JPH11318488A - Immobilization of nucleic acid and immobilized nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for immobilizing a nucleic acid for detection, purification or the like of a target nucleic acid in high reactivity by allowing the nucleic acid having functional groups A at both terminals, and a crosslinkable compound having plural functional groups B capable of binding to the functional group A to act on a carrier having the functional groups B immobilized on the surface thereof. SOLUTION: A nucleic acid having functional groups A at both terminals, and a cross-linkable compound having three or more functional groups B capable of binding to the functional group A are allowed to act on a solid carrier having the functional groups B immobilized on the surface thereof to provide the objective immobilized nucleic acid in the method for immobilizing the nucleic acid. The immobilized nucleic acid has a substantially infinitely formed three-dimensional network obtained by the bond of the nucleic acids constituting chain parts and the crosslinking compounds constituting branching parts, and immobilized on the solid carrier by the bond of some terminals of some of the nucleic acids constituting the network to the solid carrier. The immobilized nucleic acid can be utilized for various kinds of detection methods, purification methods, or the like, by utilizing hybridization of the nucleic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for immobilizing a nucleic acid and an immobilized nucleic acid. INDUSTRIAL APPLICABILITY The present invention can be used for various detection methods and purification methods utilizing nucleic acid hybridization.

[0002]

2. Description of the Related Art In a method using nucleic acid hybridization, for example, a method in which a target DNA is captured and detected using a probe DNA immobilized on a solid support, generally, a target in a solution is generally used. Nucleic acids are at low concentrations,
In addition, there is a problem that the single-stranded target nucleic acid easily anneals to its complementary nucleic acid before hybridizing with the immobilized nucleic acid.

[0003] For this reason, various attempts have been made to increase the reaction rate with the target nucleic acid by amplifying the immobilized nucleic acid, thereby increasing the sensitivity of target nucleic acid detection and the efficiency of target nucleic acid purification.

[0004] For example, in the invention of "anchors for immobilizing nucleic acid on a solid phase and a method for preparing the same" disclosed in Japanese Patent Application Laid-Open No. Hei 5-260972, a plurality of bonding groups provided at branched ends of an amplification group bonded to a solid support are disclosed. Discloses a solid-phased anchor in which nucleic acids are amplified in a so-called Christmas tree shape by binding nucleic acid probes to the respective anchors.

In the invention of "Large comb-shaped branched polynucleotide" according to Japanese Patent Publication No. 5-508862, a polynucleotide in which a plurality of polynucleotide probes are bonded in a comb-like manner and the probe is amplified in a so-called comb-like manner. Is disclosed.

[0006]

However, in the amplification of the immobilized nucleic acid probe according to these inventions, after all, the development range of the amplified nucleic acid probe is defined by the chain length of the amplification group or the polynucleotide skeleton. Yes, it is essentially finite. More practically, the development range of the nucleic acid probe is stopped near the surface of the solid phase carrier in the hybridization reaction solution.

For this reason, especially when high sensitivity is required,
For example, in a case where diagnosis and treatment are required as soon as possible in a bacterial test or the like in the medical field, it cannot be said that the enhancement of the sensitivity is sufficient.

Accordingly, the present invention solves the problem of further improving the sensitivity for detecting a target nucleic acid and the efficiency of purifying a target nucleic acid by expanding and amplifying an immobilized nucleic acid to be subjected to hybridization essentially infinitely in a reaction solution. Tasks to be done.

[0009]

Means for Solving the Problems (Structure of the First Invention) The structure of the first invention of the present application (the invention of claim 1) for solving the above-mentioned problems is that a nucleic acid having a functional group A at both ends is provided. Immobilizing the nucleic acid by allowing a cross-linking compound having three or more functional groups B that binds to the functional group A to act on a solid support having the functional group B immobilized on the surface thereof; It is a chemical method.

(Structure of the Second Invention) The structure of the second invention (the invention described in claim 2) for solving the above-mentioned problem is as follows.
The bond between the nucleic acid constituting the chain portion and the cross-linking compound constituting the branch portion forms an essentially infinite three-dimensional network, and one end of a part of the nucleic acid constituting the network. Is an immobilized nucleic acid in which the network is immobilized on the solid-phase carrier by binding to the solid-phase carrier.

[0011]

Operation and Effect of the Invention (Operation and Effect of First Invention) In the first invention, the following processes 1) to 4) are performed.

1) First, a functional group A at one end of a nucleic acid binds to a functional group B immobilized on the surface of a solid support, and then a cross-linking compound is attached to the functional group A at the other end of the nucleic acid. Is bonded to one of the three or more functional groups B. In addition, there is a possibility that the functional groups A at both ends of one molecule of nucleic acid are bonded to the two functional groups B and do not react with the cross-linking compound, but as a practical matter, the probability is small.

2) Next, the functional group A at one end of the other two or more nucleic acids binds to the remaining two or more functional groups B of the cross-linking compound, and the functional group A at the other end of these nucleic acids binds to the functional group A at the other end. On the other hand, one of the three or more functional groups B of the crosslinking compound is bonded. In addition, there is a possibility that the functional groups A at both ends of the other one molecule of nucleic acid bind to the remaining two or more functional groups B of the cross-linking compound and do not react with the next cross-linking compound,
The probability is small.

3) In the process in which the nucleic acid and the cross-linking compound are extended in a substantially infinite chain bond by such a reaction, the cross-linking compound has three or more functional groups B. Nucleic acids linked from functional groups B having different surfaces are linked via the same cross-linking compound, and a network is formed in a three-dimensional network.

4) Thus, the formation of a three-dimensional network, which is immobilized on the solid support and is essentially infinitely developed, is performed until the nucleic acid and the crosslinking compound released in the reaction solution are consumed. By adjusting the dose of the nucleic acid and the cross-linking compound, it is easy to cover the network throughout the hybridization reaction solution.

(Function / Effect of the Second Invention) The immobilized nucleic acid of the second invention can contain an essentially infinite amount of a nucleic acid portion to be subjected to hybridization with a target nucleic acid, or incorporates such a nucleic acid portion. Since the entire network can be covered in the entire hybridization reaction solution, the reaction rate with the target nucleic acid can be maximized.

Further, since the nucleic acids are fixed in a three-dimensional network, a space between the nucleic acids can be secured even if the nucleic acids are fixed at a high concentration as compared with the Christmas tree type or comb type structure. Thus, the target nucleic acid can easily enter the network and hybridize with the immobilized nucleic acid.

As a result, the sensitivity of target nucleic acid detection and the efficiency of target nucleic acid purification can be further improved as compared with the above-mentioned conventional techniques.

[0019]

Next, embodiments of the first invention and the second invention will be described. In the following, the term “the present invention” simply refers to the first invention and the second invention collectively.

[Use of the Present Invention] The present invention relates to a method for detecting a target nucleic acid in a sample solution, capturing the target nucleic acid in a sample solution for other purposes by utilizing a hybridization reaction of a nucleic acid immobilized on a solid support. , Can be used without limitation. As a detection label for detecting a target nucleic acid, any known labeling method may be used.

A typical application of the present invention is a technique for detecting the presence or absence of a nucleic acid fragment having a specific base sequence in a prepared sample solution by using a hybridization reaction of a probe DNA for a bacterial test, genetic diagnosis, or the like. And D
Examples include separation / purification techniques for NA-binding proteins.

[Nucleic acid] The nucleic acid used in the present invention must have at least a complementary base sequence portion to be subjected to hybridization with a target nucleic acid and functional groups A at both ends. However, it may contain an arbitrary constituent part, for example, a compound part for introducing the functional group A, and the like.

As a typical nucleic acid, DNA is used.
NA can also be used.

The functional group A provided at both ends of the nucleic acid may be a functional group having a property of forming a covalent bond, an ionic bond or a hydrogen bond with the functional group B provided in the cross-linking compound. In particular, it is preferable that they form a covalent bond with each other in terms of bond strength that can withstand the washing operation of the solid support after the hybridization reaction. When the functional groups A and B bind under specific reaction conditions, it is necessary to adjust the reaction conditions in the first invention.

As a typical example of the functional group A, when the functional group B is an amino group, a carboxyl group bonded to the amino group by a carbodiimide reaction, and when the functional group B is a carboxyl group, Each of the amino groups bonded by the carbodiimide reaction is exemplified.

For the introduction of a carboxyl group into a nucleic acid, a carboxyl group is linked to the 3 'end of the nucleic acid by, for example, reacting a mononucleotide with a carboxyalkylamine hydrochloride or the like in advance, and connecting the mononucleotide to the 3' end of the nucleic acid. It can be carried out by a method of binding to the terminal. The method can be carried out by introducing an amino group into the 5 'end of the nucleic acid and then bonding one carboxyl group of the dicarboxylic acid compound to the amino group.

In addition, as a functional group A forming a covalent bond with the functional group B, an amino group bonded to the functional group B (thiol group) using N-succinimidyl-3- (2-pyridyldithio) propionate; A carboxyl group or the like bonded to the functional group B (hydroxyl group) by a carbodiimide reaction can also be used.

[Cross-Linking Compound] The cross-linking compound of the present invention is not particularly limited in chemical structure as long as it has three or more functional groups B bonded to the functional group A. Functional group B
Is selected without any limitation in correspondence with the functional group A. When the functional group B is, for example, an amino group, typical examples of the crosslinking compound include 1,4,7,10-tetraazacyclododecane-tetrahydrochloride and the like.

[Solid Phase Carrier] The solid phase carrier is arbitrarily determined depending on the use of the present invention, for example, the wall of a plastic plate or other container, or solid beads. The functional group B needs to be fixed on the surface of the solid support.
There is no limitation on the fixing means. For example, the functional group B can be chemically bonded to the surface of the solid phase carrier. Organic compounds can be adsorbed and immobilized by hydrophobic binding force.

The reaction of the first invention, that is, a solid support having a functional group B immobilized on its surface, a nucleic acid having a functional group A on both ends, and three or more functional groups B binding to the functional group A The reaction for reacting with the provided cross-linking compound can be performed in a liquid phase,
Although it is more preferable because of high action efficiency, it is not limited to this.

[Network] The network of the second invention is a three-dimensional network which is essentially infinitely developed by the bond between the nucleic acid constituting the chain and the cross-linking compound constituting the branch. It is immobilized on a phase carrier. These bonding and immobilization are based on the bonding between the functional groups A and B.

The network only needs to be a three-dimensional structure having an essentially infinitely expandable structure. In practice, the network may be expanded almost infinitely, or may be covered over the entire hybridization reaction solution. You don't have to.

[0033]

Next, an embodiment of the present invention will be described.

( 1. Introduction of carboxyl group to dUTP )
ON) in a recovery flask 10 mL, of water 1mL carboxymethoxylamine hydrochloride 26 mg (0.23 m mo
l) was dissolved. To this, 42 mg (0.09 mmol) of dideoxyuridine triphosphate (ddUTP) was added, and 5
By heating and stirring at 0 ° C. for 3 hours, ddUTP into which a carboxyl group had been introduced was generated as shown in the following “Formula 1”.

[0035]

Embedded image Next, the reaction solution was subjected to high performance liquid chromatography, and the peak of the target substance was collected. The yield is 29 mg (60
%)Met.

( 2. 3 'end, 5' end of probe DNA
Introduction of a carboxyl group to the end ) Using a Boehringer's 5 'end labeling kit "DIG oligonucleotide 5' end label set", use a DNA synthesizer to prepare sample DN.
A having a base sequence capable of hybridizing to A and 5 ′
A probe DNA whose terminal was substituted with an amino group was synthesized.

Next, the above-mentioned carboxyl group-introduced ddUTP was bound to the 3 ′ end of the probe DNA, which is an oligonucleotide, using a Boehringer 3 ′ end labeling kit “DIG oligonucleotide 3 ′ end label set”. .

Further, one of the carboxyl groups of sodium glutamate, which is a dicarboxylic acid compound, is bonded to the amino group at the 5 'end of the probe DNA by a carbodiimide reaction, thereby converting the functional group at the 5' end into a carboxyl group. did.

( 3. Netting on plastic plate container wall
A ) A solution of 1 g of n-hexylamine, a saturated primary amine, dissolved in 20 mL of water was added to a 96-well plastic plate at 100 μm each, and the plate was allowed to stand overnight at 37 ° C. The above n-
Hexylamine was adsorbed. Thereafter, it was washed with water and dried.

(4. Formation of three- dimensional network ) Into the plastic plate of 2. Probe D
10 μL of a 10 μg / mL aqueous solution of NA, 1,4,7,10-tetraazacyclododecane as a crosslinking compound
10 mg / mL aqueous solution of tetrahydrochloride
μL, 1 mg each of dicyclohexylcarbodiimide and N-hydroxysuccinimide as crosslinking agents
10 μL / water solution and 50 μL of water were added, and the mixture was allowed to stand at room temperature overnight to form a three-dimensional network of nucleic acids according to the second invention. Thereafter, it was washed with water and dried.

( 5. Detection of Target Nucleic Acid ) Immobilized probe DNA that forms a three-dimensional network formed on a plastic plate
To DIG by PCR.
(Digoxigenin) -labeled sample DNA was prepared as shown in Table 1.
As shown in Table 7, the dosage was divided into seven doses in the range of 0.1 to 20 ng / plate to cause a hybridization reaction.

Thereafter, an anti-DIG antibody labeled with alkaline phosphatase was reacted, and the immobilized alkaline phosphatase was detected by measuring the absorbance at 405 nm using paranitrophenyl phosphate.

As a control, a detection test similar to that described above was conducted using Nucleolink (trade name) manufactured by Nunc, which is a product related to the conventional nucleic acid amplification solid phase technology. Table 1 shows the test results. In Table 1, ○ indicates that there was a significant difference in absorbance with respect to the blank (water), and X indicates that there was no significant difference in absorbance with respect to the blank.

From the results shown in Table 1, it was found that the examples of the present invention exhibited a detection sensitivity five times that of the control example.

[0045]

[Table 1]

Claims (2)

[Claims] 1. A nucleic acid having a functional group A at both ends and a cross-linking compound having three or more functional groups B binding to the functional group A are mixed with a solid support having the functional group B immobilized on the surface thereof. A nucleic acid immobilization method, wherein the nucleic acid is immobilized by allowing the nucleic acid to act. 2. The bond between the nucleic acid constituting the chain portion and the cross-linking compound constituting the branch portion forms an essentially infinite three-dimensional network, and the nucleic acid constituting the network. Immobilized nucleic acid, wherein the network is immobilized on the solid-phase carrier by binding one end of the nucleic acid to the solid-phase carrier.