequencing a DNA template, the method comprising:
(a) forming a primed DNA template by annealing a primer to the DNA template;
(b) forming a polymerase-template complex by incubating a primed DNA template with a molar excess of a DNA polymerase in a solution having up to 2.2 μM nucleotide polyphosphate for a period of time sufficient to allow the DNA polymerase and the DNA template to form the polymerase-template complex, wherein the incubation is performed at or above the melting temperature of the free polymerase and below the melting temperature of the DNA polymerase complexed with the DNA template;
(c) forming a nanopore-sequencing complex comprising:
(c1) a nanopore deposited in a membrane, wherein the nanopore is positioned adjacent to or in proximity to a sensing electrode of a biochip; and
(c2) the polymerase-template complex, wherein the DNA polymerase of the polymerase-template complex is linked to the nanopore;
(d) providing tagged nucleotides to the nanopore sequencing complex to initiate a primer extension reaction that polymerizes the tagged nucleotides into a nucleic acid complementary to the DNA template (“complementary strand”); and
(e) detecting tags associated with tagged nucleotides incorporated into the complementary strand by measuring a current flowing through the nanopore; and
(f) deriving the sequence of the DNA template from the measured current flowing through the nanopore.

### Claim 11 (depends on 10)

The method of claim 10, wherein the nanopore comprises a protein selected from the group consisting of outer membrane protein G, alpha-hemolysin, MspA, and variants thereof.

### Claim 12 (depends on 10)

The method of claim 10, wherein the solution comprises at least 75 mM monovalent salt.

### Claim 13 (depends on 12)

The method of claim 12, wherein the salt is selected from the group consisting of potassium glutamate, potassium chloride (KCl), potassium sulfate (K2SO4), potassium nitrate (KNO3), cesium chloride (CsCl), and cesium nitrate (CsNO3), and mixtures thereof.

### Claim 14 (depends on 10)

The method of claim 10, wherein the solution does not comprise a magnesium salt or a calcium salt.

### Claim 15 (depends on 10)

The method of claim 10, wherein the solution is essentially free of nucleotide polyphosphate.

### Claim 16 (depends on 10)

The method of claim 10, wherein the DNA polymerase comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 14.

### Claim 17 (depends on 10)

The method of claim 10, wherein said DNA polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 14.

### Claim 18 (depends on 10)

The method of claim 10, wherein the nanopore comprise alpha-hemolysin or a variant thereof, the DNA polymerase comprises an amino acid sequence having at least 75% identity with SEQ ID NO: 14, and the solution comprises at least 75 mM of a salt selected from the group consisting of potassium glutamate, potassium chloride (KCl), potassium sulfate (K2SO4), potassium nitrate (KNO3), cesium chloride (CsCl), and cesium nitrate (CsNO3), and mixtures thereof.

### Claim 19 (depends on 18)

The method of claim 18, wherein the solution is essentially free of nucleotide polyphosphate.

### Claim 20 (depends on 10)

The method of claim 10, wherein the primer extension reaction is performed at a salt concentration of at least 100 mM.