# US 2014/0154748 A1 · Thermostable chimeric nucleic acid polymerases and uses thereof

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- Number: US 2014/0154748 A1
- Document ID: US20140154748A1
- Status: Published application
- Kind: A1
- Application number: 14/108,863
- Filed: 2013-12-17
- Published: 2014-06-05
- Earliest claimed: 2000-02-17
- Assignees: Qiagen GmbH
- Applicants: Qiagen GmbH
- Inventors: Dirk Löffert; Andreas Missel; Jie Kang
- Web page: https://patentgrep.com/patent/US20140154748A1

## 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}
- C07K 2319/00: Fusion polypeptide
- 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)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Novel thermostable chimeric nucleic acid polymerases and methods for their generation and use are disclosed. It is shown that these chimeric nucleic acid polymerases, such as DNA polymerases, can be constructed using enzymatically active domains, isolated from different proteins or chemically synthesized. It is demonstrated that chimeric nucleic acid polymerases of the present invention possess the chemical and physical properties of their component domains (e.g., exonuclease activity, thermostability) and that the chimeric polymerases are thermostable.

## Claims

### Claim 1

A method of synthesizing a recombinant nucleic acid encoding a thermostable chimeric nucleic acid polymerase having two, non-naturally associated, enzymatically active domains, the method comprising:
(a) isolating a first nucleic acid encoding a first enzymatically active domain;
(b) isolating a second nucleic acid encoding a second enzymatically active domain, wherein said second enzymatically active domain is non-naturally associated with said first enzymatically active domain; and
(c) combining said first nucleic acid and said second nucleic acid to form said recombinant nucleic acid encoding said thermostable chimeric nucleic acid polymerase.

### Claim 2 (depends on 1)

The method of claim 1, wherein said first enzymatically active domain is a 3′-5′ exonuclease domain.

### Claim 3 (depends on 1)

The method of claim 1, wherein said second enzymatically active domain is a 5′-3′ polymerase domain.

### Claim 4 (depends on 1)

The method of claim 1, wherein said isolating in (a) and (b) comprises amplifying the first nucleic acid and the second nucleic acid by polymerase chain reaction (PCR) with a PCR primer comprising a first nucleotide sequence complementary to a terminal region of a 3′-5′ exonuclease domain of said first nucleic acid and a second nucleotide sequence complementary to a terminal region of a 5′-3′ polymerase domain of said second nucleic acid.

### Claim 5 (depends on 1)

The method of claim 1, wherein said combining comprises hybridizing said first nucleic acid to said second nucleic acid to form a composite polynucleotide template, and amplifying said composite polynucleotide template to form said recombinant nucleic acid encoding said thermostable chimeric nucleic acid polymerase.

### Claim 6 (depends on 2)

The method of claim 2, wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Pho DNA polymerase.

### Claim 7 (depends on 6)

The method of claim 6, wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 396 of Pho DNA polymerase (SEQ ID NO:3).

### Claim 8 (depends on 2)

The method of claim 2, wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Pwo DNA polymerase.

### Claim 9 (depends on 8)

The method of claim 8, wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 396 of Pwo DNA polymerase (SEQ ID NO:4).

### Claim 10 (depends on 8)

The method of claim 8, wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 421 of Pwo DNA polymerase (SEQ ID NO:5).

### Claim 11 (depends on 2)

The method of claim 2, wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Sso DNA polymerase.

### Claim 12 (depends on 11)

The method of claim 11, wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 508 of Sso DNA polymerase (SEQ ID NO:6).

### Claim 13 (depends on 2)

The method of claim 2, wherein said 3′-5′ exonuclease domain comprises a 3′-5′ exonuclease domain of Tpac DNA polymerase.

### Claim 14 (depends on 13)

The method of claim 13, wherein said 3′-5′ exonuclease domain comprises amino acid residues 1 to 395 of Tpac DNA polymerase (SEQ ID NO:16).

### Claim 15 (depends on 3)

The method of claim 3, wherein said 5′-3′ polymerase domain is a 5′-3′ polymerase domain of Taq DNA polymerase.

### Claim 16 (depends on 3)

The method of claim 3, wherein said 5′-3′ polymerase domain is a 5′-3′ polymerase domain of Tth DNA polymerase.

### Claim 17 (depends on 15)

The method of claim 15, wherein said 5′-3′ polymerase domain comprises amino acid residues 281 to 832 of Taq DNA polymerase (SEQ ID NO:1).

### Claim 18 (depends on 15)

The method of claim 15, wherein said 5′-3′ polymerase domain comprises amino acid residues 271 to 832 of Taq DNA polymerase (SEQ ID NO:7).

### Claim 19 (depends on 16)

The method of claim 16, wherein said 5′-3′ polymerase domain comprises amino acid residues 273 to 834 of Tth DNA polymerase (SEQ ID NO:2).