# US 10,501,813 B2 · Enzyme preparation containing thermostable DNA polymerase, method for producing same, and method for detecting subject organism to be detected

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- Number: US 10,501,813 B2
- Document ID: US10501813B2
- Status: Granted
- Kind: B2
- Application number: 15/688,473
- Filed: 2017-08-28
- Published: 2018-03-01
- Granted: 2019-12-10
- Earliest claimed: 2009-01-15
- Assignees: Hokkaido Mitsui Chemicals Inc.; National University Corporation University of Toyama
- Applicants: Hokkaido Mitsui Chemicals Inc.; National University Corporation University of Toyama
- Inventors: Homare Tabata; Hiroshi Minami; Hideki Niimi; Isao Kitajima; Tomohiro Ueno; Shiroh Hayashi; Masashi Mori
- Web page: https://patentgrep.com/patent/US10501813B2

## CPC

- C12Q 1/689: for bacteria
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/68: involving nucleic acids
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6895: for plants, fungi or algae
- 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)
- 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)}
- C12Y: ENZYMES

## Abstract

Disclosed is a thermostable DNA polymerase preparation which can illimitably reduce the risk of false positivity in the detection of a subject microorganism utilizing a gene amplification reaction and therefore enables the selective amplification of DNA for detecting the subject microorganism even when the amount of the subject microorganism is small and therefore the amount of DNA collected therefrom is extremely small, and can be produced at a reduced cost. Also disclosed is a method for quantifying or quantifying/identifying a subject organism to be detected rapidly, conveniently and with high sensitivity using the preparation of the present invention.

## Claims

### Claim 1

A production method for a thermostable DNA polymerase preparation, comprising the steps of:
(1) culturing transformant eukaryotic cells selected from the group consisting of fungi and plant cells carrying a gene encoding the thermostable DNA polymerase,
(2) obtaining an extract containing the thermostable DNA polymerase from the cultured transformant eukaryotic cells, followed by subjecting the extract to heat treatment, or
subjecting the cultured transformant eukaryotic cells to heat treatment, followed by obtaining an extract containing the thermostable DNA polymerase from the heat-treated transformant eukaryotic cells,
wherein the gene encoding the thermostable DNA polymerase comprises DNA consisting of the base sequence of SEQ ID NO:1.

### Claim 2 (depends on 1)

The production method according to claim 1, wherein the heat treatment is performed at a temperature of 50° C. or more.

### Claim 3 (depends on 1)

The production method according to claim 1, wherein the gene encoding the thermostable DNA polymerase used for the transformation is derived from a thermophilic bacterium or a hyperthermophilic bacterium.

### Claim 4 (depends on 1)

The production method according to claim 1, wherein the thermostable DNA polymerase preparation is that in which:
(1) the thermostable DNA polymerase has contamination with 10 fg or less of bacterially-derived nucleic acid other than the gene encoding the thermostable DNA polymerase based on 1 unit of the thermostable DNA polymerase, and
(2) no amplification products of the bacterially-derived nucleic acid are detected even when 32 cycles or more of gene amplification reaction are performed in the preparation under conditions containing no template using primers capable of amplifying only the bacterially-derived nucleic acid other than the gene encoding the thermostable DNA polymerase.

### Claim 5 (depends on 1)

The production method according to claim 1, wherein the fungi are yeasts or filamentous fungi.