JPH03130068A - Color reaction device of nucleic acid hybrid and method of color reaction - Google Patents

Abstract

PURPOSE:To reduce the labor in a process to detect biotin by color reaction and to improve the detection accuracy by successively carrying out an enzymic color reaction with a labeled substance immobilized on a substrate film. CONSTITUTION:Various kinds of reaction solutions in containers 2-1 to 2-8 are sent by a supply pump 2-17 through a feed system 2-27 to a treating tank 2-25. The pump 2-17 is linked to solenoid valves 2-9 to 2-16 by a controller 2-20 corresponding to an order from cpu 2-21 and a necessary amount of a given solution is sent to the treating tank in a desired timing. A heater 2-22 to be switched on-and-off depending upon signals from a drain pump 2-24, a stirrer 2-18 and a temperature sensor 2-19 is regulated by the cpu and the controller. A treating filter 2-23 is fixed to a holder 2-26 and the filter is sandwiched between meshes.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an apparatus and method for visualizing and detecting a nucleic acid hybrid formed between a non-radioactive probe and a single-stranded DNA.

More specifically, various reaction steps for detecting the labeled substance on the nitrocellulose filter after hybridization reaction using a non-radioactive probe are performed using a system in which temperature, time, etc. are controlled by a microcomputer or the like. The object of the present invention is to provide an apparatus and method for automatically and accurately determining the optimum conditions for a reaction process.

[Conventional technology]

A hybridization reaction is a typical method for detecting a DNA fragment having a specific sequence from among nucleic acids. The outline of this method is to convert the separated DNA fragments into single-stranded DNA by alkali treatment, etc.
After transferring this to a nitrocellulose membrane etc. as a stationary phase, some labeled specific DNA or RN
A hybrid is formed by treatment with a solution containing the A probe, and after washing and removing unreacted probes, the hybridized DNA fragments are detected.

These methods have been used to screen desired transformant clones during cloning (colony hybridization, plaque hybridization, Southern hybridization, spot hybridization), but Southern hybridization and Spot hybridization technology has recently attracted attention in medical fields such as genetic diagnosis.

Furthermore, radioactive isotopes have generally been used to label probes. However, these radioactive compounds have the disadvantage of being expensive and inconvenient in their handling and storage.

For this reason, labeling of nucleic acids with various non-radioactive substances including biotin reagents has recently been developed. In addition to being used for cloning, it has also been applied to genetic diagnosis, and DNA has already been used for diagnosis of infections and genetic diseases.
Probes have also been developed and are now commercially available.

The main point of hybridization reactions using non-radioactive probes is to use methods such as nick translation.
For example, a DNA probe labeled with biotin or the like is used to form a hybrid with a single-stranded DNA fragment to be detected immobilized on a nitrocellulose filter.
After blocking with biotin (SA), etc., avidin, which specifically binds to biotin, is first reacted, then biotinylated alkaline phosphatase, etc. is reacted with avidin, the amount of reaction is determined by color reaction, etc., and specific DNA fragments are detected. This is the way to do it.

This method is also applicable to methods using biotin derivatives (for example, JP-A-59-148798, JP-A-63-152999), or when -galactosidase complex (JP-A-6L-219400) is used instead of alkaline phosphatase. Detected through a similar process.

Furthermore, there are various non-radioactive substances for labeling DNA other than biotin derivatives, such as dinitrophenyl derivatives (Japanese Patent Application Laid-Open No. 59-204200), acetylaminofluorein derivatives (Japanese Patent Application Laid-open Nos. 58-502165 and 6O-231693). However, in either case, a secondary antibody is used that binds the respective labeling substance and its antibody, and then reacts with these antibodies, and whose activity toward a specific substrate can be detected by color reaction, etc. The various reactions leading to visualization are basically the same as in the case of biotin, in that the visualization operation is carried out using biotin.

[Problem that the invention seeks to solve]

The reactions leading up to the visualization described above were initially performed manually in the laboratory. This manual method involves multiple repeated liquid handling steps and incubations at controlled temperatures. These reaction processes require a lot of time and complicated handling.

In particular, in this reaction, quickly bringing the reactants, including the reaction support, to an appropriate temperature has a large effect on the accuracy of the subsequent detection reaction. This further risks giving rise to incorrect genetic diagnoses.

Therefore, the purpose of the present invention is to automatically perform the steps from the hybridization reaction to the detection of biotin and the like by color reaction without relying on manual labor, thereby obtaining faster, error-free and reproducible results.

[Means for solving problems]

The present invention was achieved as a result of extensive studies into automating the color reaction in order to solve these problems. That is, one point is to provide an automated device for color reaction.

The apparatus of the present invention sequentially performs a coloring reaction of a labeling substance immobilized on a support membrane using an enzyme. Its structure is as follows. - consists of a reactor vessel, the vessel being covered with a thermally insulating material. and means for installing the sample membrane in the tank; an enzyme solution that specifically binds to the labeling substance, a second enzyme solution that selectively binds to the enzyme, a dye solution, a color stop solution, and a plurality of containers; Means for separately storing the cleaning liquid; Feeding means for sorting these multiple types of reaction liquids and controlling the amount of reaction liquid flowing into the tank; Equipped with a heating means and a temperature detection means; Means for controlling the temperature of the tank; Means for stirring or shaking the solution in the tank; Means for discharging the reaction liquid in the tank; It is characterized by having a means for setting by the user.

FIG. 1 shows the concept of the apparatus of the present invention as a block diagram. In this figure, l-1 is a reaction tank in which a color reaction is carried out, and a reaction liquid supply system 1-2 and a discharge system 1-
3 is connected. The feeding system 1-2 feeds the reaction liquid from the storage container system 1-6 for a plurality of reaction liquids to the reaction tank 1-1, and during the feeding, the temperature control system 1-4 controls the temperature of the reaction liquid. It can be raised. The reaction solution flowing into the tank is operated by stirring or shaking means 1-5 attached to the reaction tank. Furthermore, I-2゜1-3. 1-4. 1-5 are controlled by parameters and 0N10FF by the CPU and controller, and the parameters and timing at this time are set by the user.

It goes without saying that the device of the invention includes a power supply for operation and a keyboard to allow user control, and may also be provided with a display for confirming user input signals.

In the sample used in the device of the present invention and subjected to a color reaction, a substance (ligand) bound to a labeling substance that can be colored by an enzymatic reaction is immobilized at a specific location on a support membrane and is unique to the ligand. substance that binds to (receptor)
Examples include those held on the support membrane via a support film.

However, it goes without saying that a sample in which a labeling substance is simply immobilized on a support membrane may be used. To explain more specifically with an example, as described in the prior art, a single-stranded DNA fragment immobilized on a nitrocellulose membrane,
Examples include those prepared by hybridization with an oligonucleotide probe incorporating biotin. Such samples are nothing but those prepared when searching for a specific base sequence by spot hybridization or Southern hybridization using a DNA probe, especially in genetic diagnosis.

Another aspect of the present invention is that it has been discovered that the above-mentioned apparatus can achieve a desired color reaction continuously and uniformly.

That is, a substance (ligand) bound to a labeling substance that can be colored by an enzymatic reaction is immobilized at a specific location on the support membrane and transferred onto the support membrane via a substance (receptor) that specifically binds to the ligand. This is a method in which one or more retained samples undergo a color reaction. This method is
In one reaction tank covered with a heat insulating material, an enzyme solution that specifically binds to the labeling substance, a second enzyme solution that selectively binds to the enzyme, a dye solution, a color stop solution, and multiple types of cleaning solutions. A step of sequentially immersing in water to cause a color reaction,
The sequential reaction is automatically achieved and colored by a device that controls the order of reaction liquids fed into the tank, total flow rate, reaction time interval, and temperature of reaction liquid during feeding according to signals set by the user. This is a color reaction method characterized by:

As mentioned above, examples to which the method of the present invention is applied include those prepared by hybridization of a single-stranded DNA fragment immobilized on a nitrocellulose membrane and an oligonucleotide probe incorporating biotin. .

Moreover, as described in the prior art, the labeling substance can be selected from biotin derivatives, dinitrophenyl derivatives, acetylaminofluorin derivatives, etc. in addition to biotin.

[Example 1] Examples of the present invention will be described below with reference to the accompanying drawings.

First, the basic configuration of the present invention will be explained with reference to FIG.

In the figure, containers 2-1 to 2-8 contain various reaction solutions. Standardly, 2XSSC10,1%S in 2-1 containers
0.2xSSC10.1%S for containers with DS of 2-2
Buffer (0, IM
Tris-HCl7 (pH 7,5) 10.15M
NaC! ) is in the container 2-4.
r2 (0,1M Tris-HCl (pH 9,5)
10,15M NaCf150mM MgCl! 2・
6H20) is in the Buffer in the container 2-5.
Containers 2-6 contain 3% BSA solution for Bu
f f e r 1 A solution of 5A-AP dissolved at a ratio of 1 μl to 1 ml was added to the container 2-7.
NBT 44p for offer210 m j7
i, BCIP A solution dissolved at a rate of 32 μi is
Furthermore, in containers 2-8, 20mM Trfs-HCl2
(pH 7.5) The required amount of 10.5mM EDTA solution is sealed. Each reaction is sent to the reaction tank 2-25 by the supply pump 17 via the feeding system 2-27, and the liquid volume and timing are determined by, for example, the central processing unit (
Controller 2- in response to commands from CPU) 2-21
determined by 20.

In accordance with this directive, supply pump 2-17 and solenoid valve 2-
9 to 2-16 work together to feed a desired solution in a required amount to the reaction tank 2-25 at a desired timing.

Reference numeral 2-24 is a drain pump for the reaction tank, which consists of a suction pump that sucks the solution from the reaction tank 2-25. The timing of this sucking bow is controlled by the CPU 2-21 and the controller 2-20 similarly to the above-mentioned feeding of the solution. Supply pump 2-17
By controlling the suction amount of each of the drain pumps 2-24, the time each reaction liquid stays in the reaction tank and the flow rate can be freely controlled. These control methods are controlled by CPU2-21.
A logic controller may also be used.

2-18 is a stirrer, which mixes the liquid in the reaction tank;
Any form may be used as long as it plays a role in promoting uniformity. The stirring is started and stopped by a signal from the CPU 2-21.

The reaction tank 2-25 consists of a closed container made of heat insulating material.
The reaction is carried out in a well-protected state. Feeding system 2-2
7 is provided with a temperature sensor 2-19 for measuring the temperature of the solution passing through the feeding system and a heater 2-22 for heating the solution inside the feeding system. The heater is turned ON and OFF according to a signal from the temperature sensor, thereby preheating the solution sent into the reaction tank to bring it to a temperature appropriate for the reaction. These controls are controlled by the CPU 2-21 and the controller 2-20.

No. 2-26 is a filter fixing device that holds a coloring filter, and is capable of processing a large number of filters at once, and also allows the filters to be stacked by sandwiching them between meshes so that the filters do not come into contact with each other.

FIG. 3 shows the sea fence of the CPU 2-21 in this embodiment.

FIG. 4 shows an embodiment in which a shaker 2-18 is provided in the reaction tank. Shaking can be used in any form, such as shaking the filter fixing device or shaking the reaction tank itself, as long as it plays a role in promoting mixing and homogenization of the solution in the reaction tank and promoting cleaning and various reactions. Anything is fine. Start of shaking,
Stopping is performed by a signal from the CPU 2-21. FIG. 5 shows the sea fence of the CPU 2-21 in this embodiment.

Next, a more specific embodiment will be described based on FIGS. 2 and 3.

In order to confirm the degree of color development by this device, an experiment was conducted using a system that reliably produces color. First, host JM109
Plasmid pUc19 and plasmid pBR322 were each transformed (Mo
Regular Cloning) A plate containing the same number of both colonies (25 colonies) was prepared.

Colonies from this plate were transferred to a nitrocellulose filter according to a conventional method and denatured with alkali or the like to immobilize single-stranded DNA on the filter. Next, nick translation (Mole cu l a
A biotin-labeled probe (having a sequence complementary to the base sequence of the mallotiple cloning site of pUc19) was prepared according to a conventional method such as rCloning, and colony hybridization was performed.

The 10 filters described above were attached to the filter fixture 2-26, and the reaction tank 2-25 (capacity: 150 mJ) was housed.

Next, a reaction was carried out according to Table 1. Table 1 summarizes the set values for opening and closing of the solenoid valve, total amount of reaction liquid, reaction temperature, and reaction time in each step according to the procedure shown in FIG. In addition, containers 2-1 to 2-8 correspond to the numbers in FIG.
The composition of the solution is shown in Table 1.

When I reacted according to the above matters, 2 out of 50
Five similar colors were obtained. These 25 cells all corresponded to the locations of pUc19/JM109 colonies on the plate. Further, regardless of the position of the filter in the reaction tank or the position of the colonies on the filter, uniform color development was obtained, and good results were obtained.

[Example 2] pUc19/JM109 and pBR produced in Example 1
Double-stranded DNA was extracted from the colony of 322/JM109 according to a conventional method. This double-stranded DNA solution was denatured with an alkali or the like to make single-stranded DNA, and then this solution was spotted on a nitrocellulose filter in 10 spots each.
Double-stranded DNA was immobilized on the filter.

Next, the biotin-labeled probe (pU
When spot hybridization was carried out and the reaction was carried out in the same manner as in Example 1,
The 10 spots containing the DNA extracted from the pUC C19/JM 109 colonies showed good results with high reproducibility and uneven coloring (the color was developed to the same extent).

[Example 3] Double-stranded DNA was extracted from the pUc19/JM109 colonies prepared in Example 1 according to a conventional method. this DNA
The solution was simultaneously cleaved with two types of enzymes, Eco RI and Sca I (1781 bp, 905 bp), 0.8
% agarose gel electrophoresis (constant current of 50 mA for 90 minutes) was performed. After electrophoresis, Southern blotting was performed according to a conventional method, and the double-stranded DNA was immobilized on a nitrocellulose filter.

Next, the biotin-labeled probe (pUc
When Southern hybridization was carried out and the reaction was carried out in the same manner as in Example 1.2, a colored fragment of 1781 bp containing the multiple cloning site was obtained. Ta.

In addition, three types of filters subjected to colony hybridization, spot hybridization, and Southern hybridization were mixed, attached to the filter fixture 2-26, placed in the reaction tank 2-25, and the same reaction was performed. Good color development was obtained with both filters.

〔Effect of the invention〕

When washing and color reaction after hybridization were performed using the apparatus of the present invention, the work that conventionally required a full-time operator over a day was completed without any manual intervention (good color reaction was obtained). As a result, a) the labor force was significantly reduced.

(Example) It became possible to work at night, which shortened experiment time.

c) Human error was suppressed, the color reaction proceeded uniformly, and at the same time, high reproducibility was obtained.

The following effects were obtained.

[Brief explanation of the drawing]

FIG. 1 is a block diagram showing the configuration of an apparatus according to the present invention. Figure 2 is a configuration diagram of a more specific embodiment, and Figure 3 is a diagram of the second embodiment.
3 is a flowchart showing an example of the operation of the control unit (CPU/controller) shown in the figure. FIG. 4 is a block diagram of another specific embodiment, and FIG. 5 is a flowchart showing one embodiment of the operation of the control section (CPU/controller) in FIG. 4. Symbols in the figure: 1-1...Reaction tank 1-2...Feeding system 1-3...Discharge system 1-4...Temperature control system 1-5...Stirring/shaking system 1-6... Storage system 1-7... CPU/controller 2-1 to 2-8... Reaction liquid storage container 2-9 to 2-16... Solenoid valve 2-17... Supply Pump 2-18... Stirrer 2-19... Temperature sensor 2-20... Controller 2-21... CPU 2-22... Heater 2-23... Filter 2-24... - Drain pump 2-25... Reaction tank 2-26... Filter fixture 2-27... Represents a feeding body. Figure 5 Gin-no-1 1 Operate the supply pump for a predetermined time, solenoid valve 2-1 that introduces Buff 1 from container 2-3.
Close 0 ↓ Stirrer ON Wait for the specified time ↓ Stirrer OFF Suction and waste from the reaction tank ↓ Operate the supply pump for the specified time and introduce the BSA solution from container 2-5 ↓ Turn on the temperature maintenance port 111 to the desired temperature ↓ Turn off the temperature maintenance circuit to the desired 1 degree ↓ Close solenoid valve 2-13 ↓ Turn on the stirrer ↓ Wait for the specified time ↓ Turn off the stirrer ↓ Operate the drain pump for the specified time and turn on the supply pump that sucks from the reaction tank and discards it. A solenoid valve 2-1 that operates for a predetermined time and introduces the 5A-AP solution from the container 2-6.
4 Close the stirrer ON Standby for a predetermined time Stirrer OFF Operate the drain pump for a predetermined time, Operate the supply pump for a predetermined time, Solenoid valve 2 to introduce Buffer 2 solution from container 2-4
-9 Close ↓ Stirrer ON Wait for the specified time ↓ Stirrer OFF Operate the drain pump for the specified time. Suction and disposal from the reaction tank ↓ Operate the supply pump for the specified time. Introduce NBT-BCIP solution from container 2-7 ↓ Close solenoid valve 2-15 ↓ Turn on the stirrer ↓ Wait for the specified time ↓ Turn off the stirrer ↓ Operate the drain pump for the specified period of time to suction from the reaction tank and discard it ↓ The supply pump is operated for a predetermined period of time to supply Trts to the reaction tank.
-1-1cI ・Close the solenoid valve 2-16 that introduces the EDTA solution from the container 2-8 ↓ Turn on the stirrer ↓ Wait for the specified time ↓ Turn off the stirrer ↓ Operate the drain pump for the specified time, suck it out from the reaction tank, and discard it. Do↓ εNO Figure 5 Part 2 ↓ Operate the supply pump for the specified time and fill the Buffer! ! Introduce from 2-3 ↓ Close solenoid valve 2-10 ↓ Turn on the shaker ↓ Wait for the specified time ↓ Turn off the shaker ↓ Operate the drain pump for the specified time, and operate the supply pump that sucks and discards from the reaction tank for the specified time Turn on the temperature maintenance circuit to the desired temperature to introduce the BSA solution from container 2-5 Turn off the temperature maintenance circuit to the desired temperature ↓ Turn on the shaker that closes the solenoid valve 2-13 For a predetermined time Standby shaker OFF Drain pump is operated for a predetermined period of time, and the suction and disposal are carried out from the reaction tank. 1. The supply pump is operated for a predetermined period of time, and the 5A-AP solution is introduced from volume M2-6. ↓ Close the solenoid valve 2-14. ↓ Turn on the shaker for a predetermined period of time. Standby ↓ Operate the drain pump for a specified period of time, suction from the reaction tank, 1! ! ! The solenoid valve 2 operates the supply pump for a predetermined period of time to introduce the Buffer 2 solution from the container 2-4.
-9 Close shaker ON for a predetermined time Standby shaker OFF Operate the drain pump for a predetermined time to suck suction from the reaction tank and discard S. Next, operate the supply pump for a predetermined time and transfer the NBT-BCIP solution to container 2- Close the solenoid valve 2-15 introduced from 7, turn on the shaker, turn on standby shaking for a predetermined time, turn off the drain pump, operate the drain pump for a predetermined time, and suck and discard from the reaction tank.

Claims (5)

[Claims] (1) An apparatus for carrying out a color reaction in a single reaction tank covered with a heat insulating material, and a means for mounting a sample on a support membrane containing a labeling substance that can be colored by an enzymatic reaction; Separate storage of an enzyme solution that specifically binds to the labeling substance, a second enzyme solution that selectively binds to the enzyme, a dye solution, a color stop solution, and multiple types of washing solutions (hereinafter collectively referred to as reaction solutions) feeding means for sorting out a plurality of types of reaction liquids and controlling the amount of reaction liquid flowing into the tank; equipped with a heating means and a temperature detection means to control the temperature of the reaction liquid during feeding; Means for stirring or shaking the solution in the tank; Means for discharging the reaction liquid in the tank; Users can set the control temperature in the tank, the type of reaction liquid to be fed, the inflow amount, and the timing of supply and discharge. A color reaction automation device comprising: means for: (2) A substance (hereinafter referred to as a ligand) bound to a labeling substance that can be colored by an enzyme reaction is immobilized at a specific location on the support membrane, and a substance that specifically binds to the ligand (hereinafter referred to as a receptor) is One or more samples held on the support membrane via the membrane are selectively treated with an enzyme solution that specifically binds to the labeling substance in a reaction tank covered with a heat insulating material. In the process of selectively immersing in the second enzyme solution to be combined, the dye solution, the color stop solution, and multiple types of cleaning solutions to cause a color reaction, the material is fed into the tank according to a signal set by the user. A method of color reaction, characterized in that a sequential reaction is achieved by a device that controls the species and total flow rate of the reaction solution, the time interval for maintaining the reaction solution in the tank, and the temperature of the reaction solution during feeding. (3) The method according to claim 2, wherein the substance to which the labeling substance is bound is an oligonucleotide having a specific base sequence. (4) The method for retaining the oligonucleotide at a specific location on the membrane is by hybridization with a single-stranded DNA fragment that is complementary to the base sequence of the oligonucleotide and fixed on the membrane. A method according to claims 2 and 3, characterized in that. (5) The method according to claim 2, wherein the labeling substance is one selected from biotin, biotin derivatives, dinitrophenyl derivatives, and acetylaminofluorein derivatives.