JPH03133370A - Color reaction unit for nucleic acid hybrid and method for color reaction - Google Patents

Abstract

PURPOSE:To automatically carry out in high reproducibility the process from nucleic acid hybridization to the detection of a marker substance by color reaction by using an automated system having a specified makeup. CONSTITUTION:One or more sheets 2-23 of sample where a substance (ligand) bound to a marker substance capable of color development by enzymatic reaction is immobilized at a specified position on a supporting film and held on this film through another substance (receptor) capable of specifically binding to the ligand, are successively immersed, within a reaction tank 2-25 covered with a thermal insulating material, in an enzyme solution capable of specifically binding to said marker substance, the second enzyme solution capable of selectively binding to the above enzyme, a dye solution, a color development-stopping solution and multiple cleaning solutions 2-1 to 2-8 to make color reaction. In this process, successive reactions are accomplished using units (2-20: controller, 2-21: CPU) to control, in accordance with signals set by a user, kinds of reaction fluids 2-1 to 2-8 and 2-27 to be fed to the reaction tank 2-25, the total flow, the maintaining and lasting time intervals within the reaction tank 2-25, and the respective temperatures of the above reaction fluids during feed.

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 hitherto been used to screen desired transformant clones during cloning (colony hybridization, plaque hybridization, Southern hybridization, spot hybridization); 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. It is not only used for cloning, etc. (it is also 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 using 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). In either case, each labeling substance and its antibody are combined, and then a secondary antibody that reacts with these antibodies and whose activity against a specific substrate can be detected by color reaction etc. is used. The various reactions leading to visualization are basically the same as in the case of biotin, in that the visualization operation is performed 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 are time-consuming and complex, require operator skill, are susceptible to human error, and the resulting low reproducibility results in a loss of reagents and labor. Otherwise, there is a risk of losing valuable NA, making future research difficult, or causing incorrect genetic diagnosis.

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 solution in the tank; means for discharging the reaction solution in the tank and selecting for reuse; It is characterized by having a means for using the reaction liquid again; a means for the user to set the control temperature in the tank, the type of reaction liquid to be fed, the inflow amount, and the timing signals for feeding and discharging.

FIG. 1 shows the concept of the apparatus of the present invention as a block diagram. In this figure, 1-1 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 reaction liquid is kept in a liquid state by the temperature control system 1-4. Can increase temperature. The reaction solution flowing into the tank is operated by stirring or shaking means 1-5 attached to the reaction tank. 1-
8 is a resupply system for reusing the solution once used in the reaction for the reaction. Furthermore, 1-2. 1-3. 1-4. 1-
5 is 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 specific 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 pre-diagnosis i1.

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 is colored by an enzyme 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 a 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 reactions are automatically achieved by a device that controls the selection and order of reaction liquids to be 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 causing color development.

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 (0,1M
Tris-HCI! (pH 7,5) 10.15M N
The solution aC1) is in Buffer2 in container 2-4.
(0,1M TrisHCl (pH 9,5) 10,
15M NaCf150mM MgCl! 2.6H2
0), 3% BSA solution in Buffer in container 2-5 and Buffer in container 2-6.
l 1m! ! 11. A solution of 5AAP dissolved at a ratio of tl is placed in Buffer210 in container 2-7.
NBT 44μl, BCIP 32μl for mn
The solution dissolved in the ratio of A is further added to container 2-8.
0mM Tris-HCI (pH 7,5)10.
A 5mM EDTA solution is included. Each reaction is carried out by a supply pump 2-17 in a feeding system 2-3.
The liquid amount and timing are determined by the controller 2-20 in response to commands from the central processing unit (CPU) 2-21, for example. According to this command, the supply pump 2-17 and the electromagnetic valves 2-9 to 2-16.2-29 work in conjunction to feed a desired solution in a required amount to the reaction tank 2-25 at a desired timing. 2-24 is the drain pump of the reaction tank 2-2
It consists of a suction pump that suctions the solution No. 5. The timing of this suction is controlled by the CPU 2-21 and the controller 2-20, similar to the above-mentioned feeding of the solution. 2-28 is a solenoid valve that selects whether the reaction solution is to be reused or discarded. 2-27 is a storage container that temporarily holds a reaction solution to be reused.

By controlling the suction amounts of the supply pump 2-17 and the drain pump 2-24, the time each reaction liquid stays in the reaction tank and the flow rate can be freely controlled. That is, by operating the supply pump and drain pump at desired timing to circulate the reaction solution in the reaction tank and the reaction solution to be reused in the storage container of 2-27, the capacity of the reaction solution can be increased and a good presentation can be achieved. Can promote color reaction. These control methods may use a logic controller without depending on the CPU 2-21. 2-18 is a stirrer, but any type of stirrer may be used as long as it plays the role of promoting mixing and uniformity of the liquid in the reaction tank. Start and stop stirring with C
This is done using a signal from PU2-21.

The reaction tank 2-25 consists of a sealed container made of heat insulating material,
The reaction is carried out in a well-protected state. Feeding system 2-3
0 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 in the feeding system.

The heater turns on according to the signal from the temperature sensor.

By turning off the solution, the solution sent into the reaction tank is preheated to a temperature appropriate for the reaction. These controls are CP
It is controlled by U2-21 and controller 2-20. Reference numeral 2-26 is a filter fixing device that holds a filter for color development, which allows processing of multiple layers at a time, and also allows stacking of filters 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 is provided in the reaction tank.

Shaking is a method of shaking the filter fixture, a method of shaking the reaction tank itself, etc. Mixing of the solution in the reaction tank,
Any form may be used as long as it plays the role of promoting uniformity, cleaning and various reactions. The shaking is started and stopped 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) L/, 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 (Molecular CIonin)
Biotin-labeled probe (pUc1
9) having a complementary sequence to the base sequence of the mallotible cloning site of No. 9 was prepared, and colony hybridization was performed.

The ten filters described above were attached to a filter fixture 2-26, and placed in a reaction tank 2-25 (capacity: 150 mf).

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. In addition, regardless of the position of the filter in the reaction tank or the position of colonies on the filter, good results were obtained with uniform color development.

,%-to・, [Example 2] pUC19/JM109 produced in Example 1
Double-stranded DNA was extracted from the colonies of pBR322/JM109 according to a conventional method. This double-stranded DNA solution was denatured with an alkali or the like to form single-stranded DNA, and then this solution was spotted on a nitrocellulose filter at 10 spots each to immobilize the double-stranded DNA 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 DNA extracted from the colony of pUc19/JM109 was detected.
The 10 spots including A were colored to the same extent with high reproducibility and no color unevenness, giving good results.

[Example 3] Double-stranded DNA was extracted from the pUc19/JM109 colonies prepared in Example 1 according to a conventional method. This DN
A solution was simultaneously cleaved with two types of enzymes, Eco R1 and Sea I (1781 bp, 905 bp), 0
．． 8% agarose gel electrophoresis (90% at constant current 50 mA)
minutes). 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 in the same manner as in Example 1.2, a 1781 bp fragment containing the multiple cloning site was colored. Obtained.

In addition, when a mixture of three types of filters subjected to colony hybridization, spot hybridization, and Southern hybridization was attached to the filter fixing device 2-26 and placed in the reaction tank 2-25, a similar reaction was carried out. Good color development was obtained with all filters.

〔Effect of the invention〕

When washing and color reaction after hybridization were carried out using the apparatus according to 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 achieved). 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.

2) Reactions can now be carried out efficiently with a small amount of reaction solution.

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, Figure 3
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■-4...Temperature control system 1-5...Stirring/shaking system 1-6...Storage system 1-7...CPU/controller 1-8...Refeed system 2-1 to 2-8...Reaction liquid storage container 2-9 to 2-16. 2-28. 2-29... 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 2-30...Represents the feeding system. The start drain pump is operated for a predetermined period of time, the standby drain pump is operated for a predetermined period of time, and suction is carried out from the reaction tank, and the waste is disposed of. ↓ The drain pump is operated for a predetermined period of time, and the drain pump that performs disposal is waited for a predetermined period of time. ↑ ↓ Continued supply pump is operated for a predetermined time, the 8SA solution is introduced from the container 2-5, the circuit to maintain the desired temperature is turned on, the temperature maintenance circuit to the desired temperature is turned off, the stirring HON is closed by the solenoid valve 2-13, the standby stirrer is turned off for the predetermined time, and the drain pump is turned on. A solenoid valve 2-1 that operates a drain pump for a predetermined period of time and waits for a predetermined period of time for sucking and discarding from the reaction tank.A solenoid valve 2-1 that operates a supply pump for a predetermined period of time and introduces the 5A-AP solution from the container 2-6.
4 is closed for a predetermined time Standby stirrer OFF Operate the drain pump for a predetermined time, and suck and discard from the reaction tank ↓ Operate the supply pump for a predetermined time, and pump the Buffa+2 solution from container 2-4. Solenoid valve 2 to be introduced
Close -9 ↓ Stirrer 1 is turned on, standby stirrer is turned off for a predetermined time, the drain pump is operated for a predetermined time, the supply pump that performs suction from the reaction tank and a-type is operated for a predetermined time, and the lower ris-HCl/EDTA solution is filled. Introduce from 112-8↓ Stir to close solenoid valve 2-16! The cell is turned on for a specified time, the standby stirrer is turned off ↓ The drain pump is operated with TNB for a specified period of time, then the reactor is drained and disposed of. ↓ εNO The supply pump is operated for a specified period of time, and the NBT-BCIP solution is filled! Close the solenoid valve 2-15 introduced from 2-7, turn on the stirrer, turn off the standby stirrer for a predetermined time, operate the drain pump for a predetermined time, operate the drain pump for a predetermined time, and turn on the drain pump that sucks and discards from the reaction tank. ↑ Wait for a predetermined time ↓ Turn off the shaker ↓ Operate the drain pump for a predetermined time, and supply pump for suction and disposal from reaction 1 for a predetermined time. The temperature maintenance circuit is turned ON to the desired temperature to introduce the SA solution from the container 2-5.The temperature maintenance circuit is turned OFF to the desired temperature.Shaking HON closes the solenoid valve 2-13.For a predetermined time, the standby shaker is turned OFF.Drain pump The solenoid valve 2-1 operates for a predetermined time to suck and discard from the reaction tank ↓ Next, the supply pump is operated for a predetermined time and Buffer is introduced from the container 2-3.
Predetermined time to close 0 Standby shaking HOFF Operate the drain pump for a predetermined time, suction from the reaction tank and perform FM element ↓ Continue to operate the supply pump for a predetermined time. 5A-Solenoid valve 2-1 for introducing AP solution from 8 parts 2-6
4 Close Shaking RON Predetermined time Standby shaker OFF Drain pump is operated for a predetermined time, suction is taken from the reaction tank and waste is carried out ↓ Next drain pump is on standby for a predetermined time ↑ ↓ Continued supply pump is operated for a predetermined time 4 Suction and disposal are carried out from the standby reaction tank introduced from the reactor. Run the pump for the specified time. Introduce the Tris-HCI/EDTA solution from the container 2-8.Close the 1i magnetic valve 2-16.Turn on the shaking.For a predetermined time.Turn off the standby shaking.Drain pump is operated for a predetermined time, and suction is carried out from the reaction tank and disposed of.↓ N.D.

Claims (5)

[Claims] (1) An apparatus for carrying out a color reaction in an insulated reaction tank, and a means for mounting a sample on a support membrane containing a labeling substance that can be colored by an enzymatic reaction; specifically binding to the labeling substance. A plurality of containers for separately storing an enzyme solution, a second enzyme solution that selectively binds to the enzyme, a dye solution, a color stopper solution, and a plurality of types of washing liquids (hereinafter collectively referred to as reaction solutions); Liquid supply means for sorting the reaction liquid and adjusting the flow rate of the reaction liquid into the reaction tank; equipped with a heating means and a temperature detection means; Means for controlling temperature; Means for stirring or shaking the solution in the tank; Means for discharging or reusing the reaction solution in the tank; Means for temporarily retaining the discharged reaction solution and using it again as a reaction solution; Reaction in the tank A color reaction automation device characterized by comprising: a means for discharging a liquid; a means for a user to set a control temperature in a tank, a type of feed reaction liquid, an inflow amount, and a signal for timing of supply and discharge; (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.