# US 7,745,188 B2 · Thermostable Y-family polymerases and chimeras

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- Number: US 7,745,188 B2
- Document ID: US7745188B2
- Status: Granted
- Kind: B2
- Application number: 11/596,783
- Filed: 2005-05-20
- Published: 2008-08-14
- Granted: 2010-06-29
- Earliest claimed: 2004-05-20
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Inventors: Roger Woodgate; John P. McDonald; Wei Yang
- Web page: https://patentgrep.com/patent/US7745188B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/62: DNA sequences coding for fusion proteins
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure is related to thermostable Y-family polymerases, in particular several novel Y-family polymerases and chimeras made therefrom, as well as methods of identifying other Y-family polymerases, methods of generating other chimeric Y-family polymerases, methods of amplifying ancient or damaged DNA, and methods of incorporating fluorescent or modified nucleotides into a DNA molecule.

## Claims

### Claim 1

An isolated chimeric Y-family polymerase comprising the amino acid sequence as set forth in
SEQ ID NO: 57 (AiLFSte (AiDpo4/SteDpo4LF)) or a sequence having at least 95% sequence identity to SEQ ID NO: 57
wherein the chimeric Y-family polymerase has polymerase activity.

### Claim 2 (depends on 1)

The polymerase of claim 1, comprising the amino acid sequence as set forth in SEQ ID NO: 57.

### Claim 3 (depends on 1)

The polymerase of claim 1, comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57.

### Claim 4

A method of using a chimeric Y-family polymerase to add a nucleotide to a polynucleotide, the method comprising incubating a polynucleotide and a nucleotide with a chimeric Y-family polymerase, thereby adding the nucleotide to the polynucleotide, wherein the chimeric Y-family polymerase comprises SEQ ID NO: 57 (AiLFSte (AiDpo4/SteDpo4LF)).

### Claim 5 (depends on 1)

A method of amplifying a DNA molecule, the method comprising:
thermocycling the DNA molecule with a first primer, a second primer and an amount of the chimeric Y-family polymerase of claim 1 sufficient to amplify the DNA molecule, wherein the first primer hybridizes with a DNA sequence at the 3′-end of the DNA molecule, and the second primer hybridizes with a DNA sequence at the 5′-end of the DNA molecule, thereby amplifying the DNA molecule.

### Claim 6 (depends on 5)

The method of claim 5, wherein the DNA is ancient or damaged DNA.

### Claim 7 (depends on 5)

The method of claim 5, wherein amplifying the DNA molecule comprises incorporating fluorescent or modified nucleotides.

### Claim 8 (depends on 5)

The method of claim 5, wherein the method further comprises thermocycling the DNA molecule with an amount of a high-fidelity polymerase sufficient to amplify the DNA molecule.