JPH02215399A - Detection of nucleic acid - Google Patents

Abstract

PURPOSE:To accomplish the title detection simply, readily and in high sensitivity by modifying the target DNA into single stranded DNA, binding DNA primer to this single stranded DNA, and by immobilizing the resultant target DNA replicated product on a water-insoluble carrier. CONSTITUTION:(A) A chemically modified oligonucleotide is first prepared by acting a diamino compound of the formula (m and n are each 1-12) on 10-30 short stranded oligonucleotides having both base sequence of the target DNA such as in vivo etiologic gene and the complementary base sequence. Second, (B) DNA primer is prepared by reaction between the component A, a DNA- immobilizing compound and a crosslinking agent. Third, the component B is added to (C) modified single stranded DNA formed by alkali treatment in an aqueous medium to produce (D) replicated DNA. Fourth, (E) a DNA-immobilized water insoluble carrier is obtained hy reaction of the component D with a carrier such as acrylamide gel. Fifth, the component E is stained with a light- absorptive or fluorescent DNA-staining agent (e.g. ethidium bromide) followed by determining the extinction coefficient or fluorescence intensity of the resulting stained DNA, thus accomplishing the objective detection of nucleic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application This invention relates to a method for detecting nucleic acids. For more details,
The present invention relates to a detection method that can easily detect target DNA and is useful for, for example, detection of pathogenic genes that may exist in living organisms, genetic research, and the like.

(b) Conventional technology In order to detect specific DNA, oligonucleotides having a complementary base sequence to the target DNA are used and labeled with fluorescent substances, radioactive isotopes, enzymes, etc. Methods using nucleotide probes are known.

Such conventional DNA detection methods basically involve a step of subjecting a DNA-containing sample such as a specimen or a cell disruption solution to alkali treatment or heat treatment to denature the target DNA into single-stranded DNA.
The process of immobilizing wood fiber DNA on a support such as nylon or nidomiculose membrane, and the immobilized single mlD
After the step of hybridization by reacting the above-mentioned oligonucleotide probe with NA and washing the membrane after hybridization, the immobilized DNA is removed.
It consists of the step of detecting DNA based on the fluorescence intensity, radioactivity, oxygen activity, etc. of the labeled site of A.

Recently, as a highly sensitive detection method for trace amounts of target DNA, a short chain (usually 10 to 30
A DNA primer consisting of an oligonucleotide (nucleotides) is bound to this, and d
ATP, dGTP.

The target DNA is replicated by applying a nucleotide reagent such as dCTP or dTTP, and this process is repeated as necessary to amplify the target DNA.The amplified target DNA is denatured to single strand again and detected as described above. A method has also been proposed for manipulating the so-called PCR (poly
This method is known as a DNA detection method based on the merase chain reaction method.

(c) Problems to be solved by the invention However, in any case, in the conventional detection method described above, when immobilizing a single DNA on a support, it is necessary to perform heat treatment or ultraviolet irradiation treatment, and special equipment is required. Not only is this necessary, but it also has the disadvantage of being complicated and time-consuming to handle. Furthermore, since hybridization is required after immobilization, it is necessary to maintain the immobilized DNA at an optimal temperature, and as mentioned above, a dedicated device is required.
Handling was complicated.

This invention was made under such circumstances, and the purpose is D.
NA can be easily detected, especially without hybridization or conventional immobilization on a support.
The present invention attempts to provide a detection method that can perform NA detection.

(d) Means for solving the problem Thus, according to this invention, (a) target DNA and one
1 DNA, (b) the above - wood fiber DNA,
(c) binding a DNA primer consisting of a short oligonucleotide having a base sequence complementary to a part of the base sequence of the target DNA and having a functional group for fixing DNA via a disulfide bond; A step of producing a replica of the target DNA modified with a DNA fixing functional group by reacting a nucleotide reagent with a single-stranded DNA bound to a DNA primer in the presence of a polymerase (
d) immobilizing the target DNA on the water-insoluble carrier by causing the replicated target DNA to act in an aqueous medium on a water-insoluble carrier having a functional group capable of reacting and bonding with the DNA-fixing functional group; (e) a step of staining the DNA by applying a light-absorbing or fluorescent DNA stain to the DNA-immobilized water-insoluble carrier in an aqueous medium; (r) staining the DNA;
A nucleic acid detection method is provided, which comprises the step of detecting a target DNA based on the absorbance or fluorescence intensity of the target DNA.

The first feature of this invention is that when detecting DNA, target DNA is replicated using a DNA primer having a functional group for fixation at the end via a disulfide bond. The second feature is that the replicated DNA containing a functional group for fixation is fixed on a water-insoluble carrier and then stained, and the target DNA is detected based on the absorbance or fluorescence of the stained DNA. That is.

(t) NA primer) The DNA primer used in this invention (a short oligonucleotide having a base sequence complementary to a part of the base sequence of the target DNA, and a functional group for fixing the DNA through a disulfide bond) It can be produced by combining.

Here, the short oligonucleotide is about the same length as the DNA primer used in the PCR method, usually 10
It is suitable to use something with a length of about 30 nucleotides, and it is suitable that the base sequence corresponds to the end of the target DNA.

When introducing the DNA fixing functional group into such an oligonucleotide, chemical modification with a disulfide group is first performed. This chemical modification can be carried out by treating the oligonucleotide with a diamino compound such as cystamine, which has a disulfide bond and amino groups at both ends, in an aqueous system. The diamino compounds used here include the following formula (I): HJ (CHt)--35-(CHt), -NH*
It is suitable to use a compound represented by (1) (where m and n each represent an integer of ! to 12) 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.

Introduction to the 5' end is usually achieved by treating the oligonucleotide with polynucleotide kinase in an aqueous solution.
This can be carried out by the so-called phosphoramidate method, in which the terminal hydroxyl group is converted into a phosphoric acid group, and then the phosphoric acid group is reacted with a diamino compound under mild conditions using, for example, a carbodiimide-based condensing agent. (Chu, B.
F,etal,! fuel.

Aclds, Res, 1l (1983) 6513). This reaction causes a dehydration condensation reaction between the OH group of the phosphoric acid group at the 5' end and the amino group at one end of the diamino compound,
The diamino compound residue is introduced into the oligonucleotide as shown in the following formula.

On the other hand, the introduction into the nucleobase is the well-known cytosine 4
This can be carried out by utilizing an amino group rearrangement reaction at the position. This reaction causes a dehydration condensation reaction between the oxy group in the nucleic acid base of the oligonucleotide and the amino group at one end of the diamino compound, and a diamino compound residue is introduced into the oligonucleotide as shown in the following formula, for example.

The chemically modified oligonucleotide obtained in the reaction solution in step 2 is usually purified by liquid chromatography, electrophoresis, etc., and used in the step of binding a DNA fixing functional group.

Here, the binding of the functional group for DNA immobilization is carried out by easily reacting the chemically modified oligonucleotide obtained above with the functional group of the water-insoluble carrier described below in an aqueous medium using a crosslinking agent as necessary. Compounds with other functional groups that can be used (DN
This can be carried out by reacting with A fixing compound). Examples of the combination of the NA fixing compound and the functional group of the water-insoluble carrier include a biotin/avidin combination and various antibody/antigen combinations. When using biotin, biotin derivatives that are reactive with amino groups or thiol groups are suitable. Also,
When using a hapten (antigenic group), one having a thiol group or an amino group is suitable, or a crosslinking agent having reactivity with an amino-reactive group [for example, N-succinimide-3-( 2-pyridylthio)propionate and maleimide-N-hydroxysuccinimide derivatives].

Such a binding reaction is usually suitably carried out by reacting the above-mentioned DNA fixing compound with a crosslinking agent as necessary in an aqueous system, and then reacting it with the above-mentioned chemically modified oligonucleotide in an aqueous system.

Either reaction can be allowed to proceed by mixing under mild conditions.

Note that the amount of the crosslinking agent and the DNA fixing compound to be used is preferably 5 to 25 equivalents relative to the chemically modified oligonucleotide.

(DNA Replication) The thus obtained DNA immobilizing functional group-containing D
Target DNA from one mj DNA using NA primer
is copied. Such a DNA primer is (+)
Two types (-) can also be used at the same time. This replication procedure and method can be performed in the same manner as the replication procedure in the so-called PCR method. In other words, one target DNA is isolated by alkali treatment or heat treatment in an aqueous medium @DN
After denaturation into A, the above DNA primers were added and annealed at a mild temperature, followed by polymerase and sequentially a nucleotide reagent (dATP).

dGTP, dCTP, dTTP) to make one 11D
This can be done by growing complementary polynucleotides using NA as a template. Note that the target DNA itself may be one that has been amplified by the PCR method, and it is preferable to use one that has been amplified in terms of high-sensitivity detection.

(Fixation) As the carrier for immobilizing the replicated DNA, a water-insoluble carrier having another functional group capable of reacting and bonding with the DNA fixing functional group in the replicated DNA is used. As a material for such a carrier, it is suitable to use a granular material that can be dispersed in an aqueous medium, such as a granular material made of an organic polymer such as acrylamide gel or Sepharose gel, or a granular material made of an inorganic material such as silica gel. One example is the body.

Further, as the functional group of the above-mentioned pond, avidin residues and antibodies as described above can be used, and these can be used in a state in which they are fixed to the above-mentioned carrier by chemical fixation, physical fixation, etc. by known methods. .

The immobilization of the replicated DNA on the carrier is quickly carried out by mixing them in an aqueous system at a mild temperature, and there is no need for heat treatment, ultraviolet irradiation, or the like.

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

(Staining) In this invention, a DNA stain refers to a water-soluble substance that has light absorption or fluorescence and is rapidly incorporated into DNA double strands in an aqueous medium, and in particular has light absorption in visible light. It doesn't have to be something you have. Generally, from the viewpoint of high sensitivity detection, it is suitable to use a fluorescent material, such as ethidium bromide, acridine orange, etc.

After such a staining step, DNA detection is performed, but before this detection, washing is usually performed to efficiently remove excess staining agent.

(DNA Detection) Detection is performed optically based on absorbance or fluorescence measurements of stained DNA. This optical detection involves transferring the carrier on which the stained DNA is immobilized to a suitable support (e.g.
It can also be carried out with the stained DNA supported on filter paper, glass filters, etc.), but from the viewpoint of high sensitivity detection,
A reducing agent is applied to the immobilized water-insoluble carrier in an aqueous medium to cleave the disulfide bonds of the immobilized DNA to separate the stained DNA from the water-insoluble carrier, and the aqueous medium containing the separated stained DNA is optically analyzed. It is preferable to subject it to analysis and measure its absorbance or fluorescence. Examples of the reducing agent used in this case include dithiothreitol, mercaptoethanol, and mercaptoethylamine.

(e) Function: By using a DNA primer having a functional group for immobilizing DNA that specifically binds to a water-insoluble carrier, replication D
It should be possible to efficiently immobilize NA on the carrier. DNA is detected using a staining method, and the stained D
Since NA is fixed on an insoluble carrier, it can be easily separated from contaminant components by washing with water.

Therefore, DNA can be easily detected and detected with high sensitivity without performing complicated fixation treatment on a support or heat treatment as in the conventional methods, and without performing complicated hybridization.

(to) Example 1, Chemical modification of oligonucleotides (1) Oligonucleotides As oligonucleotides, 1, primer for M13mp8 (5'-GTAAAACGACGGCCAGT
-3' and 5' -TTGTGTGGAATTGTG
AGC-3'') was chemically synthesized using a Shimadzu automatic synthesizer N5-1 and purified by HPLC.
17-mer or 18-mer with a sequence complementary to 13mp8RF-DNA (target DNA) (+) strand or (-)@
It is a quantity.

In addition, as a template, ML311)8 RF ID
When the PCB reaction was carried out using the two types of primers described above as NA primers, an electrophoretic pattern corresponding to the position of about 120 mer was obtained. This result agreed with the predicted position.

(2) Reaction ■Introduction of phosphate group Dissolution of the above oligonucleotide? &(50D, 30Mg)
treated with 30 units of T4 polynucleotide kinase (
37°C for 2.5 hours), and a phosphoric acid residue was introduced into the 5' position. The reaction solution was purified using a SEP PAK C" column to obtain three eluates. The eluates were concentrated in vacuo under shaking.

■Introduction of cystamine residue The 5'-position phosphorylated probe (IOD) was added to lutidine buffer (pH 7, 5,0,75M, 88m
12) In cystamine [HtN-(Cut)! -5
-3-(CHI) Jut] dihydrochloride (IM, 2μQ
) was reacted at room temperature overnight. The reaction solution was subjected to HPLC to collect a target peak, and concentrated in vacuo under a tube.

As a result, a chemically modified (introduced cystamine) oligonucleotide shown in the following formula was obtained.

2. Introduction of functional group for DNA fixation Cystamine-introduced oligonucleotide obtained above (0
, 700) in PRSLGO village, add 25 times equivalent amount of biotin-ε-aminocaproic acid-N-hydroxysuccinimide under stirring, and react at room temperature for 2 hours to obtain the following 5°-biotinylated primer. was synthesized and purified by HPLC or gel electrophoresis.

(Left below) Be/\3, Replication of target DNA M13mp 8 RF-DNA A 1 amol (I
The 5'-biotinylated primer (50 μmol) and unmodified primer (50 μmol) synthesized above, Taq polymerase, and the nucleotide reagent dNTP (dATP% dCT) were used as target DNA.
P1dGTP, dTTP) was added thereto, and this was amplified 40 times by PCR. This meant that the 5°-biotinylated primer was bound to the double-stranded DNA fragment amplified by the PCR reaction.

4. Immobilized avidin on a water-insoluble carrier and directly cross-linked agarose beads with lx ss
After washing twice with C, it was added to the above biotin-modified DNA-containing aqueous solution. Since the binding constant between biotin and avidin is very large, the biotin-modified DNA is quickly transferred to the beads (water-insoluble carrier) at room temperature by adding and mixing the above gel.
It will be fixed at

5. DNA staining After the above fixation, the immobilized product was stained with a pore size of 0.45μ.
- Collect unreacted single-stranded DNA by filtering it with a filter and washing thoroughly with buffer.

The primer and its impurities were removed.

Next, staining was performed by bringing the immobilized product into contact with an aqueous solution of acridine orange. Excess acridine orange was removed by washing thoroughly with water after staining.

6. Detection of DNA The stained DNA carried on the filter as described above
The immobilizer was treated with mercaptoethylamine (0
, 1M) was added, and the mixture was reacted at 37°C for 90 minutes. As a result, the disulfide bonds in the immobilized stained DNA were rapidly cleaved, the stained DNA was dispersed and dissolved in the liquid phase, and was separated from the water-insoluble carrier.

When the fluorescence intensity (Ex 490 nm) of this DNA-containing aqueous solution was measured using a fluorometer, it was found to be 5.
Clear fluorescence peaks were confirmed at 30 nm and 640 nm. The intensity ratio of both peaks and peaks is 530ns>6
4011, and the intensity ratio of the acridine orange aqueous solution itself was reversed, so it was confirmed that this was due to acridine orange trapped in the double strands of DNA.

On the other hand, when ethidium bromide is used as a staining agent, a fluorescence peak of 590ns is exhibited at Ex 300n, but this fluorescence peak intensity is increased compared to the aqueous solution of ethidium bromide bound to the present JiDNA. It was also confirmed that this was caused by ethidium bromide entrapped in double-stranded DNA.

(g) Effects of the invention According to the nucleic acid detection method of the present invention, there is no need for complicated fixation or hybridization as in conventional methods.
The target DNA can be detected easily, automation and deviceization are easy, and detection with higher sensitivity than conventional methods is also possible.

June 30, 1989 1. Display of the case 1999 Patent Application No. 35670 2. Name of the invention Nucleic acid detection method 3. Person making the amendment Relationship to the case Patent applicant address Nishinokyo Kurihara, Nakagyo-ku, Kyoto City Town number 1 name
(199) Shimadzu Corporation Representative Yohebee Minoru 4, Agent address: 5-1-3 Nishitenma, Kita-ku, Osaka 530 Quarter One Building Contents of amendment 1, 2nd line from the bottom of page 4 of the specification: Target DNA and”
Correct the target DNA to ``.

2. In the same book, page 6, lines 15-16, "It is suitable to use, and the base sequence should correspond to the end of the target DNA." was corrected to "It is suitable to use." do.

3. r 100m12J in the bottom third line of the formula on page 16 of the circular
is corrected to "100μi".

4. In the fourth line from the bottom of page 19 of the same book, "double-stranded DNA" is corrected to "double-stranded DNA."

5. In the bottom line of the same page of the same book, "(g) Effect of the invention" is corrected to "(g) Effect of the invention."

6. Scope of patent claims subject to amendment 1. (a) A step of denaturing each target DNA into 11 DNA; (b) The above-mentioned wood fiber DNA is made of a short oligonucleotide that develops a base sequence complementary to a part of the base sequence of the target DNA. a step of binding a DNA primer having a functional group for DNA fixation via a disulfide bond; (c) fixing the DNA by causing a nucleotide reagent to act on the single-stranded DNA bound to the DNA primer in the presence of a polymerase; (d) producing a replica of the target DNA modified with a functional group; (d) the replicated target DNA;
A step of immobilizing the target DNA on the water-insoluble carrier by causing A to act in an aqueous medium on a water-insoluble carrier having a functional group capable of reacting and bonding with the DNA-immobilizing functional group, (e) the DNA-immobilized water; (r) detecting the target DNA based on the absorbance or fluorescence intensity of the stained DNA; A method for detecting nucleic acids.

Claims (1)

[Claims] 1. (a) A step of denaturing the target DNA into single-stranded DNA; (b) adding a short nucleotide sequence to the single-stranded DNA that is complementary to a part of the base sequence of the target DNA; (c) bonding a DNA primer consisting of a stranded oligonucleotide and having a functional group for fixing DNA via a disulfide bond; (d) producing a replica of the target DNA modified with a DNA fixing functional group by causing the target DNA to act on the target DNA;
a step of immobilizing the target DNA on the water-insoluble carrier by causing A to act in an aqueous medium on a water-insoluble carrier having a functional group capable of reacting and bonding with the DNA-immobilizing functional group; (e) the DNA-immobilized water; (f) detecting the target DNA based on the absorbance or fluorescence intensity of the stained DNA; A method for detecting nucleic acids.