# US 11,111,526 B2 · Buffer composition for hybridization and hybridization method

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- Number: US 11,111,526 B2
- Document ID: US11111526B2
- Status: Granted
- Kind: B2
- Application number: 16/322,363
- Filed: 2017-08-02
- Published: 2019-05-30
- Granted: 2021-09-07
- Earliest claimed: 2016-08-03
- Assignees: Toyo Kohan Co., Ltd.; Yamaguchi University
- Applicants: Toyo Kohan Co., Ltd.; Yamaguchi University
- Inventors: Toshiya Tsuda; Shuichi Kamei; Mitsuyoshi Oba; Hirofumi Yamano; Ryouichi Tsunedomi; Shoichi Hazama; Hiroaki Nagano
- Web page: https://patentgrep.com/patent/US11111526B2

## CPC

- C12Q 1/6832: Enhancement of hybridisation reaction
- C12N 15/09: Recombinant DNA-technology
- C12Q 1/68: involving nucleic acids
- C12M 1/00: Apparatus for enzymology or microbiology
- 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)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}

## Abstract

In the case of using a blocking nucleic acid to prevent non-specific hybridization of a target nucleic acid with a nucleic acid probe, further excellent efficiency of detecting the target nucleic acid is achieved. A buffer composition used in hybridization of a target nucleic acid with a nucleic acid probe, wherein the buffer composition for hybridization contains a blocking nucleic acid comprising a nucleotide sequence complementary to a region comprising at least a non-detection target nucleotide in a non-target nucleic acid, in a concentration of one or more times higher than the concentration of a nucleic acid in a nucleic acid mixture consisting of the target nucleic acid and the non-target nucleic acid.

## Claims

### Claim 1

A method of hybridizing a target nucleic acid comprising a detection target nucleotide with a nucleic acid probe comprising a nucleotide sequence complementary to a region comprising at least the detection target nucleotide in the target nucleic acid, wherein
the method comprises: mixing a solution comprising a nucleic acid mixture consisting of the target nucleic acid and a non-target nucleic acid comprising a non-detection target nucleotide corresponding to the detection target nucleotide, with a buffer composition for hybridization containing a blocking nucleic acid comprising a nucleotide sequence complementary to a region comprising at least the non-detection target nucleotide in the non-target nucleic acid, in a concentration 1 to 3 times higher than the concentration of both the target and non-target nucleic acids in the nucleic acid mixture; and then hybridizing the nucleic acid probe with the target nucleic acid,
wherein the solution comprising the nucleic acid mixture contains the target nucleic acid at a percentage of 0.5% to 10%, when the total percentage of the target nucleic acid and the non-target nucleic acid is set at 100%, and
wherein the nucleic acid mixture contains the target nucleic acid in a concentration of 0.66 nM to 3.3 nM.

### Claim 2 (depends on 1)

The hybridization method according to claim 1, wherein a mixed solution prepared by mixing the buffer composition for hybridization with the solution comprising the nucleic acid mixture is allowed to come into contact with a microarray formed by immobilizing the nucleic acid probes on a substrate.

### Claim 3 (depends on 1)

The hybridization method according to claim 1, wherein the solution comprising the nucleic acid mixture is a reaction solution obtained after completion of a nucleic acid amplification reaction for amplifying the target nucleic acid, and the reaction solution is mixed with the buffer composition for hybridization.