# US 2018/0023116 A1 · Method for preparing single-stranded dna product

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- Number: US 2018/0023116 A1
- Document ID: US20180023116A1
- Status: Published application
- Kind: A1
- Application number: 15/547,442
- Filed: 2016-01-29
- Published: 2018-01-25
- Earliest claimed: 2015-01-30
- Assignees: Kurashiki Boseki Kabushiki Kaisha
- Applicants: Kurashiki Boseki Kabushiki Kaisha
- Inventors: Hiroki YAMASHITA
- Web page: https://patentgrep.com/patent/US20180023116A1

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/68: involving nucleic acids
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6862: Ligase chain reaction [LCR]
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 37/00: Details not covered by any other group of this subclass
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The purpose of the present invention is to provide the technique of a method for preparing a highly precise single-stranded DNA that makes it possible to obtain accurate results even by a simple test in a clinical sites or the like. The invention provides a highly precise single-stranded DNA product that can be utilized even in more simple and rapid genetic analyses by taking as the detection sample a single-stranded nucleotide product amplified by ligation, preferably by cycling ligation reaction, without PCR or LCR amplification.

## Claims

### Claim 1

A method for preparing a single-stranded DNA used for a genetic test, the method comprising:
hybridizing a common oligo and a query oligo on a target nucleic acid to perform a ligation reaction.

### Claim 2 (depends on 1)

The method for preparing a single-stranded DNA according to claim 1, wherein in the reaction, a cycle step of hybridization and ligation is repeated 2 to 100 times.

### Claim 3 (depends on 2)

The method for preparing a single-stranded DNA according to claim 2, wherein in the reaction, hybridization is performed about 10 seconds to 2 minutes and ligation is performed about 10 seconds to 2 minutes in one cycle.

### Claim 4 (depends on 1)

The method for preparing a single-stranded DNA according to claim 1, wherein a target DNA region is amplified by PCR, and then hybridization is performed on a PCR-amplified target DNA product to perform a ligation reaction.

### Claim 5 (depends on 1)

The method for preparing a single-stranded DNA according to claim 1, wherein the query oligo has (a) and (b) below:
(a) a first single-stranded oligonucleotide comprising an oligonucleotide complementary to the target nucleic acid; and
(b) a tag sequence or labeling moiety on the 5′-end of the first single-stranded oligonucleotide.

### Claim 6 (depends on 5)

The method for preparing a single-stranded DNA according to claim 5, wherein the number of bases of the first single-stranded oligonucleotide is 10 to 60 bases.

### Claim 7 (depends on 5)

The method for preparing a single-stranded DNA according to claim 5, wherein the common oligo has (a) and (b) below:
(a) a second single-stranded oligonucleotide comprising an oligonucleotide complementary to the target nucleic acid, and
(b) a tag sequence or labeling moiety on the 3′-end of the second single-stranded oligonucleotide.

### Claim 8 (depends on 7)

The method for preparing a single-stranded DNA according to claim 7, wherein the 5′-end of the common oligo is phosphorylated.

### Claim 9 (depends on 7)

The method for preparing a single-stranded DNA according to claim 7, wherein the number of bases of the second single-stranded oligonucleotide is 20 to 70 bases.

### Claim 10 (depends on 7)

The method for preparing a single-stranded DNA according to claim 7, wherein a length of a single-stranded DNA product to which the first single-stranded oligonucleotide and the second single-stranded oligonucleotide are ligated is 30 to 150 bases.

### Claim 11 (depends on 1)

A genetic testing method comprising using the single-stranded DNA prepared by the method for preparing a single-stranded DNA probe according to claim 1 to a strip.

### Claim 12 (depends on 1)

A genetic testing method comprising using the single-stranded DNA prepared by the method for preparing a single-stranded DNA probe according to claim 1 to a microarray.