JP2727625B2 - Nucleic acid detection method - Google Patents

Description

The present invention relates to a method for detecting a nucleic acid. More specifically, the present invention relates to a detection method that can easily detect a target DNA and is useful for, for example, detection of a pathogenic gene that may exist in a living body, genetic research, and the like.

(B) Conventional technology For detection of a specific DNA, an oligonucleotide having a base sequence complementary to the target DNA is used and labeled with a fluorescent substance, a radioisotope, an enzyme, or the like. Methods using nucleotide probes are known.

Such a conventional DNA detection method basically comprises a step of subjecting a DNA-containing sample such as a specimen or a cell lysate to alkali treatment or heat treatment to denature the target DNA into single-stranded DNA.
Was immobilized on a support such as nylon or nitrocellulose membrane, the step of allowing the oligonucleotide probe described above to act on the immobilized single-stranded DNA to perform hybridization, and washing the hybridized membrane. Thereafter, the method comprises the steps of detecting DNA based on the fluorescence intensity, radioactivity, oxygen activity and the like of the labeling site of the immobilized DNA.

Recently, as a highly sensitive detection method for a trace amount of target DNA, a short-chain (usually 10 to 30 nucleotides) oligo having a base sequence complementary to a part of the base sequence of the target DNA before the immobilization step is used. A DNA primer consisting of nucleotides was bound, and dATP, dG
TP, dCTP, replicate the target DNA by acting nucleotide reagents such as dTTP, repeat this as necessary to amplify the target DNA, denature the amplified target DNA to single-stranded again A method for performing the above detection operation has also been proposed, which is called PCR (polymerase chain reactio).
It is known as a DNA detection method by the n) method.

(C) Problems to be Solved by the Invention However, in any case, in the above-mentioned conventional detection method, heat treatment or ultraviolet irradiation treatment must be performed when single-stranded DNA is immobilized on a support, and a dedicated device is required. , And the handling is complicated and time-consuming. Furthermore, since hybridization is required after immobilization, it is necessary to maintain the immobilized DNA at an optimum temperature. As in the above case, a dedicated apparatus is required, and handling is complicated.

The present invention has been made under such circumstances,
DNA can be easily detected, and especially without the need for hybridization or conventional immobilization to a support.
It is intended to provide a detection method capable of performing A detection.

(D) Means for Solving the Problems Thus, according to the present invention, (a) the target DNA is a single-stranded D
Denaturing to NA, (b) the single-stranded DNA is composed of a short-chain oligonucleotide having a base sequence complementary to a part of the base sequence of the target DNA, and via a disulfide bond
A step of binding a DNA primer having a functional group for DNA immobilization, (c) a single-stranded DNA to which the DNA primer has been bound,
A step of preparing a copy of the target DNA modified with the functional group for DNA immobilization by allowing a nucleotide reagent to act in the presence of a polymerase, (d) reacting the duplicated target DNA with the functional group for DNA immobilization The target DNA is allowed to act on a water-insoluble carrier having a functional group capable of binding
Is immobilized on a water-insoluble carrier, (e) a step in which a DNA-absorbing or fluorescent DNA stain is applied to the DNA-immobilized water-insoluble carrier in an aqueous medium to stain the DNA, (f) the stained DNA Detecting the target DNA based on the absorbance or the fluorescence intensity of the nucleic acid.

The first feature of the present invention is to replicate a target DNA using a DNA primer having a functional group for immobilization via a disulfide bond at the end when detecting DNA. Then, staining was performed with the replicated fixing functional group-containing DNA fixed to a water-insoluble carrier, and the stained
The second feature is that the target DNA is detected based on the absorbance or the fluorescence of the DNA.

(DNA primer) The DNA primer used in the present invention is obtained by bonding a DNA-immobilizing functional group via a disulfide bond to a short-chain oligonucleotide having a base sequence complementary to a part of the base sequence of the target DNA. Can be made.

Here, as the short oligonucleotide, the same length as the DNA primer used in the PCR method, usually 10-30
Suitable are those of the order of nucleotides.

In introducing the above DNA-immobilizing functional group into such an oligonucleotide, first, chemical modification with a disulfide group is performed. This chemical modification can be performed by allowing a diamino compound such as cystamine having a disulfide bond and having an amino group at each end to act on the oligonucleotide in an aqueous system. As the diamino compound used herein, the following formula (I): H ₂ N— (CH ₂ ) _m —SS— (CH ₂ ) _n —NH ₂ (wherein, m and n are each Or a salt thereof.

In this chemical modification, the diamino compound residue is introduced into the oligonucleotide, and the introduction position can be selected either at the 5 'end of the oligonucleotide or at the nucleobase.

The introduction to the 5 'end is usually carried out by reacting the oligonucleotide with a polynucleotide kinase in an aqueous solution to convert the hydroxyl group at the 5' end into a phosphate group, and then under mild conditions, for example, carbodiimide-based condensation. The phosphoric acid group is reacted with a diamino compound using a solubilizing agent, so-called phosphoramidate method (Chu.BF
etal, Nucl. Acids. Res. 11 (1983) 6513). By such a reaction, a dehydration condensation reaction between the OH group of the phosphate group at the 5 ′ terminal and the amino group at one end of the diamino compound occurs, and the diamino compound residue is introduced into the oligonucleotide as shown in the following formula. Become.

On the other hand, introduction into a nucleobase can be carried out by utilizing a well-known amino group transposition reaction at the 4-position of cytosine. Such a reaction causes a dehydration condensation reaction between the oxy group in the nucleobase of the oligonucleotide and the amino group at one end of the diamino compound, thereby introducing a diamino compound residue into the oligonucleotide, for example, as shown in the following formula.

The chemically modified oligonucleotide thus obtained in the reaction solution is usually used in a step of purifying by a liquid chromatography, an electrophoresis method or the like and binding a DNA-immobilizing functional group.

Here, the bonding of the functional group for DNA immobilization is carried out by easily reacting the chemically modified oligonucleotide obtained above with a functional group of a water-insoluble carrier, which will be described later, in an aqueous medium using a crosslinking agent as necessary. Compounds with other functional groups (DNA
(Fixing compound). Examples of such a combination of the DNA fixing compound and the functional group of the water-insoluble carrier include a biotin / avidin combination and various antibody / antigen combinations. When using bition, a bition derivative having reactivity with an amino group or a thiol group is suitable. When a hapten (antigen group) is used, those having a thiol group or those having an amino group are suitable, or a crosslinking agent having a reactivity with an amino-reactive group [for example, N
-Succinimide-3- (2-pyridylthio) propionate and maleimide-N-hydroxysuccinimide derivatives].

It is suitable that such a binding reaction is usually carried out by reacting the above-mentioned compound for immobilizing a DNA with a crosslinking agent in an aqueous system, if necessary, and then reacting the compound with the above-mentioned chemically modified oligonucleotide in an aqueous system. Either reaction can proceed by mixing under mild conditions.

The amount of the cross-linking agent and the compound for immobilizing DNA is preferably 5 to 25 equivalents to the chemically modified oligonucleotide.

(Replication of DNA) DNA having functional group for DNA immobilization thus obtained
The target DNA is replicated from the single-stranded DNA using the primers. Such DNA primers can also be used simultaneously (+) and (-). This replication procedure and method can be performed in the same manner as the replication procedure in the so-called PCR method. That is, after denaturing the target DNA into single-stranded DNA by alkali treatment or heat treatment in an aqueous medium, the DNA
Annealing is performed under mild temperature by adding primers, and then polymerase and nucleotide reagents (dAT
P, dGTP, dCTP, dTTP) and growing a complementary polynucleotide chain using single-stranded DNA as a template. The target DNA itself may be amplified by the PCR method, and it is preferable to use the amplified DNA in terms of high-sensitivity detection.

(Immobilization) As the carrier for immobilizing the replicated DNA, a water-insoluble carrier having another functional group capable of reacting with and binding to the DNA immobilization functional group in the replicated DNA is used. As the material of such a carrier, it is suitable to use a granular material that can be dispersed in an aqueous medium, for example, a granular material composed of an organic polymer such as acrylamide gel or sepharose gel, or a granular material composed of an inorganic substance such as silica gel. Body. Also,
As the other functional group, as described above, an avidin residue or an antibody can be used, and these can be used in a state of being fixed to the above-mentioned carrier by a known method, such as a chemical fixing method or a physical fixing method.

The immobilization of the replicated DNA on the above-mentioned carrier is carried out promptly by mixing them in an aqueous system at a moderate temperature, and it is not particularly necessary to perform a heat treatment or an ultraviolet irradiation treatment.

After this immobilization, contaminant components and unreacted components can be efficiently removed from the system by washing with water, and the following dyeing step is usually performed after the washing.

(Staining) In the present invention, the DNA staining agent refers to a water-soluble substance which has a light absorbing property or a fluorescent property and is rapidly incorporated into a DNA double strand in an aqueous medium. It does not need to have. Usually, it is suitable to use a substance having fluorescence in terms of high sensitivity detection, and examples thereof include ethidium bromide and acridine orange.

After such a staining step, detection of DNA is performed. Usually, washing is performed before this detection, whereby excess dye can be efficiently removed.

(DNA detection) Detection is performed chemically based on the absorbance or fluorescence measurement of the stained DNA. This chemical detection was stained
A carrier on which DNA is immobilized is placed on a suitable support (eg, filter paper,
It can be carried out in the state of being supported on a glass filter, etc., but from the viewpoint of high-sensitivity detection, a reducing agent is allowed to act on the stained DNA-immobilized water-insoluble carrier in an aqueous medium, and the disulfide bond of the immobilized DNA is Is preferably cleaved to separate stained DNA from the water-insoluble carrier, and the separated stained DNA-containing aqueous medium is subjected to optical analysis to measure its absorbance or fluorescence. Examples of the reducing agent used at this time include dithiothreitol, mercaptoethanol, mercaptoethylamine and the like.

(E) Operation By using a DNA primer having a DNA-immobilizing functional group capable of specifically binding to a water-insoluble carrier, replicated DNA can be efficiently immobilized on the carrier. DNA is detected by a staining method. Since the stained DNA is fixed on an insoluble carrier, contaminants and the like can be easily separated by washing with water.

Therefore, simple detection of DNA and high-sensitivity detection can be performed without performing complicated fixing treatment or heat treatment on a support as in the related art and without performing complicated hybridization.

(F) Example 1. Chemical modification of oligonucleotides (1) Oligonucleotides Oligonucleotides include primers for M13mp8 (5'-GTAAAACGACGGCCAGT-3 'and 5'-TTGTGTG
GAATTGTGAGC-3 ') was chemically synthesized with Shimadzu NS-1 and then purified by HPLC. This primer is
3mp8RF-DNA (target DNA) 17-mer or 18-mer having a sequence complementary to the (+) or (-) strand.

When a PCR reaction was performed using the above two primers as the M13mp8RF I DNA primer as a template, an electrophoresis pattern corresponding to the position of about 120 mer was obtained. This result was consistent with the expected position.

(2) Reaction Introduction of phosphate group A solution (50D, 30μ) of the above oligonucleotide was added to T ₄
Treated with 30 units of polynucleotide kinase (37 ℃ 2.5
Time) to introduce a phosphate residue at the 5'-position. Reaction solution
Purification was performed using a SEP PAK ^C18 column to obtain 3 ml of an eluate. The eluate was concentrated under vacuum with shaking.

Introduction of cystamine residue The 5′-position phosphorylated probe (10D) was added to a lutidine buffer (pH 7.5) in the presence of 1-ethyl-3- (dimethylaminopropyl) carbodiimide (ECDI) (1M 10 μ) as a condensing agent. , Cystamine [H ₂ N
_{- (CH 2) 2 -S-} S- (CH 2) 2 2 hydrochloride NH _2] (1M,
2μ) and room temperature overnight. The reaction solution was used to collect the desired peak by HPLC, and concentrated under vacuum with shaking.

Thus, a chemically modified (cystamine-introduced) oligonucleotide represented by the following formula was obtained.

2. Introduction of DNA-immobilizing functional group The cystamine-introduced oligonucleotide (0.7 OD) obtained above was dissolved in 100 µ of PBS, and 25-fold equivalent of biotin-ε-aminocaproic acid-N-hydroxysuccinimide was added with stirring. By reacting at room temperature for 2 hours, the following 5'-biotinylated primer was synthesized and purified by HPLC or gel electrophoresis.

3. Using the replication M13mp8RF-DNA1a mol object DNA (10 ^-18 mol) purposes DNA, synthetic and 5'-biotinylated primer above (50 pm
ol) and unmodified primer (50 pmol), Taq polymerase, nucleotide reagent dNTP (dATP, dCTP, dGTP, dTTP)
Was added and amplified 40 times by the PCR method. This means that the 5'-bitionized primer has been bound to the double-stranded DNA fragment amplified by the PCR reaction.

4. Immobilization on water-insoluble carrier Agarose beads directly cross-linked with avidin are 1 × SSC
Washed twice with was added to the aqueous solution containing biotin-modified DNA. Since the binding constant between biotin and avidin is very large, biotin-modified DN
A is immediately fixed to the beads (water-insoluble carrier) at room temperature.

5. Staining of DNA After the above-mentioned immobilization, this immobilized product was
The unreacted single-stranded DNA, primers, and other contaminants were removed by filtering with a filter described above and washing thoroughly with a buffer.

Then, the immobilized product was brought into contact with an aqueous solution of acridine orange to make contact. Excess acridine orange was removed by washing thoroughly with water after staining.

6. Detection of DNA Stained DNA carried on filter as described above
Mercaptoethylamine (0.
A phosphate buffer solution containing 1M) was added and reacted at 37 ° C. for 90 minutes.
As a result, the disulfide bonds in the stained DNA immobilized product are rapidly cleaved, and the stained DNA is dispersed and dissolved in the liquid phase,
Separated from water-insoluble carrier.

When the fluorescence (Ex490nm) of this DNA-containing aqueous solution was measured using a fluorimeter, it was 530nm and 640nm.
A clear fluorescence peak was confirmed. The intensity ratio of both peaks is 530 nm> 640 nm, and since the intensity ratio of the acridine orange aqueous solution itself is reversed, it may be due to acridine orange trapped in the double strand of DNA. confirmed.

On the other hand, when ethidium bromide was used as the staining agent, a fluorescence peak of 590 nm was exhibited at Ex300 nm, and the intensity of the fluorescence peak was increased as compared with the ethidium bromide aqueous solution bound to single-stranded DNA. It was also confirmed that it was due to ethidium bromide trapped in double-stranded DNA.

(G) Effects of the Invention According to the nucleic acid detection method of the present invention, the target DNA can be easily detected without performing a complicated immobilization treatment or hybridization as in the conventional method, and furthermore, the automation and the apparatus Therefore, detection with higher sensitivity than before can be performed.

Claims (1)

(57) [Claims] 1. A step of (a) denaturing a target DNA into a single-stranded DNA; and (b) a short-chain oligonucleotide having a base sequence complementary to a part of the base sequence of the target DNA in the single-stranded DNA. A step of binding a DNA primer consisting of nucleotides and having a functional group for DNA immobilization via a disulfide bond, (c) allowing a nucleotide reagent to act on the single-stranded DNA bound to the DNA primer in the presence of a polymerase (D) water-insoluble having a functional group capable of reacting and binding the replicated target DNA with the DNA fixing functional group. Immobilizing the target DNA on a water-insoluble carrier by allowing the carrier to act on the carrier in an aqueous medium; and (e) absorbing or fluorescent D on the DNA-immobilized water-insoluble carrier.
A nucleic acid detection method comprising: a step of causing the NA stain to act in an aqueous medium to stain the DNA; and (f) a step of detecting a target DNA based on the absorbance or fluorescence intensity of the stained DNA.