# US 12,215,383 B2 · Polymerase compositions and methods of making and using same

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- Number: US 12,215,383 B2
- Document ID: US12215383B2
- Status: Granted
- Kind: B2
- Application number: 17/725,369
- Filed: 2022-04-20
- Published: 2022-09-29
- Granted: 2025-02-04
- Earliest claimed: 2014-12-16
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Daniel Mazur; Eileen Tozer; Sihong Chen; Peter Vander Horn; Tommie Lincecum
- Web page: https://patentgrep.com/patent/US12215383B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12Q 1/6869: Methods for sequencing
- C12Q 2521/101: DNA polymerase
- 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 compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, modified polymerases and biologically active fragments thereof, such as modified Taq polymerases, are provided that allow for improved nucleic acid amplification. In some aspects, the disclosure provides modified polymerases having improved thermostability, accuracy, processivity and/or read length as compared to a referenceTaq polymerase. In some aspects, the disclosure relates to modified polymerases or biologically active fragments thereof, useful for amplification methods, and in practically illustrative embodiments, emulsion PCR.

## Claims

### Claim 1

A composition comprising:
a modified polypeptide having at least 95% identity to SEQ ID NO: 1, wherein the modified polypeptide exhibits polymerase activity and an improvement relative to a reference polymerase of SEQ ID NO:1 and/or SEQ ID NO:34, in one or more property comprising thermostability and/or at least one sequencing property, and
wherein the modified polypeptide comprises one or more amino acid substitutions selected from A77E, A97V, K240I, L287T, E295F, E295N, and E794C, and
wherein the modified polypeptide comprises the naturally occurring DNA polymerase motifs A and C.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the modified polypeptide comprises an amino acid substitution selected from A77E, A97V, K240I, L287T, or E794C.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the modified polypeptide exhibits an improvement as measured by sequencing nucleic acid templates generated using the modified polymerase in an emulsion PCR template amplification reaction under high ionic strength conditions.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the high ionic strength conditions comprise ionic strength conditions of between 75 mM salt to about 150 mM salt.

### Claim 5 (depends on 3)

The composition of claim 3, wherein the at least one sequencing property exhibiting improvement comprises increased AQ20 mean read length reads, reduced strand bias, increased base coverage, increased accuracy, increased sequencing throughput (Mb), and increased uniformity of coverage.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the modified polypeptide further comprises one or more amino substitutions selected from E794C+E805I, P6N+E295F, E397V+P6N, or E745T+E794C.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the one or more property exhibiting the improvement comprises thermostability and/or at least one sequencing property selected from increased AQ20 mean read length reads, reduced strand bias, increased base coverage, increased accuracy, increased sequencing throughput (Mb), and increased uniformity of coverage.

### Claim 8 (depends on 1)

A method for amplifying a nucleic acid, comprising:
contacting the nucleic acid with the composition of claim 1 under suitable conditions for amplifying the nucleic acid, and
amplifying the nucleic acid.

### Claim 9 (depends on 8)

The method of claim 8, wherein the modified polypeptide comprises amino acid substitutions selected from A77E, A97V, K240I, L287T, or E794C.

### Claim 10 (depends on 9)

The method of claim 9, wherein the modified polypeptide exhibits an improvement as measured by sequencing nucleic acid templates generated using the modified polypeptide in an emulsion PCR template amplification reaction under high ionic strength conditions.

### Claim 11 (depends on 10)

The method of claim 10, wherein the high ionic strength conditions comprise ionic strength conditions of between 75 mM salt to about 150 mM salt.

### Claim 12 (depends on 10)

The method of claim 10, wherein the at least one sequencing property exhibiting improvement comprises increased AQ20 mean read length reads, reduced strand bias, increased base coverage, increased accuracy, increased sequencing throughput (Mb) and increased uniformity of coverage.

### Claim 13 (depends on 8)

The method of claim 8, wherein the modified polypeptide further comprises one or more amino substitutions selected from E794C+E805I, P6N+E295F, E397V+P6N, or E745T+E794C.

### Claim 14 (depends on 13)

The method of claim 13, wherein the one or more property exhibiting an improvement comprises thermostability and/or at least one sequencing property selected from increased AQ20 mean read length reads, reduced strand bias, increased base coverage, increased accuracy, increased sequencing throughput (Mb), and increased uniformity of coverage.

### Claim 15 (depends on 8)

The method of claim 8, wherein the suitable conditions comprise suitable conditions for performing a polymerase chain reaction, an isothermal amplification reaction, a recombinase polymerase amplification reaction, a proximity ligation amplification, a rolling circle amplification, or a strand displacement amplification.

### Claim 16 (depends on 8)

The method of claim 8, wherein the suitable conditions comprise suitable conditions for performing a polymerase chain reaction.

### Claim 17 (depends on 16)

The method of claim 16, wherein the polymerase chain reaction is an emulsion polymerase chain reaction.

### Claim 18 (depends on 8)

The method of claim 8, wherein the amplifying comprises clonally amplifying the nucleic acid in solution or on a solid support.

### Claim 19 (depends on 8)

The method of claim 8, further comprising determining a nucleic acid sequence of at least a portion of the nucleic acid.

### Claim 20 (depends on 1)

A method for performing a polymerization reaction comprising:
contacting a nucleic acid template with the composition of claim 1 in the presence of one or more nucleotide triphosphates and under suitable conditions for a polymerization reaction.

### Claim 21 (depends on 20)

The method of claim 20, wherein the modified polypeptide comprises an amino acid substitution selected from A77E, A97V, K240I, L287T, K292C, or E794C.

### Claim 22 (depends on 21)

The method of claim 21, wherein the modified polypeptide exhibits an improvement as measured by sequencing nucleic acid templates generated using the modified polymerase in an emulsion PCR template amplification reaction under high ionic strength conditions.

### Claim 23 (depends on 22)

The method of claim 22, wherein the high ionic strength conditions comprise ionic strength conditions of between 75 mM salt to about 150 mM salt.

### Claim 24 (depends on 22)

The method of claim 22, wherein the at least one sequencing property exhibiting an improvement comprises increased AQ20 mean read length reads, reduced strand bias, increased base coverage, increased accuracy, increased sequencing throughput (Mb), and increased uniformity of coverage.

### Claim 25 (depends on 16)

The method of claim 16, wherein the modified polypeptide further comprises one or more amino substitutions selected from E794C+E805I, P6N+E295F, E397V+P6N, or E745T+E794C.

### Claim 26 (depends on 25)

The method of claim 25, wherein the one or more property exhibiting an improvement includes thermostability and/or at least one sequencing property selected from increased AQ20 mean read length reads, reduced strand bias, increased base coverage, increased accuracy, increased sequencing throughput (Mb), and increased uniformity of coverage.