JPH04325091A - Immobilizing nucleic acid to membrane - Google Patents

Abstract

PURPOSE:To efficiently immobilize not only a long-chain nucleic acid but also a short-chain nucleic acid to a membrane. CONSTITUTION:A basic acid, a mercapto group-containing compound and a nucleic acid are made to exist on a membrane of cellulose or polyamide-based synthetic resin, etc., and heat-treated at 30-80 deg.C. Since a short-chain nucleic acid can be immobilized to the membrane, a material having a probe for hybridization previously immobilized to a membrane can be prepared to simplify operation of hybridization.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for immobilizing nucleic acids onto cellulose or polyamide synthetic resin membranes.

0002

[Problems with the Prior Art] Conventionally, the homology of base sequences of nucleic acids has been investigated by techniques such as so-called colony hybridization, dot hybridization, Southern hybridization, and Northern hybridization. In these methods, the nucleic acid of the sample to be examined is first immobilized on a membrane made of nitrocellulose, nylon, or polyvinylidene fluoride, and a nucleic acid called a probe forms a hydrogen bond with the nucleic acid immobilized on these membranes. The presence or absence of the same or complementary sequence as the probe is determined by whether or not the probe hybridizes.

[0003] In these methods, a predetermined amount of nucleic acids such as genes to be tested is obtained, they are immobilized on a membrane, and a probe is then hybridized to the nucleic acids immobilized on the membrane. This is commonly done.

However, when performing a test using this procedure, it is necessary to immobilize the target nucleic acid on the membrane each time the test is performed or for each test target, and the operation is complicated.

[0005]

[Problem to be solved by the invention] On the other hand, if the probe is immobilized on the membrane in advance, it is possible to omit the operation of immobilizing the nucleic acid on the membrane each time a test is performed, and the examiner only has to carry out the hybridization operation. As a result, the above-mentioned complications are eliminated.

[0006] However, this method has been difficult to implement because the probe is usually composed of a short nucleic acid chain, which makes it difficult to immobilize it on a membrane or the like using conventional methods. As an improved method, a method has been adopted in which a base sequence that is not involved in the hybridization reaction is attached to the probe, thereby increasing the chain length and making it easier to adsorb onto the membrane. However, these methods use an enzymatic reaction or a nucleic acid elongation reaction by chemical synthesis, but these methods are not necessarily the best in terms of operation, time, and cost.

[0007]Furthermore, fixation by heating or other methods results in incomplete fixation, and even for long nucleic acids of about 1000 bases in length, only about 30 to 40% of the nucleic acids are fixed; In most cases, it is not fixed.

[0008] In order to overcome the drawbacks of such conventional methods, the present invention provides a method for efficiently immobilizing not only long-chain nucleic acids but also short-chain nucleic acids onto membranes, and which is simple and easy. The aim is to enable manual hybridization.

[0009]

[Means for Solving the Problems] As a result of intensive research to solve the above problems, the present inventors have found that nucleic acids can be immobilized on membranes by using a basic substance and a compound having a mercapto group. We have discovered this and have arrived at the present invention.

[0010] That is, the present invention is a method for immobilizing a nucleic acid on a membrane, and is characterized in that a basic substance, a compound having a mercapto group, and a nucleic acid are allowed to coexist on the membrane, and heat treatment is performed at a temperature of 30 to 80°C. The present invention relates to a method for immobilizing nucleic acids on a membrane, and a membrane on which nucleic acids are immobilized using this method.

The present invention will be explained in detail below. 1. Types of Membrane Used The membrane used in the present invention has a functional group capable of condensation, and has a continuous membrane for the solution of basic substances, nucleic acids, and compounds having mercapto groups to permeate and adhere. A membrane having a three-dimensional network structure with fine pores is preferred, but a film-like membrane may also be used. For example, a membrane containing cellulose or polyamide-based synthetic resin as a component is preferably used.

As the cellulose membrane, a filter paper can be used, and as the polyamide synthetic resin membrane, a commercially available nylon membrane for hybridization can be used. Examples include Photogene film (manufactured by BRL) and Hybond N film (manufactured by Amersham). 2. Condensation Reagent In the operation of fixing nucleic acids to a membrane, it is preferable that the nucleic acids be in a solution, and as a condensation reagent, it is preferable to use a reagent that can withstand reaction in an environment where nucleic acids are easily dissolved, that is, in an aqueous solution. Desirably, water-soluble substances are preferred.

For example, basic substances include metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, magnesium hydroxide, and calcium hydroxide;
(Trishydroxymethylaminomethane), amylamine, pyridine and other amine-based substances, preferably alkali metal hydroxides, and compounds having mercapto groups such as dithiothreitol, dithioerythritol, 1,2
Examples include -dimercaptoethane, 1,6-dimercaptohexane, 1,10-dimercaptodecane, 3,4-dimercaptotoluene, and dimercaptosuccinic acid.

[0014] At this time, the basic substance and the substance having a mercapto group are used in a molar ratio of 0.01 or more to the nucleic acid. The molar ratio of the basic substance to the substance having a mercapto group is not particularly limited, and the basic substance is usually used in excess. 3. Linker Although the use of a linker is not necessarily necessary for the present invention, the amount of nucleic acid immobilized can be increased by coexisting with a basic substance, mercapto compound, and nucleic acid when nucleic acid is immobilized on a membrane.

As the linker, any reagent capable of condensation reaction can be used, and examples thereof include compounds having a hydroxyl group, an amino group, a carboxyl group, a sulfonic acid group, etc. as a functional group.

Among these functional groups, hydroxyl groups are preferably used. It is estimated that hydrogen is extracted from these compounds by S radicals, generating radicals, acting as a chain transfer agent, and making it possible to increase the amount of radicals generated by the reaction between a basic substance and a substance having a mercapto group. be done.

Examples of compounds having a sulfonic acid group include benzenedisulfonic acid, P-hydroxybenzenesulfonic acid, and P-oxycarbonylbenzenesulfonic acid; examples of compounds having an amino group or polyamino group include amino acids such as leucine and glycine, or ethylenediamine. , as a compound having a carboxyl group, succinic acid,
Phthalic acid, oxycarboxylic acid, glycol carboxylic acid,
P-hydroxybenzoic acid, thioacetic acid, amino alcohol, P-hydroxyphenyl phosphoric acid as a compound having an amino group or phosphoric acid group in addition to a hydroxyl group, ethylene glycol, as a compound having a hydroxyl group,
Diethylene glycol, polyethylene glycol, resorcinol, hydroquinone, α-hydroxymethyl-P-
Polyhydric alcohols such as cresol can be mentioned.

A compound having a mercapto group in addition to the above functional group, such as 2-mercaptophenol, can serve as a linker as well as a condensing agent.

The amount of these compounds used can be varied depending on the amount of nucleic acid to be immobilized or the amount of condensing agent, but 0.0 to each amount of nucleic acid and condensing agent.
It is used in a range of 1 to 1000 times the mole. Preferably 0
．． It is used in a range of 0.01 to 100 times the mole, more preferably 0.01 to 5 times the mole. 4. Solvents to be used As mentioned above, in the operation of fixing nucleic acids to membranes,
In order to drop the nucleic acid onto the membrane, it is preferable that the nucleic acid is in the form of a solution, and water is suitable as the solvent, but other solvents may be used to increase the solubility of basic substances or compounds having mercapto groups. They can also be used together.

Examples of these solvents include alcohols such as methanol and ethanol, and amines such as triethylamine. These solvents can be used in a range that does not denature the membrane used. For example, alkali metal hydroxides such as sodium hydroxide and amines can be used at temperatures around room temperature;
In the above case, when polyamide resin is used for the membrane, it is desirable to avoid using strong alkaline compounds such as sodium hydroxide, since there is a risk that the polyamide bonds may be cleaved.

[0021] In the fixation operation, nucleic acids can be immobilized even if the membrane is kept in a moist state, such as by immersing it in a solution, or in a dry state. 5. Method of immobilization treatment A method of immobilizing nucleic acids on a membrane using the above reagents will be explained.

[0022] As the nucleic acid used in the present invention, DNA (
Both deoxyribonucleic acid (deoxyribonucleic acid) and RNA (ribonucleic acid) can be used, and there are no restrictions on the type of nucleic acid.

[0023] There is no restriction on chain length for immobilization;
Although it is possible to use a nucleic acid with a length of 1 base or less, if it is intended to be used for hybridization, a nucleic acid with a length of several bases to several thousand bases will be used.

[0024] A common method for attaching nucleic acids to a membrane is to drip a nucleic acid solution while suctioning it from the opposite side of the membrane, but with this method, short-chain nucleic acids are not attached to the membrane and are removed from the membrane. This is not necessarily a suitable method for attaching a predetermined amount to a membrane. In this method, it is desirable to spot while making adjustments so that it does not slip out, and to avoid waste.

Specifically, the method includes picking up the membrane with tweezers or the like, dropping the liquid, forming a spot of a predetermined size, drying it, and dropping the liquid again, or placing the membrane under heating and/or reduced pressure. However, a method is used in which a very small amount of solution is continuously dropped.

[0026] In order to adsorb the nucleic acid to the membrane, the order of an aqueous solution of a basic substance such as sodium hydroxide, a nucleic acid solution, and a compound having a mercapto group, or a nucleic acid solution, a basic substance, and a compound having a mercapto group is used. How to drip,
Any of the following methods can be adopted: a method in which a nucleic acid is mixed with a basic substance and then added dropwise, and then a compound having a mercapto group is added dropwise therein, or the reverse method is used, or a solution in which all of the components are mixed is added dropwise.

Furthermore, when using a linker, the linker can be bound in advance with a condensing reagent, and the nucleic acid can be immobilized by the method described above.

Among these methods, a method is preferred in which a basic substance is first attached to the membrane, and then a nucleic acid and a compound having a mercapto group are attached.

The condensation reaction is carried out at room temperature to 120°C, preferably room temperature to 80°C, over a period of 30 minutes to 24 hours. Alternatively, a membrane to which a nucleic acid is attached is immersed in a solution of a basic substance such as an aqueous sodium hydroxide solution, and a compound having a mercapto group is added to this solution. A method of immersing the membrane in a solution of a basic substance such as an aqueous sodium oxide solution, and a method of immersing a membrane to which a nucleic acid and a basic substance such as sodium hydroxide are attached in a solution of a compound having a mercapto group can also be adopted.

In these cases, the amount of solution should be at least enough to keep the membrane in a wet state;
It is used in a range of 0 μl to 500 μl. Also, the concentration of a solution of a basic substance such as sodium hydroxide is 0.01
M to 10M, and the concentration of the compound having a mercapto group is from 0.01M to 10M if this compound is not a liquid.
In the case of a liquid, it is used in a range of 0.01M to undiluted.

In conventional fixation methods, after nucleic acids are adsorbed onto a membrane, heat treatment or ultraviolet irradiation treatment is performed to fix the nucleic acids onto the membrane. In the present invention, a basic substance is used and nucleic acids are immobilized on a membrane through a condensation reaction that proceeds even at room temperature, so heat treatment or ultraviolet irradiation is not necessary. However, by using these methods in combination, the amount of immobilization can be reduced. In some cases, it may be possible to increase the number and secure the fixation more securely.

For example, a method in which treatment with a basic substance or mercapto compound is performed while heat treatment or ultraviolet irradiation treatment is performed, or a method in which nucleic acid is attached to the membrane in advance and treatment with a basic substance or mercapto compound is performed after heat treatment or ultraviolet irradiation treatment. Alternatively, after treatment with a basic substance or mercapto compound, heat treatment or ultraviolet irradiation treatment can be used.

[0033] When a membrane with immobilized nucleic acids is used for hybridization, and the nucleic acids to be immobilized on the membrane are double-stranded, it is also possible to denature the nucleic acids before adsorbing them to the membrane. However, it may be modified after adsorption and before the condensation reaction.

After fixing the nucleic acids to the membrane, the membrane was coated with Tris(
trishydroxymethylaminomethane) buffer, SSC
Buffer (succinic acid 90mM, sodium chloride 0.9M
By washing with an appropriate buffer such as pH 7.0), residual dehydration condensation agents, linkers, etc. can be removed from the membrane, so even if another nucleic acid is applied to the membrane after fixation, it will not be further immobilized. It never happens. 6. Method of using a membrane on which nucleic acids are immobilized by the method of the present invention (hybridization) As mentioned above, since the membrane can be washed after the fixation treatment, non-specific binding of nucleic acids other than the immobilized nucleic acids can be eliminated. The membrane on which nucleic acids are immobilized according to the present invention can be used for necessary operations such as hybridization operations.

[0035] In conventional fixation by adsorption, reagents used for hybridization such as SDS (sodium dodecyl sulfate) tend to peel off from the membrane, making it difficult to use. However, by using the fixation method of the present invention, Even with short-chain nucleic acids, such peeling becomes difficult to occur, and hybridization operations can be performed more reliably.

This hybridization operation is a commonly performed operation, and for example, T. Mani
The method of T. Maniatis et al.
Regular Cloning, A Labor
Atory Manual, Cold Spring
Harbor Laboratory (1982
), pages 387 to 389), the hybridization temperature or the salt concentration of the buffer used is changed as appropriate depending on the base length of the nucleic acid to be tested.

For example, TMAC solution (50mM Tr
is-HCl (pH 8.0), 3M tetramethylammonium chloride), 2mM EDTA, 5x Denhardt's solution (1x concentration is 0.02W/V% Ficoll, 0.02W/V% polyvinylpyrrolidone, 0. 0
Aqueous solution containing 2W/V% bovine serum albumin), 0.1
%SDS (sodium dodecyl sulfate), 100μg/m
The test is carried out in an aqueous solution containing denatured herring sperm DNA at an appropriate temperature.

According to the method of the present invention, in addition to immobilizing a nucleic acid to be a test specimen and hybridizing it with a probe nucleic acid, which has been generally done in the past, conversely, the probe nucleic acid is immobilized on a membrane, It also becomes possible to hybridize nucleic acids that serve as specimens.

Furthermore, by preparing a membrane on which nucleic acids are immobilized, especially a membrane on which probes are immobilized in advance, according to the method of the present invention, a simple hybridization operation becomes possible.

[0040]

EXAMPLES The present invention will be explained in more detail by way of examples below.

[0041]

[Example 1] 1 μl of α32P-dCTP (Amers
Manufactured by ham life Products, 15T
Bq/mmol, 470MBq/ml Used approximately one month after purchase) and 100μl of 10mM Tris-1
Dissolve in mM EDTA solution (pH 7.4), and transfer 0.7 μl of this solution to a 0.64 cm 2 nylon membrane (BRL Co., Ltd.,
(PhotoGene membrane) (spot: diameter 5
mm circle).

[0042] Further, 1.5 μl of a 0.1M aqueous sodium hydroxide solution was added dropwise to the same place, followed by 1.5 μl droplet of a dithiothreitol aqueous solution having a concentration of 2% by weight. The film thus obtained was heat treated at 50° C. for 2 hours.

After the heat treatment, this film was treated with 6 times the concentration of SS.
After soaking in 5 ml of C solution and shaking at room temperature for 5 minutes, the membrane was washed three times by exchanging the solution.

[0044] Radioactivity on the membrane before and after washing was measured using a survey meter (β(γ) survey meter, TG, manufactured by Aloka).
S-136 type).

As a result, the concentration was 5000 cpm before washing and 300 cpm after washing. Subtracting the background value of 50 cpm from this value revealed that 5% of radioactivity remained.

[0046]

[Comparative Example 1] The same procedure as in Example 1 was carried out except that the sodium hydroxide aqueous solution and the dithiothreitol aqueous solution were not added dropwise in Example 1, and the radioactivity was examined in the same manner.

As a result, 0.01% remained.

[0048]

[Example 2] The process was carried out in the same manner as in Example 1, except that a HybondN membrane (manufactured by Amersham, made of nylon) was used instead of the PhotoGene membrane.
The radioactivity on the membrane was examined. As a result, 5% remained.

[0049]

[Comparative Example 2] The same procedure as in Example 2 was carried out except that the aqueous sodium hydroxide solution and dithiothreitol were not added dropwise, and the radioactivity was examined in the same manner. As a result, 0.01% remained.

From the above results, it was found that by using a basic substance and a compound having a mercapto group, a nucleic acid (mononucleotide) having a length of one base can be bound to a membrane.

[0051]

[Example 3] Instead of using a Photogene membrane as a membrane, a cellulose membrane (filter paper manufactured by Advantec,
No. The membrane was treated in the same manner as in Example 1, except that 5A) was used, and the radioactivity on the membrane was examined.

As a result, 7% remained.

[0053]

[Comparative Example 3] The same procedure as in Example 3 was performed except that the sodium hydroxide aqueous solution and dithiothreitol solution were not added dropwise, and the radioactivity was measured. As a result, 0.01% remained.

From the above results, it was found that even when cellulose is used as the membrane material, nucleic acids can be bound to it in the same way as nylon.

[0055]

Example 4 The same procedure as in Example 1 was carried out except that a solution prepared by dissolving 32P-d(T)12-18 in a 1N aqueous sodium hydroxide solution was used instead of α32P-dCTP. As a result of examining the radioactivity before and after washing, 1
0% remained.

Note that the 32P label on the 5' side OH of d(T)12 to 18 is T. The method of Maniatis et al.
T. Maniatis et al., Molecular
Cloning, A Laboratory Ma
nual, Cold Spring Harbor
Laboratory (1982) 396
1 μg of d(T)12-18 was injected with γ-ATP (NEN Research Pro) using T4 polynucleotide kinase (manufactured by Takara Shuzo)
This was carried out by adding 32P using 32P (manufactured by Ducts Inc.) as a labeling agent.

[0057]

Comparative Example 4 The same procedure as in Example 4 was carried out except that 1N aqueous sodium hydroxide solution and dithiothreitol were not used. As a result of examining the radioactivity before and after washing, 0.2% remained.

From the above results, it was found that oligonucleotides as well as mononucleotides can be immobilized on membranes according to the present invention.

[0059]

[Example 5] A film obtained in the same manner as in Example 4 was
After washing with SC solution, 0.5% SDS, 6x SSC, 6x
Denhardt's solution, 100 μg/m of herring sperm denatured DNA
The treatment was carried out in a solution containing 1.5 liters of water at 37° C. for 30 minutes with shaking. Thereafter, as a result of examining the remaining radioactivity, it was found that 10% of the radioactivity dropped onto the membrane remained.

From the above results, it was found that the nucleic acid fixed to the membrane according to the present invention does not peel off from the membrane depending on the hybridization conditions.

[0061]

[Example 6] In Example 4, 32P-d(T)12~
The same procedure as in Example 4 was carried out except that d(T)12-18 not labeled with a radioisotope was used instead of 18, and 32P-d(A)12-18 was further incubated at 34°C.
Hybridization was performed in a solution containing 6x SSC, 0.5% SDS, 6x Denhardt's solution, and herring sperm denatured DNA at 100 μg/ml.

[0062] The hybridization solution was removed, and the plate was further washed twice for 5 minutes each at 36°C in 6×SSC solution. The film thus obtained was coated with an X-ray film (RX-H, Fuji Medical Systems Co., Ltd.) using an intensifying screen (Hi-Screen B-2, Fuji Photo Film Co., Ltd.).
When exposed to light at -78℃ for 5 days, d(T)12~
A clear signal was observed at the position where the 18 solution was dropped,
It has been found that hybridization by the method according to the invention is possible.

[0063] The 32P label of the 5' side OH of d(A)12-18 is as follows: d(T)12-18.
The same procedure as in Example 5 was carried out except that No. 18 was used.

[0064]

[Example 7] Same as Example 4, 32P-d(T)12~
18 was fixed and washed twice with 5 ml of 6×SSC solution for 5 minutes each. Then, 32P-d(T)12-18 Tr
The IS solution was applied and left at room temperature for 2 hours. The membrane was washed twice with 5 ml of 6xSSC solution for 5 minutes each. There was no increase or decrease in radioactivity after washing compared to the first fixation.

[0065] From this result, it was found that the nucleic acid treated after removing basic substances etc. by washing was not immobilized on the membrane.

[0066]

[Effects of the Invention] The present invention not only makes it possible to reliably immobilize short-chain nucleic acids, but also prevents the immobilized nucleic acids from peeling off from the membrane, thereby facilitating hybridization. data can be obtained more reliably than conventional methods. Furthermore, since hybridization can be performed repeatedly, efficient use of reagents and simplification of operations can be achieved.

Claims (5)

[Claims] 1. A method for immobilizing nucleic acids on a membrane, which comprises allowing a basic substance, a compound having a mercapto group, and a nucleic acid to coexist on the membrane, and heat-treating the membrane at a temperature of 30 to 80°C. How to fix it on the membrane. 2. The method for immobilizing a nucleic acid onto a membrane according to claim 1, wherein the membrane contains cellulose and/or polyamide-based synthetic resin as a component. 3. The method for immobilizing a nucleic acid on a membrane according to claim 1 or 2, wherein the substance having a mercapto group contains a plurality of mercapto groups in the same molecule. 4. The method for immobilizing a nucleic acid onto a membrane according to claim 1, wherein the basic substance is a metal hydroxide. 5. A membrane on which nucleic acids are immobilized by the method according to any one of claims 1 to 4.