# US 7,745,124 B2 · Hybridization method

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- Number: US 7,745,124 B2
- Document ID: US7745124B2
- Status: Granted
- Kind: B2
- Application number: 11/579,063
- Filed: 2005-04-28
- Published: 2008-10-09
- Granted: 2010-06-29
- Earliest claimed: 2004-04-28
- Assignees: Eisai R&D Management Co., Ltd.
- Inventors: Chikako Hakii; Mitsugu Usui
- Web page: https://patentgrep.com/patent/US7745124B2

## CPC

- C12Q 1/682: Signal amplification
- C12Q 1/6832: Enhancement of hybridisation reaction
- B01L 7/54: {using spatial temperature gradients}
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

A hybridization method is provided in which an efficient hybridization reaction can be carried out. Further, there are provided, using this hybridization method, a method for detecting a target gene with high sensitivity and a signal amplifying method for markedly improving the detection sensitivity of the target gene. There is provided a hybridization method comprising the use of oligonucleotides in a reaction solution, the method comprising forming partially a reaction temperature region in the reaction solution and performing a hybridization reaction in the reaction temperature region.

## Claims

### Claim 1

A hybridization method comprising,
performing a hybridization reaction in a reaction solution, wherein the hybridization reaction is a self-assembly reaction forming a double-stranded self-assembly substance by hybridizing plural kinds of oligonucleotide probes having complementary base sequence regions hybridizable to each other to self-assemble the oligonucleotide probes, and
wherein the reaction solution has a high temperature region and a low temperature region and the hybridization reaction preferentially occurs in the high temperature region.

### Claim 2 (depends on 1)

The hybridization method according to claim 1, wherein the plural kinds of oligonucleotide probes are a pair of oligonucleotide probes consisting of a first probe and a second probe, the first probe having at least three nucleic acid regions, a nucleic acid region X, a nucleic acid region Y and a nucleic acid region Z in order from 5′-end and having the structure of the following chemical formula (1), and the second probe having at least three nucleic acid regions, a nucleic acid region X′, a nucleic acid region Y′ and a nucleic acid region Z′ in order from 5′-end and having the structure of the following chemical formula (2)
[Chemical formula 1]
(1)
[Chemical formula 2]
(2)
(In the above chemical formulae (1) and (2), each of X-X′, Y-Y′ and Z-Z′ is complementary nucleic acid regions hybridizable to each other).

### Claim 3 (depends on 1)

The hybridization method according to claim 1, wherein the plural kinds of oligonucleotide probes comprise:
a dimer forming probe series having n groups of plural pairs of dimer forming probes consisting of a first group to, optionally, a (2n−1)-th (n≧1) group in order, in which each of a pair of No. 1 and No. 2 oligonucleotides is divided to 3 regions of a 3′-end region, a middle region and a 5′-end region, a base sequence of the middle region of each oligonucleotide is complementary to each other, and a base sequence of the 3′-end region and a base sequence of the 5′-end region of each oligonucleotide are not complementary to each other; and
a cross-linking probe series having n groups of plural pairs of cross-linking probes consisting of a second group to, optionally, a 2n-th group in order, in which each of a pair of No. 1 and No. 2 oligonucleotides is divided to 2 regions of a 3′-end region and a 5′-end region, and a base sequence of the 3′-end region and a base sequence of the 5′-end region of each oligonucleotide are not complementary to each other; and
wherein the base sequence of the cross-linking probe is cross-linkable with plural pairs of dimers formed from the dimer forming probes.

### Claim 4 (depends on 3)

The hybridization method according to claim 3, wherein in the case of n=1 each of the following pairs of the base sequences of the probes is complementary to each other: the 3′-end region of No. 1 oligonucleotide of the first group and the 3′-end region of No. 1oligonucleotide of the second group, the 5′-end region of No. 2 oligonucleotide of the first group and the 5′-end region of No. 2oligonucleotide of the second group, the 3′-end region of No. 2 oligonucleotide of the second group and the 3′-end region of No. 2oligonucleotide of the first group, and the 5′-end region of No. 1 oligonucleotide of the second group and the 5′-end region of No. 1oligonucleotide of the first group.

### Claim 5 (depends on 4)

The hybridization method according to claim 4, wherein in the case of n =1 each of the following pairs of the base sequences of the probes is complementary to each other: the 3′-end region of No. 1 oligonucleotide of the first group and the 3′-end region of No. 1oligonucleotide of the second group, the 5′-end region of No. 2 oligonucleotide of the first group and the 5′-end region of No. 1oligonucleotide of the second group, the 3′-end region of No. 2 oligonucleotide of the first group and the 3′-end region of No. 2 oligonucleotide of the second group, and the 5′-end region of No. 1 oligonucleotide of the first group and the 5′-end region of No. 2oligonucleotide of the second group.

### Claim 6 (depends on 3)

The hybridization method according to claim 3, wherein in the case of n≧2 each of the following pairs of the base sequences of the probes is complementary to each other: the 3′-end region of No. 1 oligonucleotide of the (2n−3)-th group and the 3′-end region of No. 1-oligonucleotide of the (2n−2)-th group, the 5′-end region of No. 2 oligonucleotide of the (2n−3)-th group and the 5′-end region of No. 2-oligonucleotide of the (2n−2)-th group, the 3′-end region of No. 2 oligonucleotide of the (2n−2)-th group and the 3′-end region of No. 2-oligonucleotide of the (2n−1)-th group, the 5′-end region of No. 1 oligonucleotide of the (2n−2)-th group and the 5′-end region of No. 1-oligonucleotide of the (2n−1)-th group, the 3′-end region of No. 1 oligonucleotide of the last group of the dimer forming probe series and 3′-end region of No. 1 oligonucleotide of the last group of the cross-linking probe series, the 5′-end region of No. 2 oligonucleotide of the last group of the dimer forming probe series and the 5′-end region of No. 2 oligonucleotide of the last group of the cross-linking probe series, the 3′-end region of No. 2 oligonucleotide of the last group of the cross-linking probe series and the 3′-end region of No. 2 oligonucleotide of the first group of the dimer forming probe series, and the 5′-end region of No. 1 oligonucleotide of the last group of the cross-linking probe series and the 5′-end region of No. 1 oligonucleotide of the first group of the dimer forming probe series.

### Claim 7 (depends on 3)

The hybridization method according to claim 3, wherein in the case of n≧2 each of the following pairs of the base sequences of the probes is complementary to each other: the 3′-end region of No. 1-oligonucleotide of the (2n−3)-th group and the 3′-end region of No. 1oligonucleotide of the (2n−2)-th group, the 5′-end region of No. 2 oligonucleotide of the (2n−3)-th group and the 5′-end region of No. 2oligonucleotide of the (2n−2)-th group, the 3′-end region of No. 2 oligonucleotide of the (2n−2)-th group and the 3′-end region of No. 2oligonucleotide of the (2n−1)-th group, the 5′-end region of No. 1 oligonucleotide of the (2n−2)-th group and the 5′-end region of No. 1oligonucleotide of the (2n−1)-th group, the 3′-end region of No. 1 oligonucleotide of the last group of the dimer forming probe series and the 3′-end region of No. 1 oligonucleotide of the last group of the cross-linking probe series, the 5′-end region of No. 2 oligonucleotide of the last group of the dimer forming probe series and the 5′-end region of No. 1 oligonucleotide of the last group of the cross-linking probe series, the 3′-end region of No. 2 oligonucleotide of the last group of the cross-linking probe series and the 3′-end region of No. 2 oligonucleotide of the first group of the dimer forming probe series, and the 5′-end region of No. 2-oligonucleotide of the last group of the cross-linking probe series and the 5′-end region of No. 1 oligonucleotide of the first group of the dimer forming probe series.

### Claim 8 (depends on 1)

The hybridization method according to claim 1, wherein the plural kinds of oligonucleotide probes comprise:
plural pairs of dimer forming probes having plural groups of dimer forming probes consisting of a first group to a k-th (k≧2) group in order, in which each of a pair of No. 1 and No. 2 oligonucleotides is divided to 3 regions of a 3′-end region, a middle region and a 5′-end region, a base sequence of the middle region of each oligonucleotide is complementary to each other, and a base sequence of the 3′-end region and a base sequence of the 5′-end region of each oligonucleotide are not complementary to each other; and
wherein each of the following pairs of the base sequences of the probes is complementary to each other:
(a) the 3′-end region of No. 1 oligonucleotide of the (k−1)-th group and the 3′-end region of No. 2 oligonucleotide of the k-th group,
(b) the 5′-end region of No. 2 oligonucleotide of the (k−1)-th group and the 5′-end region of No. 1 oligonucleotide of the k-th group,
(c) the 3′-end region of No. 1 oligonucleotide of the last group and the 3′-end region of No. 2oligonucleotide of the first group,
(d) the 5′-end region of No. 2 oligonucleotide of the last group and the 5′-end region of No. 1oligonucleotide of the first group.

### Claim 9 (depends on 1)

The hybridization method according to claim 1, wherein the plural kinds of oligonucleotide probes comprise:
plural pairs of dimer forming probes having plural groups of dimer forming probes consisting of a first group to a k-th (k≧2) group in order, in which each of a pair of No. 1 and No. 2 oligonucleotides is divided to 3 regions of a 3′-end region, a middle region and a 5′-end region, a base sequence of the middle region of each oligonucleotide is complementary to each other, and a base sequence of the 3′-end region and a base sequence of the 5′-end region of each oligonucleotide are not complementary to each other; and wherein each of the following pairs of the base sequences of the probes is complementary to each other: and
wherein each of the following pairs of the base sequences of the probes is complementary to each other:
(a) the 3′-end region of No. 1-oligonucleotide of the (k−1)-th group and the 3′-end region of No. 2-oligonucleotide of the k-th group,
(b) the 5′-end region of No. 1-oligonucleotide of the (k−1)-th group and the 5′-end region of No. 2-oligonucleotide of the k-th group,
(c) the 3′-end region of No. 1-oligonucleotide of the last group and the 3′-end region of No. 2-oligonucleotide of the first group,
(d) the 5′-end region of No. 1-oligonucleotide of the last group and the 5′-end region of No. 2-oligonucleotide of the first group.

### Claim 10 (depends on 1)

The hybridization method according to claim 1, wherein the oligonucleotide comprises at least one kind of nucleotide selected from the group consisting of DNA, RNA, PNA and LNA.

### Claim 11 (depends on 1)

The hybridization method according to claim 1, wherein at least one of the oligonucleotide is labeled with a labeling substance.

### Claim 12 (depends on 1)

The hybridization method according to claim 1, wherein the labeling substance is a radioisotope, a fluorescent substance, a luminous substance, a coloring substance, a coloring enzyme or a luminous enzyme.

### Claim 13 (depends on 1)

A signal amplifying method, wherein the detection sensitivity of a target gene in a reaction device for detecting a gene is improved using a self-assembly reaction forming a double-stranded self-assembly substance by hybridizing plural kinds of oligonucleotide probes having complementary base sequence regions hybridizable to each other to self-assemble the oligonucleotides, and wherein the self-assembly reaction is performed using the hybridization method according to claim 1.

### Claim 14 (depends on 13)

The signal amplifying method according to claim 13, wherein the reaction device is a microplate, a DNA microarray or a magnetic particle.

### Claim 15 (depends on 13)

The signal amplifying method according to claim 13, wherein the target gene is single-stranded DNA and/or RNA.

### Claim 16 (depends on 13)

The signal amplifying method according to claim 13, wherein the target gene is double-stranded DNA and/or RNA.

### Claim 17 (depends on 13)

The signal amplifying method according to claim 13, wherein the target gene includes SNPs (Single Nucleotide Polymorphisms).

### Claim 18 (depends on 13)

The signal amplifying method according to claim 13, wherein at least one of the oligonucleotide probes to be used for the self-assembly reaction has a complementary base sequence at a part of the target gene.

### Claim 19 (depends on 13)

The signal amplifying method according to claim 13, wherein there is used an assist probe having a region complementary to each base sequence of the target gene and the oligonucleotide probe to bind the target gene to the oligonucleotide probe.

### Claim 20 (depends on 13)

The signal amplifying method according to claim 13, wherein the oligonucleotide probe comprises at least one kind of polynucleotide selected from the group consisting of DNA, RNA, PNA and LNA.

### Claim 21 (depends on 13)

The signal amplifying method according to claim 13, wherein at least one of the oligonucleotide probe is labeled with a labeling substance.

### Claim 22 (depends on 21)

The signal amplifying method according to claim 21, wherein the labeling substance is a radioisotope, a fluorescent substance, a luminous substance, a coloring substance, a coloring enzyme or a luminous enzyme.

### Claim 23 (depends on 13)

The signal amplifying method according to claim 13, wherein a labeled probe is hybridized to the self-assembly substance bound to the target gene to detect the presence of the self-assembly substance.

### Claim 24 (depends on 23)

The signal amplifying method according to claim 23, wherein the labeled probe is labeled with a coloring enzyme, a luminous enzyme or a radioisotope.

### Claim 25 (depends on 13)

The signal amplifying method according to claim 13, wherein a fluorescent substance bindable to a nucleic acid is added to the self-assembly substance and the presence of the self-assembly substance is detected by a photochemical change of the fluorescent substance.

### Claim 26 (depends on 13)

A method for detecting a target gene, the method comprising the use of the signal amplifying method according to claim 13.

### Claim 27 (depends on 1)

A method for detecting a target gene, the method comprising the use of a hybridization reaction of oligonucleotides in a reaction solution to detect a gene, wherein the hybridization reaction includes a hybridization reaction carried out using the hybridization method according to claim 1 in a reaction device.

### Claim 28 (depends on 27)

The method for detecting a target gene according to claim 27, wherein the reaction device for detecting a gene is a microplate, a DNA microarray or an magnetic particle.

### Claim 29 (depends on 27)

The method for detecting a target gene according to claim 27, wherein at least one of the oligonucleotides to be used for the hybridization reaction has a complementary base sequence to a part of the target gene.

### Claim 30 (depends on 27)

The method for detecting a target gene according to claim 27, wherein the target gene is single-stranded DNA and/or RNA.

### Claim 31 (depends on 27)

The method for detecting a target gene according to claim 27, wherein the target gene is double-stranded DNA and/or RNA.

### Claim 32 (depends on 27)

The method for detecting a target gene according to claim 27, wherein the target gene has SNPs (Single Nucleotide Polymorphisms).