JPH01242965A - Film for fixing dna and dna fixing method - Google Patents

Abstract

PURPOSE:To rapidly and efficiently fix trace nucleic acid components to various carrier films by using a photoreactive crosslinking agent having plural functional groups which can be crosslinked to nucleic acid base by UV irradiation. CONSTITUTION:The material for crosslinking and fixing DNA (e.g. solalene deriv.) having the plural functional groups which can make reaction bonding to the nucleic acid base under photoirradiation and the desired DNA are brought into contact with the film (e.g. nitrocellulose film, nylon film) for depositing DNA previously immobilized with denatured DNA different from the desired DNA and the contact region is irradiated (e.g. for about 10min) by the UV rays (e.g. 320-370nm), by which the desired DNA can be crosslinked to the carrier film and is fixed thereto. The denatured DNA (e.g. the DNA formed by denaturing the DNA of a salmon and herring) signifies one-chain DNA denatured by a heat or alkaline treatment. The nucleic acid is thereby released of the complementary bond and is held exposed. The material for crosslinking and fixing the DNA is eventually crosslinkable to such nucleic base.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field This invention relates to a membrane for fixing DNA and a method for fixing DNA. More specifically, it is suitable for methods for detecting trace amounts of nucleic acid components or specific nucleic acid base sequences contained in living organisms, and for devices that automatically detect nucleic acid components or specific nucleic acid base sequences using DNA probes. Regarding the law.

(b) Conventional technology In order to detect trace amounts of nucleic acid components or specific nucleic acid base sequences in a living body by, for example, colony hybridization, it is necessary to detect nucleic acids extracted from the living body (hereinafter referred to as target DNA).
It is necessary to use it by immobilizing it on a suitable carrier, such as a nitrocellulose membrane, but this immobilization requires
In order to ensure affinity between NA and a predetermined support, heating at 80° C. under vacuum for about 2 hours is usually required.

(c) Problems to be Solved by the Invention However, the method described above, which takes time for immobilization, impedes automation of the detection device and also poses a problem in terms of cost. Some attempts have been made to use nylon membranes instead of nitrocellulose membranes to solve these problems, but the non-specific adsorption of proteins to nylon membranes is greater than that of commonly used nitrocellulose membranes, so for example There are problems such as not being suitable for detection methods using enzymes.

The present invention was made in view of this situation, and aims to provide a DNA immobilization membrane and a DNA immobilization method that can efficiently immobilize trace amounts of nucleic acid components onto various supporting membranes in a short time.

(d) Means for Solving Problems Thus, according to the present invention, a DNA crosslinking and fixing substance having a functional group capable of reactively bonding with a nucleic acid base under light irradiation is attached to a DNA supporting film via a base material. A membrane for fixing DNA that is bonded with or without intervening, and a DNA of interest is brought into contact with this membrane for DNA immobilization, and this contact area is irradiated with ultraviolet rays, whereby the DNA of interest is bonded to the substance for cross-linking and fixing DNA. A method for immobilizing DNA is provided, which is characterized in that the DNA is crosslinked and immobilized on the supporting membrane.

This invention proposes that target DNA (hereinafter referred to as target DNA) can be immobilized on a supporting membrane in a short time by using a photoreactive crosslinking agent that has multiple functional groups that can form crosslinks with nucleic acid bases when irradiated with ultraviolet rays. It is characterized by

As the DNA-supporting membrane used in this invention, those commonly known in the art can be used, such as nitrocellulose membranes, nylon membranes, and the like.

In the DNA immobilization membrane of this invention, D
A substance for NA crosslinking fixation is bonded. This bond may be made directly to the supporting film chemically or physically, or may be made via a base material. Here, the base substance is one that can be easily immobilized on the supporting membrane, has a bond with the DNA cross-linking immobilization substance after immobilization, and does not inhibit DNA analysis/reaction, etc. Refers to substances such as denatured DNA. When using such denatured DNA, the denatured DNA is converted into DNA in advance.
It is also possible to provide a method for immobilizing target DNA using a membrane immobilized on a supporting membrane. In other words, the present invention provides a DNA cross-linking and immobilizing substance having a plurality of functional groups capable of reacting and bonding with nucleic acid bases under light irradiation, and a DNA-supporting membrane on which denatured DNA different from the target DNA has been immobilized in advance. It is also possible to provide a DNA immobilization method characterized in that the target DNA is crosslinked to the denatured DNA and immobilized on the support membrane by contacting the denatured DNA with the denatured DNA and irradiating the contact area with ultraviolet rays.

That is, this is a method in which denatured DNA is used as one crosslinking end when crosslinking and fixing target DNA. This denatured DNA means single-stranded DNA denatured by heat or alkali treatment.

That is, by forming single-stranded DNA, the complementary bonds of the nucleobases are broken and exposed, and these nucleobases can be crosslinked with the above-mentioned DNA crosslinking and fixing substance. As the above-mentioned denatured DNA, any DNA may be used as long as it does not inhibit the properties of the targetera l-DNA to be immobilized later and the reactions to this immobilized target DNA, such as salmon,
DNA of herring etc. can be denatured and used. When these denatured DNAs are immobilized on the supporting membrane, a conventional immobilization method is used. Regarding this, reference will be made to the description of the embodiment described later.

The above-mentioned DNA crosslinking fixing substance used in this invention includes:
A compound is selected that has two or more photoreactive active sites and a structure in which these sites can react with ultraviolet light to form a crosslink with a nucleic acid base. These compounds include coumarin derivatives, especially psoralen derivatives known as urchinos, which react specifically with pyrimidine bases.

Therefore, by using this psoralen derivative, it is possible to crosslink with each of the thymine, cytosine, and uracil nucleic acid bases of the single-stranded DNA fixed as described above.

The method of the present invention also provides that the denatured DNA is
It may be used by crosslinking the crosslinking fixing substance A with some of its functional groups and then fixing it on the supporting membrane.

In the second invention, the target DNA is immobilized on the supporting film on which the denatured DNA has been immobilized in advance by ultraviolet irradiation. As the ultraviolet rays used, those in the long wavelength range, for example, 320 to 370 nm, are selected. The irradiation time of the ultraviolet rays described above differs slightly depending on the target DNA, etc., but if the degree of fixation is equivalent to that of conventional methods, the irradiation time is short, e.g.
Achieved in about 0 minutes. Further, as an advantage of the immobilization method according to the method of the present invention, the support film on which the target DNA is immobilized by the above-mentioned ultraviolet irradiation is irradiated with ultraviolet rays in a short wavelength range, for example, 25
This fixation can be released by irradiating with 4 nm or the like. That is, it becomes possible to perform the immobilization operation reversibly.

(E) Effect According to the present invention, when the target DNA is brought into contact with the substance for DNA cross-linking fixation that has been fixed in advance on a supporting membrane and irradiated with ultraviolet rays, the functional groups of the substance for DNA cross-linking fixation are affected by the ultraviolet rays. The base of the target DNA reacts with the base of the target DNA to form a crosslink between them, thereby immobilizing the target DNA on the support membrane.

The present invention will be described in detail below with reference to Examples, but the present invention is not limited thereby.

(v) Examples Example 1 (i) Preparation of support membrane for DNA immobilization Schleicher & Schnull
Nitrocellulose membrane (IX
5 cm) is pre-wetted with 20 mM phosphate buffer.

Meanwhile, 1hg/m12 (7) salmon sperm DNA was added to 20mM
'): Prepare a denatured (single-stranded) DNA solution by heating at 100°C for 5 minutes in an acid buffer and then immediately cooling in ice water. The nitrocellulose membrane is wetted with this single MDNA solution and heated at 50'C for 1 hour.The membrane is then washed lightly with phosphate buffer and heated at 80C under vacuum for 2 hours. The membrane is lightly washed with phosphoric acid and air dried.

(i) Fixation (crosslinking treatment) M137 y-cyDNA (l μg/1hC) (
External DNA) was placed on the dried DNA-immobilized nitrocellulose membrane so that the spot had a diameter of 5III11 or less, and before the membrane was dry, a psoralen derivative solution (trimethylpsoralen:
10μe of 10mM solution) was added and left for 5 minutes. Thereafter, this film was directly subjected to UV irradiation using an ultraviolet (UV) irradiation device (maximum wavelength 366 nm) for 3 minutes at room temperature. After the irradiation treatment, the membrane was soaked in 20mM phosphate buffer (50°C
) for 10 minutes, then air-dried and stored.

(i) Detection of DNA The M13 phage DNA immobilized on the nitrocellulose membrane as described above was detected using a DNA probe (universal primer for M13) 7-di DNA whose 5' end was labeled with 0P). It could be detected by hybridization method. At this time, the detection efficiency was comparable to that of the conventional method.

The above results indicate that M13 phage DNA can be detected with an appropriate probe after immobilization with a photoreactive crosslinking agent, indicating that the immobilization method of the present invention is effective.

(ff) External DNA fixation efficiency ■, salmon DNA prepared by the same method as in (i) above
Fixed nitrocellulose membrane (6 μg/cm 2
When immobilizing llp-labeled M13 phage DNA (hereinafter referred to as external DNA) onto the membrane) using the same method as in (i) above (however, UV irradiation time: 5 minutes), the external DNA immobilization rate was varied. The results shown in FIG. 1 were obtained by using a solution of the psoralen derivative (same as above) at a certain concentration.

The above results indicate that the external DNA fixation rate is dependent on the concentration of the psoralen derivative.

■、Salmon D to nitrocellulose (hereinafter abbreviated as NO) membrane
Table 1 below shows the results of an investigation of the fixation rate of external DNA depending on the presence or absence of NA fixation (that is, the operation (i) above, hereinafter referred to as pretreatment) and the difference in the pretreatment method.

The following four types of NG membranes are used: NC membranes (No, l) pretreated using salmon DNA that has been photocrosslinked with a psoralen derivative (same as above); pretreated in the same manner as in (1) above; NC film with psoralen derivative (same as above) added thereto (No. 2): NC film pretreated in the same manner as in (1) above but without psoralen derivative added.
(No, 3); For the NC film without pretreatment (Ifo, 4), the same method as in (i) above was applied (however, U
, V irradiation time = 5 minutes)
The external DNA fixation rate was determined from the autoradiodram of the A-fixed NC membrane.

In addition, in both cases 1 and 2 above, the external DNA fixation rate - [amount of fixed DNA (UVS minute irradiation) / fixed DNA
ff1 (U unirradiated)].

[Table 1] UV fixation of external DNA onto NC membrane [Table 1]
] results show that by using psoralen derivatives, external DNA can be efficiently fixed to the NC film by ultraviolet irradiation.

(g) Effects of the invention According to this invention, DNA that can easily immobilize target DNA
A membrane for immobilization can be provided. Furthermore, the target DNA can be immobilized on the support membrane simply and in a short time. Moreover, this makes it possible to solve problems associated with automation of DNA probe testing equipment.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the relationship between the external DNA fixation rate and the concentration of the photoreactive crosslinking agent in the method of the present invention.

Claims (1)

[Scope of Claims] 1. DNA in which a DNA cross-linking fixing substance having a functional group capable of reacting and bonding with a nucleic acid base under light irradiation is bound to a DNA-supporting film with or without a base substance. Fixation membrane. 2. By bringing the target DNA into contact with the DNA fixing film according to claim 1 and irradiating this contact area with ultraviolet rays,
A method for fixing DNA, which comprises crosslinking the target DNA with the substance for crosslinking and fixing DNA and fixing it on the supporting membrane. 3. Contact the target DNA with a DNA cross-linking fixing substance having a plurality of functional groups that can react and bond with nucleic acid bases under light irradiation on a DNA-supporting membrane on which denatured DNA different from the target DNA has been immobilized in advance. A DNA immobilization method characterized in that the target DNA is cross-linked to the denatured DNA and immobilized on the support membrane by irradiating the contact area with ultraviolet rays.