JPH06233698A - Blotting method using highly water-absorbing resin and blotting-assisting apparatus - Google Patents

Abstract

PURPOSE:To increase the speed of blotting with simple operation by imparting blotting with water-absorptivity using a highly water-absorbing resin. CONSTITUTION:A gel 5 such as agarose gel or polyacrylamide gel subjected to electrophoresis, modification and neutralization is closely placed on a filter paper 4 and a blotting film 6 is closely placed on the filter paper. Other filter papers 7 are placed on the blotting film, a frame 9 of a blotting assistant device 8 is placed on the filter paper 7 and a highly water-absorbing resin 10 (e.g. starch, cellulosic material and synthetic polymer) is uniformly scattered on the filter paper 7 in the frame 9.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for blotting nucleic acids and proteins that is widely used in the fields of research such as biochemistry and molecular biology, and particularly in each field utilizing recombinant DNA technology, and the blotting used in this blotting method. Regarding auxiliary equipment.

[0002]

2. Description of the Related Art Blotting is a nitrocellulose membrane or nylon in which a nucleic acid or protein sample fractionated in an agarose gel or polyacrylamide gel by electrophoresis is adhered from the gel to the gel surface by capillary action. It is a technique of moving to a membrane. This technique is one of the basic and important techniques in research such as biochemistry and molecular biology, and in gene recombination technology. It is an essential technique in whole genome analysis research.

The blotting method currently used is the method reported by Southern (J. Mol. Biol., 98; 503, 197).
Based on 5), that is, stacking paper towels on the nitrocellulose membrane adhered to the gel to a height of several centimeters, and letting this paper towel absorb water effectively moves the nucleic acid in the gel. It is a thing. Recently, a method of using a vacuum pump for absorbing water, a method of electrically eluting nucleic acids and proteins from a gel and transferring them to a membrane, and the like have been developed, and devices for them have been commercially available.

[0004]

A problem with the Southern method is that it takes time to move the sample. For example, in the case of a DNA having several thousand base pairs, the time required is several hours, and in the case of a large molecule such as genomic DNA, it takes ten hours to twenty hours or more. Also, in order to absorb water efficiently, the paper towel containing water must be replaced with a new one. Therefore, the paper towel used at one time is
It's a significant amount, and its cost is not negligible.

On the other hand, the method of electrophoretically moving the sample from the gel to the membrane and the method of using a vacuum pump can complete the blotting in a short time, but an apparatus for carrying out these methods is available. Expensive and its operation is
Complicated compared to Southern's method. Furthermore, in the method using a vacuum pump, if the pressure is set incorrectly, the gel may be damaged and the sample may be lost.

[0006]

Means for Solving the Problems In order to solve the above problems, according to the present invention, a nucleic acid or a protein molecule fractionated in an agarose gel or a polyacrylamide gel by electrophoresis was brought into close contact with the gel by capillary action. In the blotting method of moving and adsorbing to the blotting membrane, the blotting method is characterized by utilizing water absorption by the super absorbent resin. As the super absorbent polymer, a starch-based polymer, a cellulose-based polymer, or a synthetic polymer was used.

Further, a blotting assisting device consisting of a box-shaped frame for adhering the filter paper to the blotting membrane is provided. The filter paper is sandwiched and fixed to the lower part of the frame, or is inserted and fixed through a slit formed in the lower part of the frame. Further, the frame may be composed of an inner frame and an outer frame, and the filter paper may be sandwiched and fixed between both frames to bring the filter paper into close contact with the blotting membrane.

[0008]

According to the above means, unlike the Southern method, it does not take a long time to move the sample and the paper towel is not consumed in a large amount, and the super absorbent resin and the filter paper placed on the super absorbent resin. With a simple blotting auxiliary device for fixing, the operation is simple and blotting can be completed in a shorter time.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the blotting apparatus used in the present invention will be described with reference to FIG. Put an appropriate amount of SSC solution (0.03M to 0.3M sodium citrate, 0.3M to 3M sodium chloride) or alkali blotting solution into the solution tank 1 and place the support 3 in the center of the solution tank 1. To do. A filter paper 4 is covered on the support base 3 so that both ends are immersed in the solution 2. When the filter paper 4 has sufficiently absorbed the blotting solution 2, after electrophoresis by a known method, denaturation,
The neutralized agarose gel 5 is placed so as to be in close contact with the filter paper 4. On this agarose gel 5, 1 more than this
After slicing into ˜2 mm, a suitable pretreated nitrocellulose membrane or nylon membrane is placed in close contact with the agarose gel 5 as the blotting membrane 6. On this blotting membrane 6, two to three gel-sized filter papers (which are generally used for blotting) 7 moistened with an appropriate solution are placed.

Next, a blotting auxiliary device 8 as shown in FIG. 2 is prepared in advance. It consists of a frame 9 whose base area is one size larger than a commonly used electrophoretic gel and whose height is 1-2 cm. The blotting auxiliary device 8 having only the frame 9 is directly placed on the filter paper 7 on which two or three sheets are stacked, and the filter paper 7 is brought into close contact with the blotting membrane 6. A highly water-absorbent resin 10 such as a starch-based polymer, a cellulose-based polymer, or a synthetic polymer is evenly spread over the filter paper 7 in the frame 9.

In the blotting auxiliary device 8 shown in FIG. 3, the lower portions of the two sets of facing frames 9 project inwardly to form edges 11, and both ends of the filter paper 12 are attached to the edge 11 of one set. A slit 13 for sandwiching and fixing is provided. The filter paper 12 is sandwiched and fixed in the slit 13, and the blotting assisting device 8 is placed so that the filter paper 12 comes into close contact with the filter paper 7 stacked on the agarose gel 5.
This blotting auxiliary device 8 is placed on a filter paper 7 having a stack of 2 to 3 sheets, and when the filter paper 12 in the frame 9 gets wet, a starch-based polymer, a cellulose-based polymer, a synthetic polymer or the like is uniformly applied to the entire wetted part. The highly water-absorbent resin 10 is sprinkled.
The filter paper 12 is pressed against the peripheral edge 11 by the weight of the blotting auxiliary device 8 so that the super absorbent resin 10 contained in the frame 9 does not fall.

A blotting auxiliary device 8 shown in FIGS. 5 and 6.
Is composed of an inner frame 14 and an outer frame 15 which are substantially similar to each other. The lower portions of the two sets of frames 16 facing each other of the outer frame 15 are each formed with an edge 17 that extends inward and surrounds the periphery. This edge 17
Filter paper 1 cut into a size that fits inside the outer frame 15
The filter paper 12 is fixed to the blotting auxiliary device 8 by placing 2 and fitting the inner frame 14 therein. This blotting auxiliary device 8 is placed on a filter paper 7 having a stack of 2 to 3 sheets, and when the filter paper 12 in the frame 9 gets wet, a starch-based polymer, a cellulose-based polymer, a synthetic polymer or the like is uniformly applied to the entire wetted part. The highly water-absorbent resin 10 is sprinkled.

In this state, the plate is allowed to stand at room temperature for 30 minutes to 1 hour 30 minutes for blotting. After completion of the blotting, the blotting membrane can be subjected to an experiment such as hybridization after the fragments of the agarose gel 5 adhering to the surface are washed away and subjected to an appropriate treatment.

The highly water-absorbent resin 11 is sodium polyacrylate crosslinked formed into powder, granules, or sheets at a low price, for agricultural / horticultural use, civil engineering / construction use, or as a sanitary material. Polymers, vinyl acetate maleic acid ester copolymers and the like are preferable, and those having an average particle diameter of 150 to 300 μm are preferable, but not limited thereto. The blotting auxiliary device 8 is preferably made of a material having excellent salt resistance and alkali resistance, but the material is not limited to this.

Next, blotting of DNA (Southern blotting) will be described. Example 1 : 2 μg of lambda phage DNA digested with restriction enzyme HindIII (manufactured by Nippon Gene) was recovered by ethanol precipitation, and the resulting DNA precipitate was added with 5 μl of 0.1 mM EDTA.
Dissolved in. In the dark, 5 μl of 1 mg / ml photobiotin acetate (manufactured by Bresa) was added to this DNA solution and mixed. A mercury lamp (TOSHIBA CHEMICAL LAMP: FL2
0S-BL), place 8cm away from the light source, 350-370n
It was irradiated with m light for 3 hours. This is followed by 40 μl of sterile distilled water and 10
50 μl of mM Tris-hydrochloric acid (pH 7.5) was added, and 100 ul of 2-butanol was further added and mixed. Centrifugation was performed at 5000 rpm for 1 minute. Next, the butanol layer was removed, and the same butanol extraction was repeated again. Finally, the butanol layer was removed, and then the aqueous layer was subjected to ethanol precipitation to collect DNA.
The obtained DNA precipitate was dissolved in 20 μl of 0.1 mM EDTA.

The biotin-labeled lambda phage DNA obtained here was appropriately diluted, and a part of it (0.25 ng, 0.5 ng, 1 ng, 2n
For g), electrophoresis was performed using 0.7% agarose gel. After electrophoresis, 0.4M of this gel (size 5cm x 6cm)
Shake in NaOH, 0.6M NaCl for 30 minutes at room temperature,
The DNA in the gel was denatured. After this, the gel was transferred into 0.5M Tris-HCl, pH 7.5, 1.5M NaCl and shaken at room temperature for 30 minutes. The gel was placed on Whatman 3MM filter paper (4 in FIG. 1) imbibed with 1.5M NaCl, 0.15M sodium citrate so as to be in close contact, and the gel was covered with Saran wrap.

Cut into 1 to 2 mm larger than this gel, 0.3M
Blotting membrane, Gene Screen Plus (manufactured by Du Pont) moistened with NaCl, 0.03M sodium citrate was placed on the gel so as to be in close contact, and 0.3M NaCl, 0.
Two sheets of Whatman 3MM filter paper (7 in Fig. 1), which was the same size as the gel, and which had been moistened with 03M sodium citrate were stacked. Next, as shown in FIG. 4, a filter paper 12 is fixed to the bottom surface of the blotting assisting device 8, the filter paper 12 is placed so as to be in close contact with the filter paper 7 of FIG.
0.5 to 0.6 g of 4 (manufactured by Nippon Shokubai Chemical Co., Ltd.) was evenly spread over the entire filter paper. In this state, it was left at room temperature for 1 hour and 30 minutes.

After this, the blotting membrane was taken out, and
By shaking for 5 minutes in 3M NaCl, 0.03M sodium citrate, gel fragments and the like adhering to the membrane surface were removed. Dip this membrane between paper towels or filter paper, dry it at room temperature, and irradiate it with UV light.
NA was immobilized on the membrane. This membrane is 3% bovine serum albumin
Buffer 1 (100mM Tris-HCl pH7.5, 1M NaC
1 ml, 2 mM MgCl2, 0.05% Triton X-100) and 2 ml were sealed in a polyethylene bag and kept at 65 ° C for 1 hour.

After this, the membrane was reacted in 12 ml of buffer containing 0.4 units of streptavidin-alkaline phosphatase (Boehrinnger-Mannheim) at room temperature for 10 minutes. The membrane was washed by shaking in 75 ml of buffer 1 at room temperature for 20 minutes. This washing operation was repeated 3 times. Further 75 ml of Buffer 2 (100
The cells were washed by shaking them in mM Tris-hydrochloric acid pH 9.5 1M NaCl, 5 mM MgCl 2) at room temperature for 20 minutes, and this was repeated twice.

After this, the membrane was treated with 0.3 mg / ml nitropru-tetrazolium salt, 0.2 mg / ml 5-bromo-4-chloro-
It was enclosed in a polyethylene bag together with 2 ml of buffer 3 containing 3-indolyl phosphate (100 mM Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl 2) and allowed to stand in the dark for 40 to 50 minutes to develop color. Take the membrane out of the bag, shake it in 15 ml of buffer 4 (10 mM Tris-HCl pH 7.5, 10 mM EDTA) at room temperature for 5 minutes, and repeat this 3 times.
The color reaction was stopped. As shown in FIG. 6, this membrane has a 23k fragment corresponding to a HindIII degradation product of lambda phage DNA.
Bands of b, 9.4 kb, 6.6 kb, 4.4 kb.2.3 kb, and 2.0 kb were observed, and it was confirmed that the DNA migrated from the gel to the blotting membrane.

Example 2 : 2 μg of plasmid pUC19 digested with restriction enzyme HinfI (manufactured by Nippon Gene) was recovered by ethanol precipitation, and the resulting DNA precipitate was 0.1 mM EDTA5.
It was dissolved in μl. 1 mg / m in this DNA solution in the dark
5 μl of l-photobiotin acetate (manufactured by Bresa) was added and mixed. A mercury lamp (TOSHIBA CHEMICAL LAMP: FL2
0S-BL), place it 8cm away from the light source, 350
Irradiation with light of ˜370 nm for 3 hours.

Thereafter, 40 μl of sterile distilled water and 10 mM Tris-HCl
(pH 7.5) 50 μl was added, and 2-butanol 100 ul was further added and mixed. After centrifugation at 5000 rpm for 1 minute, the butanol layer was removed, 100 μl of 2-butanol was added again, and after mixing, centrifugation was performed at 5000 rpm for 1 minute. After removing the butanol layer, ethanol precipitation was performed on the aqueous layer to recover DNA. The obtained DNA precipitate was dissolved in 20 μl of 0.1 mM EDTA.

The biotin-labeled pUC19 thus obtained was appropriately diluted, and a part of it (0.5 ng, 1 ng, 2 ng, 4 ng) was diluted to 1.5
Electrophoresis was performed using a% agarose gel. After electrophoresis, use this gel (size 5 cm × 6 cm) with 0.4N NaOH, 0.6M NaC.
DNA in gel is shaken for 30 minutes at room temperature in
Was denatured. After this time, the gel was washed with 0.5 M Tris-HCl, (pH
7.5) Transferred to 1.5M NaCl and shaken at room temperature for 30 minutes. As shown in 4 of Fig. 1, this gel was washed with 1.5M NaCl, 0.15M
The gel was placed on top of Whatman 3MM filter paper imbibed with sodium citrate in close contact and the gel was covered with Saran wrap. Cut 1-2mm larger than gel, 0.3M NaC
Gene Screen Plus (manufactured by Du Pont), a blotting membrane that had been moistened with l, 0.03M sodium citrate, was placed on the gel so as to be in close contact, and as shown in 7 of FIG. , WhatM 3MM filter paper 7 of the same size as the gel, which had been moistened with 0.3M NaCl, 0.03M sodium citrate, were stacked. As shown in FIG. 4, a filter paper 12 is fixed to the bottom surface of the blotting assisting device 8, and the filter paper 12 is placed so that the filter paper 12 is in close contact with the filter paper 7 of FIG.
AK-4 (manufactured by Nippon Shokubai Chemical Co., Ltd.) 0.5 to 0.6 g of filter paper 7, 9
Spread evenly throughout. In this state, it was left at room temperature for 1 hour.

After this, the blotting membrane was taken out, and
By shaking in 3M NaCl, 0.03M sodium citrate for 5 minutes, gel fragments and the like adhering to the membrane surface were removed. Dip this membrane between paper towels or filter paper, dry it at room temperature, and irradiate it with UV light.
NA was immobilized on the membrane. This membrane was replaced with bovine serum albumin 3
%, Buffer 1 (100 mM Tris-HCl pH 7.5, 1M Na
Cl, 2 mM MgCl2, 0.05% Triton X-100) and 2 ml were sealed in a polyethylene bag and kept at 65 ° C for 1 hour.

After this, the membrane was reacted for 10 minutes at room temperature in 12 ml of buffer containing 0.4 units of streptavidin-alkaline phosphatase (Boehrinnger-Mannheim). The membrane was washed by shaking in 75 ml of buffer 1 at room temperature for 20 minutes. This washing operation was repeated 3 times. Further, in 75 ml of buffer 2 (100 mM Tris-HCl pH 9.5, 5.1 M NaCl, 5 mM MgCl2) at room temperature, 2
Washing was performed by shaking for 0 minutes, and this was repeated twice.

After this, the membrane was buffered with buffer 3 (100 mM Tris-HCl pH 9.90) containing 0.3 mg / ml nitroblue-tetrazolium salt, 0.2 mg / ml 5-bromo-4-chloro-3-indolyl phosphate.
2100 ml of 5,100 mM NaCl, 5 mM Mgcl2) was enclosed in a polyethylene bag and allowed to stand in the dark for 40 to 50 minutes for color development. Remove the membrane from the bag and remove 15 ml of Buffer 4 (10 m
In M Tris hydrochloric acid pH 7.5, 10 mM EDTA), the mixture was shaken at room temperature for 5 minutes, and the coloring reaction was stopped by repeating this 3 times. As shown in FIG. 7, 1.4 kb, 0.5 kb, 0.4 kb and 0.2 kb bands of the HinfI degradation product of pUC19 were observed on this membrane, confirming that the DNA was transferred from the gel to the blotting membrane. .

[0027]

As described in detail above, according to the present invention, blotting can be completed by a simple operation in a short time, the running cost is low, and no special equipment is required.

[Brief description of drawings]

FIG. 1 is a sectional view of a blotting device used in the present invention.

FIG. 2 is a perspective view of a blotting auxiliary device of the present invention.

FIG. 3 is a perspective view showing another embodiment of the blotting assisting device.

FIG. 4 is a perspective view of the blotting assisting device with the filter paper also fixed.

FIG. 5 is a perspective view showing still another embodiment of the blotting auxiliary device.

FIG. 6: Biotin labeled lambda phage DNA-Hind
It is a figure which shows the blotting lane of a III degradation product.

FIG. 7: Biotin-labeled puc19DNA-Hinf I
It is a perspective view which shows the blotting of a decomposition product.

[Explanation of symbols]

 5 Agarose gel 6 Blotting membrane 8 Blotting auxiliary device 7 Filter paper 9 Frame 10 Super absorbent resin 11 Edge 12 Filter paper 13 Slit 14 Inner frame 15 Outer frame

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[Procedure amendment]

[Submission date] August 31, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 6

[Correction method] Change

[Correction content]

FIG. 6: Biotin-labeled random phage DNA-Hind
It is a photograph of a chromatograph showing the blotting lane of the III degradation product.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 7

[Correction method] Change

[Correction content]

FIG. 7: Biotin-labeled puc19DNA-Hinf I
It is a photograph of a chromatograph showing the blotting of the decomposition products.

Claims (6)

[Claims] 1. A blotting method in which a nucleic acid or a protein molecule fractionated in an agarose gel or a polyacrylamide gel by electrophoresis is moved and adsorbed to a blotting membrane adhered to the gel by capillarity. A blotting method using a super absorbent polymer characterized by utilizing water absorption by the super absorbent polymer. 2. The blotting method using a super absorbent polymer according to claim 1, wherein the super absorbent polymer is a starch-based polymer, a cellulose-based polymer, or a synthetic polymer. 3. The blotting method according to claim 1, wherein a box-shaped frame is provided on the blotting membrane to bring the filter paper into close contact with the blotting membrane. 4. The blotting auxiliary device using a super absorbent resin according to claim 3, wherein the filter paper is clamped and fixed to a lower portion of a frame. 5. The blotting auxiliary device using a super absorbent resin according to claim 3, wherein a slit for holding and fixing the filter paper is formed in a lower portion of the frame. 6. The frame according to claim 3, wherein the frame is composed of an inner frame and an outer frame, and the filter paper is sandwiched and fixed between both frames to bring the filter paper into close contact with the blotting membrane. Blotting auxiliary device using super absorbent resin.