JPH03130067A - Device of immobilizing single stranded dna and immobilization - Google Patents

Abstract

PURPOSE:To reduce the labor in a process from preparing single stranded DNA to fixing thereof to a treating film by separating a double stranded DNA into the single stranded DNA in a treating tank equipped with a specimen where target microorganisms are held 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 heat exchanger 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 preparing and immobilizing single-stranded DNA on a treatment membrane as a pretreatment for hybridization.

More specifically, in order to perform a hybridization reaction, various reactions and processing steps for immobilizing single-stranded DNA on a filter are performed using a system in which temperature, time, etc. are controlled by a microcomputer, etc. An object of the present invention is to provide an apparatus and a method for automatically and accurately determining the optimum conditions for a 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 R
Hybrids are formed by treatment with a solution containing an NA probe, and after washing and removing unreacted probes, DNA fragments that have formed hybrids are detected.

These methods have been used to screen desired transformant clones during cloning.

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, etc., it has also been applied to genetic diagnosis, and has already been used to diagnose infectious and genetic diseases.
A probe has also been developed and commercialized.

[Problem that the invention seeks to solve]

Among the above steps, the reactions up to the step of preparing the single-stranded DNA and immobilizing it on the treated membrane 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. Moreover, the resulting low reproducibility not only results in the loss of reagents and labor, but also the loss of valuable NA, complicating subsequent research and risking erroneous genetic diagnoses. In addition, in hybridization reactions using probes labeled with non-radioactive substances, which have recently been attracting attention from the viewpoint of safety, convenience, and low cost, it is especially important to improve the purity of DNA in order to compensate for the sensitivity. There is an increasing need to improve Therefore, new processing has to be added in addition to conventional processing, making the entire process more complicated, and it is no longer possible for humans to handle it.

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.

Therefore, in the present invention, the steps before the hybridization reaction, from preparing the double-stranded DNA to immobilizing it on the processing membrane, are performed automatically without relying on manual labor, thereby achieving faster, error-free and reproducible results. That is the purpose.

[Means for solving problems]

In order to solve such problems, the present invention aims to
This was achieved as a result of intensive research into automating the extraction and immobilization of That is, one point is to provide an automated device for extraction and immobilization of double-stranded DNA.

The device of the present invention separates double-stranded DNA into single-stranded DNA in a treatment tank consisting of a target microorganism immobilized on a support membrane, or a target microorganism itself, and one tank.
This is a device for fixing on a support membrane. Its configuration is as follows. It consists of one treatment tank, and the tank is covered with a heat insulating material. and a means for mounting a sample in which the target microorganisms are inoculated or the target microorganisms themselves are held on a support membrane; a solution for denaturing and dissolving the target microorganisms, and a step for neutralizing the solution in multiple containers; Second solution, dissolved solution, D
A means for separately storing a solution for NA purification and a plurality of types of washing liquids (hereinafter collectively referred to as reaction liquids); a supply system for sorting the plurality of reaction liquids and controlling the amount of reaction liquid flowing into the tank; Feeding means: Temperature control means equipped with means for heating and cooling the reaction liquid in the tank and temperature detection means: Means for stirring or shaking the solution in the tank; Means for discharging the reaction liquid in the tank; Control temperature and supply in the tank An automated device for extracting and immobilizing single-stranded DNA, comprising means for a user to set signals for the type of reaction liquid to be fed, the amount of inflow, and the timing of supply and discharge.

FIG. 1 shows the concept of the apparatus of the present invention as a block diagram. In this figure, 1-1 is a series of processing
A treatment tank for carrying out a reaction, and a treatment/reaction liquid feeding system 1-2
and discharge system 1-3 are connected. A feeding system 1-2 feeds the solution from a plurality of reaction liquid storage container systems 1-6 to the processing tank 1-1, and also controls the temperature of the reaction liquid by a temperature control system 1-4 during feeding. do.

The solution flowing into the tank is operated by stirring or shaking means 1-5 attached to the processing tank. Furthermore, 1-2゜1-3
．． 1-4. l-5 is controlled by parameters and 0N10FF by the CPU and controller,
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.

Samples used in the device of the present invention and subjected to processing reactions include plaques formed by phages, transcripts from colonies formed by bacteria such as Escherichia coli and yeast, or samples themselves retained on a support membrane. It's better to let it happen.

Another aspect of the present invention is based on the discovery that desired processing reactions can be continuously and uniformly achieved using the above-mentioned apparatus.

In other words, one or more samples inoculated with the target microorganisms, or samples in which the target microorganisms themselves are held on a support membrane, are placed in a solution that denatures the transferred material in a treatment tank consisting of one tank. The DNA is then selectively immersed in a second solution for neutralization, a solution for DNA purification, and multiple types of washing solutions to prepare single-stranded DNA as a pretreatment for hybridization and immobilize it on the treated membrane. In the process, successive reactions are achieved by a device that controls the type and total flow rate of the reaction solution fed into the tank, the duration interval for maintaining the tank, and the temperature in the tank according to signals set by the user. Features: Single-stranded DNA extraction and immobilization method.

Examples to which the method of the present invention is applied include, in addition to the general hybridization pretreatment described in the prior art, hybridization pretreatment with an oligonucleotide probe incorporating a non-radioactive substance, such as biotin. Can be done.

[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. Typically, 2 x SSC in 2-1 containers, 2
-2 container contains lX5SC, 2-3 container contains 95%
The ethanol solution was saturated phenol solution (phenol:chloroform:isoamyl alcohol 25:24:1) in container 2-4, and 0,5 M in container 2-5.
N a OH/ 1, 5 M N a Cj! The solution is 0.5M Tr, 1s-HCf in the container 2-6.
(pH8,0) / 1.5MN a C1! Container 2-7 contains 0.05M Tris・HCj
! (pH8,0)/25% 5UCRO3E/1.5
Dissolve the lysozyme solution at the rate of μg/m, f lysozyme, and add 200 μg/ml in 2-8 containers.
A required amount of protease solution called protease/1xssc is enclosed. Each reaction uses supply pump 2-1
7 to the processing tank 2-25 via the feeding system 2-27, the amount and timing of which 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 solenoid valves 2-9 to 2-16 work together to supply the required amount of the desired solution to the processing tank 2 at the desired timing.
-25. Reference numeral 2-24 is a drain pump for the processing tank, which consists of a suction pump that sucks the solution from the processing tank 2-25. The timing of this suction is the same as the above solution feeding.
U2-21. It is controlled by a controller 2-20. By controlling the suction amounts of the supply pump 2-17 and the drain pump 2-24, the time each processing liquid stays in the processing tank and the flow rate can be freely controlled. These control methods may use a logic controller without depending on the CPU 2-21.

2-18 is a stirrer, which mixes the liquid in the processing 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 processing tank 2-25 is a closed container made of a heat insulating material. The feeding system 2-27 is provided with a temperature sensor 2-19 for measuring the temperature of the solution passing through the feeding system and a heat exchanger 2-22 for adjusting the temperature of the solution in the feeding system to a desired temperature. ing.

This heat exchanger may be of any type as long as it can cool and heat the solution; for example, heating is performed by a heater, and cooling is performed by a Peltier element or a cooling coil through which a refrigerant such as Freon flows. The heat exchanger may not only be in contact with the feed system, but also may be included in the feed system. The heat exchanger is turned on according to the signal from the temperature sensor.
, OFF, the CPU 2-21 is controlled by the controller 2-20.

Reference numeral 2-26 is a filter fixing device that holds filters to be processed.It is capable of processing a large number of filters at a time, and also allows stacking of filters by sandwiching the filters 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 processing tank.

Shaking is a method of shaking the filter fixing device, a method of shaking the processing tank itself, etc. Mixing of the solution in the processing 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.

First, plasmid pUc was introduced into host E. coli JMI O9.
Transform 19 according to the conventional method (Mol
ecularCloning) L, a plate containing 16 colonies was prepared. Colonies were filtered from this plate according to the standard method.
541).

The above filter is attached to the fixing device 2-26, and the reaction tank 2-
I paid it within 25.

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.

Further, containers 2-1 to 2-8 correspond to the numbers in FIG. 2, and the compositions of the solutions are as shown in Table 1.

After carrying out the reaction according to the above-mentioned conditions, single-stranded DNA with a known concentration was spotted on the filter stepwise in order to measure the amount of DNA. After incubating at 80°C for 2 hours to fully fix the single-stranded DNA on the filter, it was stained with ethidium bromide and exposed to UV light. Main strand D
The presence of NA was confirmed.

In addition, nitrocellulose filters (Schleicher & 5
chuellBA85), reaction order 1 in Table 1
0. By performing the reaction by omitting 11, 12, and 13, the amount of DNA comparable to that of the Whatman 541 filter was confirmed.

[Example 2] First, conventional method (Molecular Cloning)
E. coli p2392 was infected with the phage according to the procedure, and plates were prepared with approximately 20 plaques per plate. Remove the plaque from this plate using the conventional method.
Transferred to a H atman 541 filter.

Next, a reaction was carried out in the same manner as in Example 1 according to Table 1, and when dyed with ethidium bromide, a sufficient amount of single-stranded D
The presence of NA was confirmed.

In addition, regarding both filters produced in Example 1.2,
When hybridization was performed using a biotin-labeled probe (having a sequence complementary to the DNA in the colonies and plaques of Example 1.2), extremely good color development was obtained in all filters.

〔Effect of the invention〕

When pretreatment prior to hybridization was performed using the apparatus of the present invention, good results were obtained without any manual intervention, a task that conventionally required a full-time operator over a day. As a result, a) the labor force was significantly reduced.

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

2) Extraction can now be carried out efficiently without a slight loss of NA.

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. Code 1-1 in the figure...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 2-1 to 2-8...Reaction liquid storage container 2 -9~2-16... Solenoid valve 2-17... Supply pump 2-18... Shaking stirrer 2-19... Temperature sensor 2-20... Controller 2-21... CPU 2-22... Heat exchanger 2-23... Filter 2-24... Drain pump 2-25... Processing tank 2-26... Filter fixture 2-27... Feeding represents a system. Start ↓ Listen to solenoid valve 2-13 ↓ Operate the supply pump for a specified period of time and fill the processing tank with 0.5M
Introduce NaOH/15M NaCl from container 2-5 ↓ Close solenoid valve 2-13 ↓ Wait for a predetermined time ↓ Operate the drain pump for a predetermined interval to suck and discard from the treatment tank ↓ Open solenoid valve 2-14 ↓ The supply pump is operated for a specified period of time, and 0.5M is added to the processing tank.
Th1s-HCI (pH8,0) / 15M Na
Introduce Cl from container 2-6 ↓ Close N-magnetic valve 2-14 ↓ Turn on the stirrer ↓ Wait for the specified time ↓ Turn off the stirrer Operate the drain pump for the specified period of time, suck it out from the treatment tank and dispose of it ↓ Dzuku The supply pump is operated for a predetermined time, and the lysozyme solution is introduced from the container 2-7 into the processing tank.The temperature maintenance circuit to the desired temperature is turned on.The temperature maintenance circuit to the desired temperature is turned off.The agitator is turned on, which closes the solenoid valve 2-15.The agitator is turned on for a predetermined time. Standby stirrer OFF Temperature maintenance circuit to maintain the desired temperature by operating the drain pump for a predetermined time, operating the supply pump for suction and disposal from the processing tank for a predetermined time, and introducing the BuO-tease solution from container 2-8 into the processing tank. ON Turns off the temperature maintenance circuit to the desired temperature Turns on the stirrer that closes the solenoid valve 2-16 Turns off the standby stirrer for a predetermined time The drain pump operates for a predetermined time to suck and dispose of water from the processing tank 3-section axis 7 Start ↓ Solenoid Open valve 2-13 ↓ Operate the supply pump for a specified time and fill the processing tank with 0.5M
Introduce NaOH/15M NaCl from container 2-5 ↓ Close solenoid valve 2-13 ↓ Wait for a predetermined time ↓ Operate the drain pump for a predetermined time. Suction and disposal from the treatment tank ↓ Open solenoid valve 2-14 ↓ Operate the supply pump for the specified time and add 0.5M ris-HCI (pH 8,0) / + 5M Nl to the treatment tank
Introduce lCl from container 2-6 ↓ Close fEa valve 2-14 ↓ Turn on the shaker ↓ Wait for the specified time ↓ Turn off the shaker ↓ Operate the supply pump for the specified time and pour the lysozyme solution into the processing tank in container 2 Turn on the temperature maintenance circuit to the desired temperature introduced from -7 Turn off the temperature maintenance circuit to the desired temperature Turn on the shaker that closes the solenoid valve 2-15 Turn off the standby shaker for a predetermined time Operate the drain pump for a predetermined time to remove the water from the processing tank Operate the mass supply pump for suction and disposal for a predetermined time, and introduce the protease solution into the processing tank from the container 2-8.Temperature maintenance port HON to the desired temperature.Ayi maintenance circuit OFF to the desired temperature.Close the solenoid valve 2-16. Shaker ON, standby shaking for specified time! OFF Operate the drain pump for the specified time, ↓ Continued

Claims (2)

[Claims] (1) Two treatment tanks consisting of one treatment tank covered with heat insulating material.
This is a device that separates double-stranded DNA into single-stranded DNA and immobilizes it on a support membrane, and it is used to separate phage plaques and bacterial colonies (hereinafter referred to as target microorganisms), or target microorganisms that have been transplanted. Means for mounting the sample held on the support membrane; a second solution for denaturing the target microorganism and neutralizing the solution, a lysis solution, a solution for DNA purification, and multiple types of washing solutions in multiple containers; Means for separately storing the reaction liquid (hereinafter collectively referred to as reaction liquid); Feeding means for sorting multiple types of reaction liquids and controlling the amount of reaction liquid flowing into the tank: heating and cooling of the reaction liquid in the tank Means for controlling the temperature of the reaction liquid during feeding, comprising:
Means for stirring or shaking the solution in the tank: Means for discharging the reaction liquid in the tank; 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; An automated device for extracting and immobilizing single-stranded DNA, comprising: (2) A solution that denatures the transferred material in a treatment tank consisting of one tank, in which one or more sheets of a sample in which the target microorganism is inoculated or the target microorganism itself is held on a support membrane; A second solution that neutralizes the solution, DNA
In the process of sequentially immersing DNA in a purification solution and multiple types of washing solutions to separate it into single-stranded DNA and immobilize it on a support membrane, the reaction is fed into the tank according to a signal set by the user. A method for extracting and immobilizing single-stranded DNA, characterized in that successive reactions are achieved by a device that controls the species and total flow rate of the liquid, the time interval for maintaining the tank, and the temperature in the tank.