# US 5,352,600 A · Purified thermostable enzyme

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- Number: US 5,352,600 A
- Document ID: US5352600A
- Status: Granted
- Kind: A
- Application number: 07/971,798
- Filed: 1992-11-05
- Granted: 1994-10-04
- Earliest claimed: 1986-08-22
- Assignees: Hoffmann-La Roche Inc.
- Inventors: David H. Gelfand; Susanne Stoffel
- Web page: https://patentgrep.com/patent/US5352600A

## 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 9/1276: {RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- 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

A purified thermostable enzyme is obtained that has unique characteristics. Preferably the enzyme is isolated from the Thermus aquaticus species and has a molecular weight of about 86,000-95,000 daltons. The thermostable enzyme may be native or recombinant and may be used in a temperature-cycling chain reaction wherein at least one nucleic acid sequence is amplified in quantity from an existing sequence with the aid of selected primers and nucleotide triphosphates. The enzyme is preferably stored in a buffer containing non-ionic detergents that lends stability to the enzyme.

## Claims

### Claim 1

A purified thermostable template-dependent DNA polymerase from the species Thermus aquaticus, wherein said polymerase is substantially free of contaminating deoxyribonucleases and retains activity at 60.degree. C. to 80.degree. C. for after exposure to a temperature of 90.degree. C. for up to four minutes and has a specific activity of about 200,000 units/mg, wherein one unit corresponds to 10 nmoles of product synthesized on activated salmon sperm DNA in 30 minutes.

### Claim 2 (depends on 1)

The polymerase of claim 1 that is produced in a recombinant host cell.

### Claim 3

A purified thermostable template-dependent DNA polymerase from the species Thermus aquaticus, wherein said polymerase is substantially free of contaminating deoxyribonucleases and retains activity at 60.degree. C. to 80.degree. C. after exposure to a temperature of 90.degree. C. for up to four minutes and has a specific activity of about 20 units/pmole enzyme, wherein one unit corresponds to 10 nmoles of product synthesized on activated salmon sperm DNA in 30 minutes.

### Claim 4 (depends on 3)

The polymerase of claim 3 that is produced in a recombinant host cell.