JPH07140141A - Organismic component purifying apparatus and gene inspection apparatus - Google Patents

Abstract

PURPOSE:To provide a system for extracting, purifying and inspecting a DNA easily and efficiently without requiring any skill in which a DNA is extracted automatically from a cell when a solution containing cells is injected, and objective DNA is separated, demarcated and inspected. CONSTITUTION:After cell components are separated by a first separating means 10, the cell is subjected to cytoclasis by applying a high voltage pulse from a field generating means 20. Genes spring out from the nucleus of cell simultaneously with cytoclasis and subjected to membrane separation by a second separating means 30 before the genes are cut into fragments by further applying high voltage pulse. The fragmental genes are guided by a gene detecting means 40 while being separated by a small quantity at a time through electrophoresis thus measuring the DNA.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for efficiently extracting and purifying biological components such as genes, proteins and enzymes, particularly genomic DNA, from test samples, particularly human and animal cells. The present invention also relates to a genetic test device used for efficiently extracting and purifying genomic DNA from a test sample, particularly human and animal cells, and identifying the obtained genomic DNA.

[0002]

2. Description of the Related Art In recent years, it has become possible to analyze various diseases at a gene level with the remarkable development of gene manipulation technology. For example, after extracting genomic DNA from leukocytes in blood collected from peripheral blood vessels by a complicated method such as Ficoll method, Dot blot method, in situ
Analysis is performed using techniques such as the Hybridization method and Southern blotting method to diagnose genetic diseases.

However, according to the Ficoll method, "an equivalent amount of phosphate buffered saline (PBS, pH 7.4) is added to blood to which an anticoagulant such as heparin has been added, and the mixture is sufficiently mixed to contain a separating agent. Gently inject into a Spitz tube, overlay the diluted blood sample on the separating agent, centrifuge at 400 g for 30 minutes, collect the intermediate layer containing white blood cells, collect this in another Spitz tube, add PBS and mix. After washing, and then centrifuging this for 160 g for 10 minutes, further removing the supernatant, adding PBS and mixing and centrifuging for 100 g for 10 minutes, repeat the procedure twice, then remove the supernatant and suspend it in the required solvent. Finally, count the number with a microscope and adjust the number of white blood cells to the required number. "It is very complicated to operate, requires a long time, and requires skill of the operator. It was

In addition, Dot blot method and in situ Hybridi
The zation method has a feature that the measurement operation is easy and the operation time is relatively short, but it has a problem that the detection sensitivity is low. Southern
Although the blotting method has high detection sensitivity, it has a problem that the operation is complicated and takes a long time, and PCR is necessary to obtain sufficient sensitivity.
DNA of the site to be inspected by
It is supposed to be amplified by 100,000 times. The PCR method is extremely sensitive, but has a problem that contamination and operating conditions are complicated.

[0005]

However, in such a conventional gene purification method, there is a problem that the operation is complicated, complicated, and time-consuming, and the gene testing device is complicated with many devices. Since it requires various operations and conditions, there is a problem that contamination and reading errors occur.

The present invention has been made in view of the above circumstances, and provides an apparatus for extracting / purifying / inspecting biological components, particularly genes, which is easy to operate, requires no skill, and is efficient. Therefore, it is an object of the present invention to provide a system for automatically taking out DNA from a cell when a solution containing cells is injected, separating and fractionating the target DNA, and performing a DNA test.

[0007]

The problem to be solved by the present invention is to provide a first separating means for separating cell components from a test sample,
There is provided an electric field generating means for crushing cells in the obtained cell components under a high electric field to extract biological components from the cells, and a second separating means for separating the extracted biological components from the cells. However, the electric field generating means is achieved by a biological component refining device, which guides the extracted biological component so as to pass through the second separating means. As the biological component to be purified by the biological component purification device, genes (particularly genomic DNA), proteins, enzymes and the like can be applied.

Another problem to be solved by the present invention is that the first separating means for separating cell components from a test sample and the nucleated cells in the obtained cell components are crushed in a high electric field to remove the nucleated cells from the nucleated cells. The electric field generating means for extracting a gene, the second separating means for separating the extracted gene from the nucleated cell, and the gene detecting means for measuring the cleaved gene are included. It can also be achieved by a gene testing device characterized in that a gene extracted by an electric field generated is cut into small fragments.

Further, the problem to be solved by the present invention is that the first separating means for separating cell components from the test sample and the nucleated cells in the obtained cell components are crushed in a high electric field to remove the nucleated cells from the nucleated cells. An electric field generating means for extracting the gene, a second separating means for separating the extracted gene from the nucleated cell, and a cutting means for cutting the extracted gene into small fragments,
The present invention can also be achieved by a gene testing device, which has a gene detecting means for measuring a cleaved gene, and the electric field generating means guides the extracted gene to pass through the second separating means.

The first separating means performs dialysis using a dialysis membrane such as a hollow fiber membrane or fractionates only cell components using a cell-adsorptive membrane and then a solution containing almost no electrolyte. It is preferable to separate only the cell components by.

It is preferable that the electric field generating means is composed of two electrodes for generating a high voltage pulse, and both electrodes are disposed so as to sandwich the second separating means, and the second separating means is the first separating means. A hollow fiber having a pore diameter larger than that of the membrane of the separating means, one pole of the electric field generating means is installed in the hollow fiber of the second separating means, and the multipole is outside the hollow fiber of the second separating means. The electrodes may be installed, or the two electrodes may be installed with an electrostimulation-responsive membrane sandwiched therebetween, the pores being opened by electrostimulation.

The above-mentioned cutting means is preferably also used by the electric field generating means by applying a high voltage pulse, but the gene may be cut by introducing a restriction enzyme.

As the above-mentioned gene detecting means, a method of hybridizing with a probe labeled with a radiolabel, an enzyme or a fluorescent dye and performing a separation test by using an electrophoretic method can be used. Is most preferably used.

Further, as a conveying means for guiding the cleaved gene to the gene detecting means, the inside of the microchannel such as a groove provided between two glass plates or the inside of the capillary is provided with the gene inlet of the microchannel or the capillary. It is preferable that a voltage generator is provided from the above to the gene detection means, the gene is electrophoresed by the pulse voltage generated by the voltage generator, and the gene is transferred while being separated from other substances. At this time, it is preferable that the microchannel or the capillary is filled with a gel so that the gene can be easily fractionated.

[0015]

According to the present invention, according to the above-mentioned constitution, after the cell components containing nucleated cells such as white blood cells and lymphocytes are separated by the first separating means, cells are applied by applying a high voltage pulse by the electric field generating means. Destroy. At this time, by making the magnitude of the electric field and the time for which the electric field is applied to be specific, the gene is ejected to the outside of the cell nucleus at the same time as the cell destruction, and the ejected gene is membrane-separated by the second separating means, and then, A high voltage pulse is applied to cut the gene into small pieces. The small fragment gene is introduced into the gene detecting means while being separated into small amounts by electrophoresis, and the DNA is measured. The gene may be cleaved using a restriction enzyme.

[0016]

Embodiments of the present invention will be specifically described below with reference to the drawings.

(Embodiment 1) FIG. 1 shows the configuration of a genetic testing apparatus including a biological component purifying apparatus according to an embodiment of the present invention. In FIG. 1, 10 is a first separating means,
Reference numeral 11 is a hollow fiber membrane, 12 is a dialysate inlet for introducing a dialysate into the hollow fiber 11, 13 is a dialysate outlet, and 20 is an electric field generating means. Reference numeral 21 denotes a negative electrode of the electric field generating means, which is a hollow fiber membrane 31 of the second separating means 30 described later.
It is installed inside. 22 is a positive electrode, which is the hollow fiber membrane 3
It is installed outside 1. Reference numeral 30 denotes a second separating means, which is a hollow fiber membrane 31 having a pore diameter larger than that of the hollow fiber membrane 11 of the first separating means 10, a gene buffer solution for collecting a gene, and a gene buffer solution for adjusting pH. And an inflow port 32. The biocomponent purification device of the present invention is configured by the above-described components. Further, 40 is a gene detecting means, 41 is a DNA detecting sensor, 41 is a resonance frequency measuring means, 50 is a microchannel through which a purified gene passes, 5
1 is a voltage generator for guiding genes in the microchannel, 52
Indicates an introducing means for introducing the cleaved gene into the microchannel 51. By continuously assembling these with the gene purification device, the gene is purified from the test sample and the gene is measured. It becomes a genetic testing device that can continuously perform a series of operations up to.

The operation of the gene testing apparatus constructed as above will be described with reference to FIG. About 1 ml of the test sample 101 containing nucleated cells such as blood is injected into the hollow fiber membrane 11 in the first separating means 10, and the dialysate flowing from the dialysate inlet 12 prevents the purification in blood. Separation of electrolytes and proteins. Here, the hollow fiber membrane 11
Used a single hollow fiber membrane having an average pore diameter of 0.1 to 1 μm and a diameter of 0.1 to 2 mm. It may be a bundle of many, or flat membranes stacked in multiple stages. The solution containing the cell component 102 containing almost no electrolyte in this way has an average pore diameter of 1 to 10 μm and the first separating means 10 described above.
Has a larger pore diameter than the hollow fiber membrane 11 of
It is transported into the second separating means 30 constituted by a single hollow fiber membrane 31 of 2 mm. Cell component 102 transported
On the other hand, the negative electrode 21 and the positive electrode 22 of the electric field generator 20 pass through the holes 33 of the hollow fiber membrane 31 and the hollow fiber membrane 3
When a high voltage pulse is applied to the inside and outside of No. 1, the electric field is concentrated in the pores, so that the nucleated cells can be disrupted with a relatively small voltage. The negative electrode 21 has a diameter of 25 μm to 1
A cylindrical platinum electrode of about mm is preferable, and the positive electrode 22 is preferably a cylindrical platinum electrode so as to surround the hollow fiber membrane. Further, in the first embodiment, since the hollow fiber membrane is used, the shape of the electrode is as described above. However, when a flat membrane as shown in a second embodiment described later is used,
A plate electrode can be used for both the positive electrode and the negative electrode.
The high voltage pulse generated by the electric field generating means 20 is
By optimizing the magnitude and application time of the electric field, it is possible to cause the DNA 103 to be ejected as a gene to the outside of the cell membrane and further to the outside of the pores at the same time as cell destruction. As for the suitable magnitude of the electric field and the application time, the magnitude of the electric field is about 100 to 1000 V, and the application time is 10 μsec to 1 ms. At a voltage of 1000 V or higher or for a long time of 1 millisecond or longer, the temperature rises, which adversely affects cellular components including genes, and there is a possibility that accurate gene measurement cannot be performed.
At a voltage of 0 V or less or a short voltage of 10 μsec or less, crushing of nucleated cells does not occur, and DNA 103 may not be extracted. Furthermore, by injecting a buffer consisting of Tris-hydrochloric acid buffer having a pH of about 6 to 8 from the gene buffer solution inlet port 32 as a gene extract solution outside the hollow fiber membrane 31, the DNA 103 jumped out by the pressure of the buffer solution is removed. It is refluxed and collected in the direction of the gene detecting means 40. The buffer solution is preferably an EDTA buffer solution containing a deproteinizing agent such as proteinase K. Since a higher voltage pulse is applied near the positive electrode 22, the DNA 103 is cut into small pieces. When the cleavage is insufficient, by injecting a restriction enzyme from the gene extract inlet 32,
If it is preferable to set the magnitude of the electric field and the application time with emphasis on the above-mentioned cell disruption and extraction of the DNA 103, the restriction may be adjusted so that only the restriction enzyme is used for cleavage. The small fragment DNA is injected into the minute channel 50 formed between the two glass plates by the negative pressure generated by the introducing means 51, and a voltage is applied to the voltage generator 51 applied to both ends of the minute channel 50. Thus, the gel in the microchannel 50 is guided to the gene detecting means 40 by the crystal oscillator while separating the gel by electrophoresis, and the change of the resonance frequency is measured as the DNA measurement.

(Embodiment 2) FIG. 2 shows the construction of a gene testing apparatus including a biological component purifying apparatus according to a second embodiment of the present invention. In FIG. 2, 10 is a first separating means, 13 is a cell adsorbing membrane, 14 is a solution inlet for introducing a washing solution upstream of the cell adsorbing membrane 13, 20 is an electric field generating means, and 21 is the electric field. It is a negative electrode of the generating means, and is installed upstream of the electrical stimulus responsive film 34 of the second separating means 30 described later. Reference numeral 22 is a positive electrode, which is installed downstream of the electrical stimulus responsive membrane 34. Reference numeral 30 denotes a second separating means, and an electric stimulus responsive membrane 34 having pores opened by stimulation of the electric field generated by the electric field generating means 20 and a gene buffer solution for collecting genes and adjusting pH are added. It comprises a gene buffer inlet 32. The gene purification device of the present invention is constituted by the above-described components.
Further, 40 is a gene detecting means, 50 is a microchannel through which a purified gene passes, and 51 is a voltage generator that guides the gene in the microchannel. By assembling, the gene testing device can purify a gene from a test sample and continuously perform a series of operations from measuring the gene.

Since the operation of the gene testing apparatus constructed as described above is basically the same as that of the first embodiment of the present invention shown in FIG. 1, the same parts are designated by the same reference numerals for description. Will be omitted and only different points will be described. The feature of the gene testing device of the second embodiment is that by using the cell-adsorptive membrane 13, the nucleated cells are selectively fixed and easily separated from the test sample 101 containing the nucleated cells such as blood. This is a point that can be done. When the nucleated cells are adsorbed by the cell-adsorptive membrane 13, a washing solution containing almost no electrolyte is then injected from the solution inlet 14 to wash the nucleated cells, so that only the nucleated cells flow to the second separating means 30. Will be sent. In the second separating means 30, a high voltage pulse is applied by the electric field generating means 20 to crush the nucleated cells. At this time, the high-voltage pulse was also applied to the DNA at the same time as the cell destruction as in Example 1.
The magnitude of the electric field and the application time are set to be suitable so that 103 can be projected to the outside of the cell membrane. With the generation of the high-voltage pulse, the holes of the electrostimulation-responsive film 34 are opened, and the protruding DNA 103 is separated. Since the pores of the electric stimulus responsive film 34 are closed when there is no electric field, the possibility of passing unnecessary substances other than the DNA 103 is low. After that, the process in which the DNA 103 is further cut into small fragments and introduced into the gene detecting means 40 while separating the inside of the minute channel 50 by electrophoresis and the DNA measurement is performed is the same as in the first embodiment.

[0021]

As is apparent from the above description, the gene purifying apparatus and the gene testing apparatus of the present invention have an integrated system, so that they are easy to handle, and contamination and reading errors are less likely to occur. In addition, since the system can be downsized, the amount of sample can be small.
Furthermore, since the operation is simple, the quick measurement as compared with the conventional method enables simultaneous use in many kinds of tests.

Further, the cost can be kept low by using a membrane for the separating means of the present invention, and further, since the detection part can be regenerated, the inspection cost can be reduced by repeatedly using it.

From a medical point of view, according to the present invention,
It is expected that the elucidation of human genes will be accelerated, contribute to the etiology diagnosis and treatment of intractable diseases at the gene level, and be useful for human welfare.

Further, since the oncogene and the tumor suppressor gene can be efficiently elucidated, the range of choice of the method for preventing recurrence and onset is broadened. Further, there is an effect that an infectious disease such as HIV virus and HCV virus can be accurately and promptly diagnosed at a gene level and a fundamental treatment can be executed at an early stage.

[Brief description of drawings]

FIG. 1 is a diagram illustrating a gene testing device including a gene purification device according to a first embodiment of the present invention.

FIG. 2 is a diagram illustrating a gene testing device including the gene purification device according to the first embodiment of the present invention.

[Explanation of Codes] 10 First Separation Means 11, 33 Hollow Fiber Membrane 12 Dialysate Inlet 14 Solution Inlet 20 Electric Field Generating Means 21, 22 Electrode 30 Second Separation Means 34 Electrical Stimuli Responsive Membrane 40 Gene Detecting Means 50 Micro Channel

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12Q 1/68 Z 9453-4B G01N 1/28 (72) Inventor Katsuaki Kikuchi Inoguchi, Nakai-cho, Ashigaragami-gun, Kanagawa Prefecture No. 1500 Terumo Corporation (72) Inventor Sadayuki Sakuma No. 1500 Inoguchi, Nakai-cho, Ashigarakami-gun, Kanagawa Prefecture Terumo Corporation

Claims (2)

[Claims] 1. A first separating means for separating cell components from a test sample, and an electric field generating means for crushing cells in the obtained cell components in a high electric field to extract biological components to be purified from the cells. And a second separating means for separating the extracted biological component from the cells, and the electric field generating means guides the extracted biological component to pass through the second separating means. A biological component purifying device. 2. A first separating means for separating cell components from a test sample, and an electric field generating means for crushing nucleated cells in the obtained cell components with a high electric field and extracting a gene from the nucleated cells. And a second separating means for separating the extracted gene from the nucleated cell, and a gene detecting means for measuring the cleaved gene, wherein the gene extracted by the electric field generated by the electric field generating means is A genetic testing device characterized by cutting into small pieces.