JP2002340858A - Device and method for gene diagnosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a method for gene diagnosis capable of easily and accurately detecting a gene aberration of one base or more in a short time and reducing the seize, weight, and costs of the device for automating the diagnosis at a low running cost. SOLUTION: This gene diagnosis device is provided with a high temperature part splitting a double helix of a DNA sample and a separation part adjusted at a temperature lower than that of the high temperature part for separating the split single stranded DNA sample between a normal DNA, an abnormal DNA, and a noise DNA. In a buffer solution inside a sealed flow passage, the DNA sample and a separating DNA conjugate, in which a base sequence and a polymer compound bondable with the single stranded DNA sample via hydrogen bonds are bonded for separating the DNA sample between the normal DNA, the abnormal DNA, and the noise DNA according to a difference of bonding strength, are charged. When a constant voltage is applied, the DNA inside the sealed flow passage is electrophoresed, and a passage amount of the normal DNA and that of the abnormal DNA are measured respectively by means of a detection part.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene diagnosis apparatus and a gene diagnosis method capable of simply and accurately detecting the presence or absence of a gene abnormality.

[0002]

2. Description of the Related Art Inherited factors and environmental factors are involved in all diseases with various probabilities. However, inborn errors of metabolism, cancer, diabetes, hypertension, Alzheimer's, autoimmune diseases, atopy, obesity, etc. Diseases such as alcohol dependence have a very large proportion of genetic factors. On the other hand, diseases that greatly contribute to environmental factors include diseases caused by infectious diseases and sequelae of trauma.

In recent years, with the rapid progress of molecular biology, the involvement of genetic elements, that is, genes, in various diseases has been elucidated quite accurately, and attention has been paid to medical treatment targeting genes. It has become to. Currently, the ones that are receiving the most attention are those called SNPs (snips). This is a single
Nucleotide polymorphism is generally translated as "single nucleotide polymorphism" and is a generic term for the difference of one code (one nucleotide) in a gene between individuals.

[0004] All living organism genes on the earth, including humans (or genomes, which mean the entire collection of genes), share a common 4
It is composed of two bases, and various proteins are produced by the sequence of these bases, and life activities unique to each organism are performed. The four bases common to all organisms are adenine (denoted as A), guanine (denoted as G), thymine (denoted as T), and cytosine (denoted as C). It is said that human genes are composed of about 3 to 3.2 billion base sequences, but each individual has a base that differs from others by about one in hundreds to 1,000 bases. Exists. Usually, SNPs in which this single base change occurs at a frequency of 1% or more in the total population of a certain human population are called SNPs.

[0005] Therefore, in all genes (genomes), 30
It is said that there are 100,000 to 10 million SNPs,
Currently, the search for SNPs is ongoing worldwide. SNP
The reason why s has attracted attention is that it is thought that the classification of SNPs can statistically estimate the morbidity for many diseases that are said to involve the gene of each individual. For example, taking breast cancer as an example, by comparing SNPs between a group of patients having breast cancer and a normal group, it is possible to specify SNPs common to persons who are likely to have breast cancer. Then, at the time of a medical examination, it is possible to investigate the gene and find a person who has the SNPs, that is, a person who is currently normal but is likely to contract breast cancer in the future.

[0006] By this diagnosis, even if a person who is susceptible to breast cancer becomes susceptible to cancer by performing frequent examinations, he / she can be treated very early to improve the possibility of survival. The same can be said for lifestyle-related diseases such as diabetes and high blood pressure, and it becomes possible to make accurate dietary and lifestyle guidance before the onset of diseases that many people worldwide suffer.

[0007] SNPs are also expected to play an important role in drugs used for treating diseases. Drugs used in treatment are not equally effective for everyone. In general, it is widely known that drugs are effective for a certain percentage of people but have no effect for other people, and rather may cause adverse results due to side effects and the like. By the way, it is well known that side effects due to drugs rank high among the causes of death in the United States. It is said that the effect of the drug is deeply related to the constitution of the person, and that the constitution can also be distinguished by the classification of SNPs. In other words, it is possible to predict the effects and sensitivities of a certain drug in advance and to make an appropriate prescription by the classification based on the analysis of SNPs, and to administer the optimum drug to the individual constitution of the patient and the risk of side effects. Avoidance is expected. Such medical treatment is called tailor-made medical treatment or tailor-made medical treatment, and its practical application in the future is surely considered.

[0008] Further, it is known that cancer is caused by mutation of a specific site on a gene which plays an important role in normal cells, for example, by the action of ultraviolet rays or mutagenic substances. By reading a mutation on a specific gene, it becomes possible to diagnose at an early stage whether or not a cell is cancerous. The SNPs are also effective in identifying a criminal in a criminal investigation, determining an ambiguous parent-child relationship, and identifying whether or not the person is a person.
As mentioned above, each individual has between 3 and 10 million S
NPs exist, and each SNP is different from parents
It is said that there is no human on the earth who has exactly the same SNPs, even if they are parents and siblings, in order to take over s. This is the reason that allows complete identification of the individual.

[0009] As described above, observing a mutation occurring at one base in a specific gene may make a great contribution to various matters including medical treatment. However, at present,
The method of observing the mutation of a specific gene requires a very complicated operation or an expensive apparatus as described below, and the running cost is very high, so that it has not yet been widely used.

The most commonly used SNPs at present
Is a method called sequencing (determination of base sequence) in which the base sequence of DNA is read directly from the end. Since a gene is a unit of DNA having base sequence information for forming one kind of protein, SNPs can be clarified by reading the base sequence from the end. There are several reports on the method of performing sequencing, but the most commonly performed method is dideoxy sequencing (Sanger method) described below. All methods including this method are based on a technique capable of separating and discriminating a difference in length of one base length by denaturing polyacrylamide gel electrophoresis or capillary electrophoresis having high separation ability.

[0011] Dideoxy Sequencing (Sanger
Method), also called enzymatic sequencing, is characterized by the use of a DNA polymerase to synthesize the complementary strand of single-stranded template DNA, and the use of four artificially made dideoxynucleotides. It is. As a sequencing operation, single-stranded DNA whose base sequence is to be sequenced
Using a synthetic base sequence complementary to the 3 'end of the primer as a primer, an operation of extending a deoxynucleotide added evenly to the DNA polymerase from the primer by an enzyme reaction is performed. At this time, four reaction vessels are simultaneously prepared. In each case, a small amount of a base analog, dideoxynucleotide, which does not have a hydroxyl group at the 3 'end of the four bases of ATGC and therefore cannot continue the DNA extension reaction, is separately mixed. This stops DNA synthesis at the time the dideoxynucleotide was added to the end of the growing DNA, and each reaction vessel had various lengths, but the ends were always added base analogs. Strand DNA is formed. The reaction vessel is reacted with S1 endonuclease to digest all the single-stranded DNA to make only double-stranded DNA. The DNA strands of the four reaction vessels thus obtained are subjected to gel electrophoresis or capillary electrophoresis, and the separated DNAs are read in order from the shorter one (the one that moved faster), and the base sequence of the DNA complementary to the template strand is found. Understand. At this time, the result of the electrophoresis is identified, for example, by radioactively labeling the phosphorus or sulfur of the added dideoxynucleotide or by binding a fluorescent substance. In the case of a radioactive label, detection is performed by exposure to a film, and in the case of a fluorescent chemical substance, a laser beam is irradiated to detect fluorescence. Recently, a method has been developed in which four types of base analogs of A, T, G, and C are labeled with four types of reagents having different fluorescence wavelengths, and the four colors of fluorescence are simultaneously detected.

As described above, conventionally, the exact nucleotide sequence of a gene isolated and purified from a subject is determined by this dideoxy sequencing (Sanger method) or other nucleotide sequencing methods, and compared with a normal or standard nucleotide sequence. By doing so, the presence or absence of SNPs or the presence or absence of mutation is confirmed, and individual identification is performed.

[0013]

As described above,
Gene diagnosis and identification of individuals using genes using conventional gene sequencing methods are performed by isolating the target gene, amplifying and purifying it, and then using a gene sequencing device to identify the target gene. Since reading was performed by reading the nucleotide sequence, the experiment required a huge amount of work, a very long time, and a large running cost. Automated equipment for base sequence determination is very expensive, occupies a large space, and requires a large amount of expensive reagents.

Therefore, in order to solve such a conventional problem, the present invention is capable of detecting a gene abnormality of one or more bases in a short time, easily and accurately, and is very small, lightweight, inexpensive, and extremely. It is an object of the present invention to provide a gene diagnosis apparatus capable of automating diagnosis at a low running cost.

It is a further object of the present invention to provide a method for diagnosing a gene, which can easily and accurately determine a genetic abnormality of one or more nucleotides in a short time.

[0016]

According to the present invention, there is provided a gene diagnostic apparatus comprising: a first container and a second container which are connected to each other by filling a buffer containing a linear polymer and a DNA binding control agent with each other; A flow path, a control section for controlling a power supply section to apply a predetermined constant voltage between the second electrode and the first electrode,
And a detection unit for detecting the amount of passing air,
A high temperature section for separating the double helix of the A sample and a separation section adjusted to a temperature lower than the temperature of the high temperature section and separating the separated single-stranded DNA sample into normal DNA, abnormal DNA, and noise DNA are provided. And the buffer in the closed channel contains D
The base sequence capable of hydrogen bonding to the NA sample and the single-stranded DNA sample and the polymer compound are bound to each other.
The sample A is filled with a separation DNA conjugate for separating normal DNA, abnormal DNA, and noise DNA, and the DNA in the closed flow path is electrophoresed by applying a constant voltage. Are measured respectively.

[0017] This makes it possible to detect a gene abnormality of one or more bases in a short time, easily and accurately, and to be small, light, inexpensive and at a very low running cost.
Diagnosis can be automated.

Further, according to the gene diagnosis method of the present invention, the buffer is contained in the first container, the first electrode is immersed in the buffer,
A buffer is also contained in the container, and the second electrode is immersed in the container. The first container and the second container are filled with a buffer containing a linear polymer and a DNA binding control agent, and are connected to each other through a closed channel. A high-temperature portion for heating the DNA sample, a base sequence capable of hydrogen bonding to a single strand of the DNA sample, and a polymer compound in a buffer solution in the closed channel. Introducing a DNA conjugate for separation, which separates the DNA sample into normal DNA, abnormal DNA and noise DNA from the difference in binding force, applying a predetermined constant voltage between the second electrode and the first electrode, Separate the double helix of the DNA sample with, and electrophorese the buffer in the closed flow path.
And noise DNA, and the ratio of normal DNA to abnormal DNA or abnormal DNA is detected.

Thus, it is possible to easily and accurately determine a genetic abnormality of one or more nucleotides in a short time.

[0020]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The first aspect of the present invention provides a first container containing a buffer solution and immersed in a first electrode, and a second container containing a buffer solution and immersed in a second electrode. Applying a positive potential to the second electrode and applying a negative potential to the first electrode, a closed flow path that connects the first container and the second container with each other by filling the buffer containing the linear polymer and the DNA binding control agent with each other; A power supply unit, a control unit that controls the power supply unit and applies a predetermined constant voltage between the second electrode and the first electrode, and a detection unit that is provided in the closed channel and detects a passing amount of DNA passing through the inside. In the closed channel, a high-temperature portion for separating the double helix of the DNA sample, and a temperature of the high-temperature portion adjusted to be lower than that of the high-temperature portion, and the separated single-stranded DNA sample are separated from normal DNA, abnormal DNA, and noise. A separation unit for separating DNA is provided, and the buffer in the closed channel contains D A DNA conjugate for separating a DNA sample into a normal DNA, an abnormal DNA and a noise DNA based on a difference in binding strength between a base sequence capable of hydrogen bonding to the A sample and a single-stranded DNA sample and a polymer compound. The gate is filled, and by applying a constant voltage, the DNA in the closed flow path is electrophoresed, and the detection unit detects normal DNA and abnormal DN.
Since the genetic diagnostic apparatus is characterized by measuring the amount of A passing through, the double helix of the DNA sample can be separated in the high temperature part, and can be automatically converted into a single-stranded DNA sample. There is no need to make troublesome preparations.

Further, a predetermined constant voltage is applied between the second electrode and the first electrode, and the DNA sample can be electrophoresed. However, since the DNA conjugate for separation is bonded to a polymer compound, The migration speed is only a few% as compared with the DNA sample (however, it depends on the type and length of the polymer compound), and can be relatively immobilized. Therefore, the DNA sample is separated by electrophoresis into D
Pass through the NA conjugate portion. At that time, the migration speed of the normal DNA and the abnormal DNA group can be reduced with respect to the noise DNA by utilizing the difference in hydrogen bonding force with the DNA conjugate for separation. Therefore, noise DNA which is not affected by the DNA conjugate for separation can be migrated at almost the same speed.

Affinity DNA is known as a method for delaying a DNA sample. Affinity DNA is generally fixed to the wall of a closed flow path such as a capillary tube, and the fixing process is difficult. Although the closed channel must be disposable, according to the present invention, it is not necessary to disposable because it is pseudo-fixed,
It becomes extremely easy to adjust the concentration of each of the DNA conjugate for separation and the sample and the mixing ratio of the DNA conjugate for separation and the sample.

The normal DNA and the abnormal DNA move while being affected by the binding force of the DNA conjugate for separation, and the noise DNA which is not affected by this is migrated first when a voltage is applied and is moved by the detection unit. Is detected. However, the abnormal DNA has a binding force with the DNA conjugate for separation (however, it is weaker by one base than that of the normal DNA), so that the effect of the conjugate cannot be easily shaken, and the abnormal DNA is migrated later than the noise DNA. , After the noise DNA is detected by the detection unit. The normal DNA exerts the maximum binding force, is migrated later than the abnormal DNA as well as the noise DNA, and is finally detected by the detection unit. This can cause a difference in the detection time of the three DNAs.

As described above, since the electrophoresis and the hydrogen bonding of the DNA are utilized, the DNA can be obtained within a short time of several minutes to several tens of minutes.
Can be easily adjusted to an optimal voltage with a high resolution by simply applying a predetermined constant voltage, and the presence or absence of abnormality in DNA can be accurately detected.
An inexpensive device with light weight and low running cost can be provided, and automation of diagnosis is extremely easy.

According to a second aspect of the present invention, there is provided the gene diagnostic apparatus according to the first aspect, wherein at least one closed flow path is provided, so that the DNA conjugate is fixed to each closed flow path. DNA conjugate and DNA binding control agent are changed so that normal DNA and abnormal DN
The separation environment of A can be changed for each closed channel.

According to a third aspect of the present invention, there is provided the gene diagnosis apparatus according to the first or second aspect, wherein the DNA conjugate is a vinylated DNA. The DNA can be clearly separated and measured.

According to a fourth aspect of the present invention, there is provided the gene diagnosis apparatus according to any one of the first to third aspects, wherein the temperature of the high-temperature portion is adjusted to 90 ° C. or more.
The double helix of DNA can be separated by adjusting the temperature.

According to the fifth aspect of the present invention, the normal DNA and the abnormal DNA are adjusted by adjusting the liquid in the closed channel to 20 ° C. to 60 ° C.
The genetic diagnostic apparatus according to any one of claims 1 to 4, wherein NA is separated, so that normal DNA and abnormal DNA are bound to or separated from the DNA conjugate for separation by temperature control. It is possible to adjust the bonding force with the temperature, and it is possible to measure each of them after the separation by the detection unit.

According to a sixth aspect of the present invention, there is provided the gene diagnostic apparatus according to the first to fifth aspects, wherein the closed flow path is a fused silica capillary tube having an inner diameter of 50 to 100 μm. Can transmit more than 90% of ultraviolet rays,
Detection of abnormal DNA can be facilitated by detecting the amount of ultraviolet light that has passed.

According to a seventh aspect of the present invention, there is provided the genetic diagnosis according to any one of the first to sixth aspects, wherein the closed flow path is a combination of a plate having a groove and a plate transmitting at least 90% of ultraviolet rays. Because it is a device, it can transmit more than 90% of ultraviolet rays,
Detection of abnormal DNA can be facilitated by detecting the amount of ultraviolet light that has passed.

According to the invention described in claim 8, the first electrode is immersed in the first container containing the buffer solution, and the second electrode is immersed in the second container also containing the buffer solution. The container and the second container are filled with a buffer solution containing a linear polymer and a DNA-binding control agent and communicate with each other via a closed channel, and a high-temperature portion for heating the inside of the closed channel to a predetermined temperature is provided. In a buffer solution, a DNA sample, a base sequence capable of hydrogen bonding to a single strand of the DNA sample, and a high molecular compound are bound, and the DNA sample is separated from normal DNA, abnormal DNA, and noise DNA based on a difference in binding strength. Then, a positive potential is applied to the second electrode and a negative potential is applied to the first electrode, and a predetermined constant voltage is applied between the second electrode and the first electrode. To separate the double helix of the DNA sample at high temperature The buffer in the flow path are electrophoresed to separate the normal DNA and abnormal DNA and noise DNA, normal D
Since this is a genetic diagnostic method characterized by detecting the ratio of NA to abnormal DNA or abnormal DNA, it is possible to separate the double helix of the DNA sample at a high temperature part and automatically convert it into a single-stranded DNA sample. Yes, without the need for cumbersome preparations for measurements.

Further, a predetermined constant voltage is applied between the second electrode and the first electrode, and the DNA sample can be electrophoresed. However, since the DNA conjugate for separation is bonded to a polymer compound, The migration speed is only a few% as compared with the DNA sample (however, it depends on the type and length of the polymer compound), and can be relatively immobilized. Therefore, the DNA sample is separated by electrophoresis into D
Pass through the NA conjugate portion. At that time, the migration speed of the normal DNA and the abnormal DNA group can be reduced with respect to the noise DNA by utilizing the difference in hydrogen bonding force with the DNA conjugate for separation. Therefore, noise DNA which is not affected by the DNA conjugate for separation can be migrated at almost the same speed.

As a method of delaying the DNA sample, there is an affinity DNA, but the affinity DNA is generally fixed on the wall of a closed flow path such as a capillary tube, and the fixing treatment is difficult. Although the closed channel must be disposable, according to the present invention, it is not necessary to disposable because it is pseudo-fixed,
It becomes extremely easy to adjust the concentrations of the DNA conjugate for separation and the sample and the mixing ratio of the DNA conjugate for separation and the sample.

The normal DNA and the abnormal DNA move while being affected by the binding force of the DNA conjugate for separation, and the noise DNA which is not affected by the DNA conjugate migrates first when a voltage is applied and is moved by the detection unit. Is detected. However, the abnormal DNA has a binding force with the DNA conjugate for separation (however, it is weaker by one base than that of the normal DNA), so that the effect of the conjugate cannot be easily shaken, and the abnormal DNA is migrated later than the noise DNA. , After the noise DNA is detected by the detection unit. The maximum binding force acts on the normal DNA, and the DNA is electrophoresed later than the abnormal DNA as well as the noise DNA, and finally detected by the detection unit. This can cause a difference in the detection time of the three DNAs.

As described above, since the electrophoresis and the hydrogen bonding of the DNA are utilized, the DNA can be obtained within a short time of several minutes to several tens of minutes.
Can be separated, and it is only necessary to apply a predetermined constant voltage, so that the voltage can be adjusted to an optimum voltage with high resolution very easily, and the presence or absence of abnormality in DNA can be accurately detected.

Hereinafter, a gene diagnosis apparatus and a gene diagnosis method according to an embodiment of the present invention will be described with reference to the drawings.

(Embodiment 1) FIG. 1 is an external view of a gene diagnostic apparatus according to Embodiment 1 of the present invention, FIG. 2 is an external view of an open top of the gene diagnostic apparatus according to Embodiment 1 of the present invention, and FIG. FIG. 4 is a configuration diagram of an apparatus including an electrophoresis unit of the gene diagnosis apparatus according to Embodiment 1 of the present invention. FIG. 4 is a main part of a control circuit of the gene diagnosis apparatus according to Embodiment 1 of the present invention. FIG. 5 is a diagram showing the introduction state of a DNA conjugate for separation and a DNA sample in a closed flow channel of the genetic diagnostic device according to the first embodiment.

In FIG. 1, reference numeral 1 denotes a power switch of the gene diagnosis apparatus, 2 denotes operation buttons for performing various operations of the apparatus, 3 denotes a display panel, 4 denotes an upper lid, and 5 denotes a housing of the apparatus. 2, 3 and 4, reference numeral 6 denotes an electrophoresis section for performing electrophoresis, 7 denotes a support for supporting the electrophoresis section 6, 8
Is a control for performing electrophoresis and a suction pump 2 described later.
0, a control calculation unit including a board that controls the detection unit 15 and calculates the detected data. 9 is a power box,
9 a is a power supply unit provided in the power supply box 9. In the present gene diagnosis apparatus, there are different optimum applied voltage values for each type, concentration, and processing condition of DNA.
Abnormal DNA, normal DNA and noise DNA from A sample 23
The control operation unit 8 controls the power supply unit 9a to adjust the voltage to a value at which the highest resolution separation can be performed, so that the electrophoresis most suitable for the separation can be performed. 10 is a double helix of DNA (hereinafter, referred to as a double helix)
High-temperature section for separating two strands) into one strand, 11
Is a heat insulating part for making the temperature of the high temperature part 10 and the separation part 12 described later independent, and 12 is adjusted to a predetermined temperature to separate normal DNA, abnormal DNA and noise DNA,
This section separates the DNA into three DNAs, 1
Reference numeral 3 denotes a first temperature controller for adjusting the temperature of the high temperature section 10, and reference numeral 14 denotes a second temperature controller for adjusting the temperature of the separation section 12.

Although the details of the configuration inside the separation section 12 will be described later, at the start of measurement, as shown in FIG. 5, the DNA conjugate for separation 21 is located near the heat insulation section 11 from the position of the separation section 12, and the DNA sample 23 is It is arranged so as to be near the high temperature part 10. By performing the arrangement and voltage control, concentration adjustment, temperature control, and the like set as described above, the present gene diagnostic apparatus converts the DNA sample 23 into normal DNA, abnormal DNA, and noise DNA for several minutes while performing electrophoresis. Separates in minutes.

In the first embodiment, the DNA sample 23 is filled and arranged in the high-temperature section 10 with the double strand in the first embodiment. (5 ° C.). The double strands of the DNA introduced by this heating are automatically separated into single strands. Subsequently, the DNA conjugate 21 for separation is subjected to an electrophoretic action from the separation part 12 to the heat insulation part 11 due to a difference in the bonding force of hydrogen bond.
At least 10 ° C. lower than the temperature of the high-temperature section 10 so that normal DNA, abnormal DNA, and noise DNA can be separated.
That is, the temperature is controlled so as to be maintained at a predetermined temperature in a temperature range of 15 ° C. to 80 ° C., preferably 20 ° C. to 60 ° C. Normal DN
In order to separate A from abnormal DNA, it is preferable to control the temperature within a range of ± 1 ° C. from the predetermined temperature suitable for DNA separation. The heat insulating portion 11 is originally provided to thermally shut off the space between the separating portion 12 and the high temperature portion 10 to adjust the temperature. Is placed at an intermediate temperature between the temperature of 20 ° C. and 60 ° C. of the part 12, and the separation force of D
When NA conjugate 21 undergoes normal migration
Separate NA and abnormal DNA from noise DNA.

In FIGS. 3, 4 and 5, reference numeral 18 denotes a buffer for transporting charge during electrophoresis and stabilization of the pH of the DNA sample, and reference numeral 18a denotes a cathode-side container (book) containing the buffer 18. The first container of the invention), 18b is an anode-side container (second container of the present invention) for storing the buffer solution 18, and 18d is DN
This is a linear polymer gel obtained by adding an A-binding control agent and a linear polymer to a buffer solution 18.
No. 3 has a function of obstructing migration so as not to migrate at a high speed. With this function, the DNA sample 23 can have a chance to sufficiently encounter the DNA conjugate 21 for separation in the capillary tube 19. In addition, 1
Reference numeral 9 denotes a capillary tube (closed flow path of the present invention) for migrating the DNA sample 23 during electrophoresis, and reference numeral 20 denotes a buffer tube 18, a DNA sample 23, and a reagent such as a linear polymer gel 18d in a capillary tube 19. It is a suction pump for injecting. The electrode 17 is contained in the buffer solution 18 of the container 18a.
Is immersed, and the electrode 16 is immersed in the buffer solution 18 of the container 18b. Buffer 18 in containers 18a and 18b
Are communicated by the buffer solution 18 in the capillary tube 19. Therefore, the buffer solution 18 is also mixed in the DNA conjugate for separation 21 as a base of the solution, and the buffer solution 18 of the same concentration is present in all parts of the capillary tube 19. When a voltage is applied between the electrodes 16 and 17, electrophoresis is induced in the capillary tube 19, and the separation DN
Since the A conjugate 21 and the DNA sample 23 are negatively charged, the DN for separation is transferred from the container 18a to the container 18b.
The A conjugate 21 and the DNA sample 23 move,
Since the separation DNA conjugate 21 has a high molecular weight polymer, the DNA sample 2
There is almost no movement compared to the movement amount of 3.

Next, the details required for performing the measurement with the gene diagnosis apparatus of the present invention will be described. The DNA sample 23 measured by the present gene diagnostic apparatus is DNA obtained from human cells, blood, or the like. It is necessary to cut out a target portion to a target length from a human genomic DNA which is said to have about 3 billion base pairs by a method such as PCR (polymerase chain reaction) and increase the number for measurement. However, since this PCR is not necessary for the description of the present gene diagnosis apparatus, a specific description is omitted.

The number of target DNAs extracted by PCR or the like is about 6 to 1000 in terms of the number of bases.
In human genomic DNA, said to be 100 million base pairs,
The number that can be considered stochastically that the same DNA sequence does not exist may be about 50. However, this means that the total number need only be about 50 pieces. Therefore, when DNA is cut out several times, it may be divided into 6 to 12 pieces. And according to our experiments,
Abnormal DN with one base difference from normal DNA with this gene diagnosis device
In the case of separating A, the result that the number of bases of 6 to 12 can be separated most efficiently is obtained. However, the separation of the abnormal DNA is performed at the concentration of the DNA sample 23 (μM,
M = mol / liter), DNA conjugate 2 for separation
Since there is a close relationship with the DNA concentration (mol%), measurement temperature, etc. of No. 1, separation will not occur unless proper conditions are set, so it should be noted whether such conditions can be set. There is a need. For example, the concentration of the DNA sample 23 is appropriately 1 to 10 (μM), and the DNA contained in the DNA conjugate for separation 21 is 0.0002 to 0.05 (molar) with respect to the mole of the basic linear polymer. %) Is appropriate, but the total amount of DNA in the DNA conjugate for separation 21 is 20 to 6 times the concentration of the DNA sample 23.
It is desirable to make it 00 times. As described above, the DNA sample of the present gene diagnostic apparatus requires a DNA sample having about 6 to about 1000 base sequences obtained by pretreatment by the above-described PCR or the like, and appropriate condition setting is required. .

Next, the capillary tube 19 shown in FIGS. 3, 4 and 5 will be described. If a gel is put in the capillary tube 19 and electrophoresis is performed, a liquid flow called an electroosmotic flow is generated. In the present gene diagnosis apparatus, it is necessary to prevent this phenomenon from occurring. Therefore, it is preferable to coat the inner wall of the capillary tube 19 with acrylamide or the like. As long as the generation of electroosmotic flow can be prevented, another coating method may be used. For example, a commercially available coated capillary tube may be used. The capillary tube 19 has an inner diameter of 50 to
A fused silica capillary tube of 100 μm is most suitable because it can transmit 90% or more of ultraviolet rays and, when detecting DNA using ultraviolet rays as described later, can easily detect the passing amount of ultraviolet rays. Even if a closed channel corresponding to the capillary tube 19 is constituted by a combination of a plate having a groove and a plate transmitting ultraviolet rays of 90% or more, the ultraviolet rays can be transmitted by 90% or more, and the amount of the ultraviolet rays is detected. Thus, abnormal DNA can be easily detected. In addition, when the plate with the groove is a plate through which ultraviolet light can be transmitted, transmitted light is detected. When the plate with a groove is a plate through which ultraviolet light is not transmitted, abnormal DNA is detected by reflected light. When detecting the reflected light, it is necessary to devise a rectangular cross section in order to make the groove shape a uniform reflecting surface.

The buffer 18 contained in the containers 18a and 18b and in the capillary tube 19 is made of Tris-Borate.
(About pH 7.2 to pH 8) It is appropriate to use a buffer or the like. Among these, as a DNA binding control agent mixed as necessary in the buffer solution 18 contained in the capillary tube 19, a DNA conjugate to the separation DNA conjugate 21
A binding promoter such as magnesium chloride for promoting the binding of
There are two types of release agents, such as urea, that promote release. This 2
Various kinds of DNA binding control agents can control various migration speeds for DNA by selecting two kinds of mixing ratios and substances (for example, other electrolytes as a binding promoter). As the linear polymer contained in the linear polymer gel 18d, polyacrylamide can be used, and this is also used for preparing the conjugate of the DNA conjugate 21 for separation.

Next, regarding the order of filling the capillary tube 19, as shown in FIG. 5, a linear polymer gel 18d containing a linear polymer, a DNA binding control agent and a buffer 18 is first introduced into the capillary tube 19. Subsequently, a DNA conjugate 21 for separation described in detail below is added. The linear polymer gel 18d may be introduced after the introduction of the DNA conjugate 21 for separation. In the case of FIG. 5, it is introduced in a separated state after the introduction. Thereafter, the DNA sample 23 diluted with the buffer solution 18 or pure water is introduced and electrophoresed. DNA
The linear polymer gel 18d may be introduced after the introduction of the sample 23, and in the case of FIG. 5, it is introduced in a separated state after the introduction.

In the first embodiment, since acrylamide and DNA are polymerized to form the DNA conjugate 21 for separation, the difference in weight and structure between the DNA and the DNA is large. Speed (0.
6 cm / min to 0.7 cm / min) is 1/20 to 1/20 of the optimal migration speed of DNA (13 cm / min to 20 cm / min).
A state is realized in which the movement is very slow and is relatively pseudo fixed. Therefore, at the start of the measurement, the DNA conjugate for separation 21 is adjusted while adjusting the position of the capillary tube 19 containing the filler among the high-temperature section 10, the heat-insulating section 11, and the separation section 12.
From the separating section 12 to the vicinity of the heat insulating section 11, and the high temperature section 10
, A DNA sample 23 described later is arranged. In this way, the double strand of the DNA sample 23 is
Can be separated into main strands. Since the DNA conjugate for separation 21 was filled from the heat insulating section 11 to the separation section 12, normal DNA and abnormal DNA were separated at a speed difference, and the noise DN was increased.
A can also be separated.

The capillary tube 1 of the gene diagnosis apparatus
9, a mechanism for exchanging each solution of the DNA sample 23, the DNA conjugate for separation, and the linear polymer, for example, a suction pump 20 shown in FIG.
It is desirable to equip a container for storing each of the solutions 3 and the DNA conjugate for separation and the linear polymer, and a mechanism for automatically exchanging the container and connecting it to the capillary tube 19.

Further, the capillary tube 19 is unitized in units of one or a plurality of tubes, and this is used as a replacement sealed channel cartridge for separation so that it can be attached to the separation unit 12, the high temperature unit 10, and the heat insulation unit 11, and the separation unit Second temperature controller 1 in 12
It is also appropriate to control the temperature by the first temperature controller 13 in the high temperature section 4 or 4. The capillary tube 19 is previously filled with the buffer solution 18 containing the linear polymer gel 18d and the DNA binding control agent, and the DNA conjugate for separation 21 and the DNA sample 23 are filled in a separated state to prepare a closed channel cartridge for separation. It will be possible to keep. With this configuration, each time the measurement is performed, the entire separation channel cartridge is replaced. Therefore, the separation DNA conjugate 2 is used for each separation channel cartridge.
1. Since the arrangement of the DNA sample 23 can be set in advance, measurement can be performed easily and accurate measurement can be performed. In this separation channel cartridge for separation, in order to separate the DNA conjugate 21 for separation and the DNA sample 23, the linear polymer gel 18d is filled between them. Each of them may be filled with a mixture of the linear polymer gel 18d.

Next, the DNA conjugate for separation will be described. FIG. 6 is a conceptual diagram showing the relationship between a DNA conjugate for separation and a DNA sample in a closed channel of the genetic diagnostic apparatus according to Embodiment 1 of the present invention. In FIG.
21 is a DNA conjugate for separation. There are two types of DNA, one that forms a double strand and one that separates it. The four bases A, T, C, and G of the DNA form hydrogen bonds between A and T, and G and C, respectively. It has an easy property, and AT and GC form a pair in the double strand of DNA. Therefore, the single-stranded DNA is ATCGCGTCTA
When it is sequenced as GC (described in SEQ ID NO: 1), the DNA of the remaining strand has a base sequence of TAGCGCAGATCG (described in SEQ ID NO: 2). This relationship is called a complementary relationship. As long as the complementary relationship is satisfied, A and T, G and C are respectively bonded by hydrogen bonds,
Form a double strand. In the present invention, in order to utilize this relationship, as shown in FIG.
Has a base sequence complementary to the normal DNA of the DNA sample. Therefore, the normal DN of the DNA sample
The nucleotide sequence of A is ATCGCGTCTAGC (SEQ ID NO: 1
Described above), and the abnormal DNA is ATCA ^* CGTCTA
In GC (described in SEQ ID NO: 3), normal D
If the bases of NA and abnormal DNA are different,
If the sequence of the DNA portion of the NA conjugate 21 (first base sequence of the present invention) is TAGCGCAGATCCG (described in SEQ ID NO: 2), the abnormal DNA is not complementary to the DNA conjugate 21 for separation in A ^* , No binding occurs, and the binding force of the entire hydrogen bond is larger by one base in the normal DNA of the DNA sample than in the abnormal DNA. As a result, the normal DNA binds to the separation DNA conjugate 21 with a stronger binding force than the abnormal DNA for a longer time during the electrophoresis described later.
A becomes relatively slower than abnormal DNA. This is because not only the binding force at the time of electrophoresis but also the separation force by electrophoresis acts, so that a large number of DNAs are in a binding state in which binding and detachment are repeated intermittently. When this migration speed is averaged, the normal DN binding for a long time is
The electrophoresis speed of A is lower than that of abnormal DNA. Among the double-stranded DNAs, some DNA samples have a normal D such as the remaining separated single-stranded DNA which is not the object of measurement.
DNA other than NA and abnormal DNA is also contained.
NA acts as noise at the time of electrophoresis described later, and this noise DNA is used as the DNA conjugate 2 for separation.
Since it does not bind to the DNA of Example 1 with almost no reaction, it is separated with the fastest migration speed. It is appropriate that the total DNA amount of the DNA conjugate for separation 21 is 20 to 600 times the concentration of the DNA sample.

Next, a method for preparing and synthesizing such a DNA conjugate for separation 21 will be described. To synthesize vinylated DNA, the separation DN
The DNA for A conjugate 21 is cut out. In the above example, TAGCGCAGATCCG (described in SEQ ID NO: 2)
Is the DNA of the DNA conjugate for separation. next,
The 5 'end of the DNA having the desired base sequence is aminated (usually via a hexyl group). The aminated DNA thus obtained is diluted by adding ultrapure water sterilized to 2.6 mM. Next, MOS
U (methacryloid oxysuccinimide) 71.3
Dilute with DMSO (dimethylsulfoxide) to 88 mM. Then, the aminated DNA thus obtained and the MOSU are added and adjusted so as to have a ratio of 1:50. Further, to this adjusted solution, a solution adjusted with sodium hydrogen carbonate and sodium hydroxide so as to have a pH of 9 for pH adjustment is added in an amount equal to the amount of the aminated DNA.

Then, the obtained solution is shaken overnight.
Then, HPLC (High Performance)
Using Liquid Chromatography (High Performance Liquid Chromatography), the vinylated DNA in the shaken solution is separated from the aminated DNA, MOSU, etc. Vinylated DNA was used as eluent (TEAA;
Triethylamine-mixed solution of acetic acid and acetonitrile)
, And further concentrated under reduced pressure using a centrifugal evaporator having a vacuum drying function.

Next, 10% AAM which is a polymerization solution
(Acrylamide) is replaced with 53 μmol nitrogen. Furthermore, 1.34% of TEMD (N, N,
N'N'-tetramethylethylenediamine) is diluted with sterilized ultrapure water degassed by ultrasound. This is diluted with sterilized ultrapure water of 1.34% APS (ammonium persulfate), which is a polymerization initiator, similarly degassed by ultrasonic waves. Further, the concentrated vinylated DNA is diluted with sterilized ultrapure water. Then, in order to synthesize 100 μl of the separation DNA conjugate, 34 μl of the above AAM, 5 μl of each of the above TEMD and APS, and 0.01% to 0.05% mol of the vinylated DNA with respect to the AAM. Add to be 1
Add sterile ultrapure water to a volume of 00 μl and leave it for about 60 minutes.
A conjugate is obtained.

Incidentally, if gel filtration with an appropriate pore size selected in order to remove unreacted vinylated DNA is carried out several tens of times, a highly pure DNA conjugate can be obtained.

Next, the operation of the gene diagnosis apparatus according to the first embodiment will be described. First, D
A capillary tube 19 into which the NA sample and each solution are introduced is set, and containers 18a and 18 are provided at both ends as shown in FIGS.
Soak buffer 18 in b. A predetermined voltage is applied between the electrode 16 and the electrode 17 by the power supply unit 9a. The optimum voltage is preferably 10 to 20 kV, but may be 100 to 30 kV depending on the state of the electrolyte and the electrodes. For example, when the electrolyte concentration is low or the electrode area is large, the voltage is set to a low voltage. Then, instead of applying a predetermined voltage from the beginning, before applying a constant voltage, a high voltage of 30 kV or more for preparation is applied, and a noise DN is applied.
If A is separated quickly, the measurement can be performed quickly.

Here, the reason why the predetermined voltage is applied will be described.
The difference between the binding force of the A sample and the separation force due to the electrophoretic force is
Because it has a great effect on the migration speed and migration difference of DNA,
This is because it is necessary to perform electrophoresis by applying a predetermined constant voltage at which the separation of normal DNA, abnormal DNA, and noise DNA is performed at the highest resolution. Therefore, the optimum voltage to be applied is checked in advance for each DNA and each condition, and a preliminary voltage of about 30 kV or more is initially applied for a short time, and then this voltage is applied. Since the DNA in the capillary tube 19 is negatively charged and proceeds to the anode side, the power supply unit 9a needs to apply the electrode 16 so as to have a positive potential and the electrode 17 to have a negative potential.

The above-mentioned predetermined constant voltage is higher than a voltage at which normal DNA which is most difficult to migrate can be electrophoresed. When a high voltage is applied, the resolution of normal DNA and abnormal DNA is reduced. Must. Therefore, the most appropriate voltage value is near the voltage at which normal DNA can be electrophoresed. Further, in the electrophoresis, if the electrophoresis time is too long, the buffer 1
Since 8 is dried, it is necessary to avoid running the electrophoresis longer than necessary.

Next, a description will be given of the high-temperature section 10, the separation section 12, and the heat insulating section 11 in which DNA separation is performed, which is a feature of the present invention. As described above, the first temperature controller 13 controls the temperature to be 90 ° C. or more (predetermined temperature ± 5 ° C.) in order to separate the DNA double strands in the high temperature section 10. Also, the separation unit 12
In this case, it depends on the condition of the DNA sample.
The temperature of the high temperature part 10 is lower than the temperature of the high temperature part 10 by about 10 ° C., that is, 15 ° C. to 80 ° C., preferably 20 ° C., so that normal DNA, abnormal DNA, and noise DNA can be separated based on the difference in binding strength between the NA conjugate and the DNA sample. It must be kept at a predetermined temperature in the temperature range of ６０60 ° C. In the gene diagnostic apparatus of the first embodiment, a silicon rubber heater, a nichrome wire or the like, and a temperature sensor such as a thermocouple or a thermal are arranged below the separation unit 12 covering the capillary tube 19.
The second temperature controller 14 manages the temperature to be equal to or lower than a predetermined temperature ± 1 ° C. However, depending on the environmental temperature, the room temperature may exceed the intended predetermined temperature, and it is preferable to use a heatable / coolable component such as a Peltier element instead of a silicon rubber heater or a nichrome wire. Further, the heat insulating portion 11 is made of a heat insulating material such as glass wool, a foaming agent, mica, porous ceramics, or the like.
You may use what evacuated the content. Heat insulation part 1
In the vicinity of 1, since the temperature becomes an intermediate temperature between the high temperature part 10 and the separation part 12, the normal DNA and the abnormal DNA can be separated from the noise DNA by the separation DNA conjugate 21 using this.

Subsequently, the DNA conjugate for separation 21
Causes a difference in migration speed due to the action of
How to detect A, abnormal DNA, and noise DNA will be described. FIG. 7 is a diagram showing the relationship between the elapsed time and the absorbance when the elapsed time from the start of electrophoresis is set on the horizontal axis and the absorbance is set on the vertical axis. The detection is performed by measuring the absorbance when the ultraviolet irradiation is shielded by normal DNA, abnormal DNA, and noise DNA. In Embodiment 1, FIG.
As shown in ( ₂₎ , the glass part is exposed at a part of the capillary tube 19, ultraviolet rays having a wavelength of 260 nm are irradiated from the D2 lamp 15a, the ultraviolet irradiation light obtained at this time is detected by the photodiode 15b, and the absorbance is measured. I have. The power supply unit 9a is controlled by the control calculation unit 8 to control the D ₂ lamp 15a.
Is emitted, and the current detected by the photodiode 15b is amplified by the preamplifier 15c, and converted into a digital amount by the A / D converter 15d and converted into an absorbance by the control operation unit 8.
The control calculation unit 8 has a built-in timer (not shown) and can measure the elapsed time from the electrophoresis start time. In FIG. 7, the earliest time (I) is noise DNA that does not bind to the DNA conjugate, the medium time (II) is abnormal DNA, and the time is late (III) is normal DNA.

When the peak height, the time, and the number of peaks are read from this relationship diagram, it can be seen from the number of peaks that the DNA sample contains abnormal DNA. That is, whether or not abnormal DNA is present can be determined from the number of peaks. The number of peaks may be changed depending on the applied voltage, the DNA concentration, and the length of DNA (the number of bases).
In this case, the abnormal DNA
Measure only the amount of passing. The comparison of the peak heights indicates the difference in absorbance between two sets having a migration speed difference in DNA electrophoresed under the same conditions.
And the normal DNA, and the existence ratio can be determined. The determination of the amount of abnormal DNA may be performed by inputting standard data obtained from the detected peak waveform of the standard DNA sample into the control calculation unit 8 in advance, comparing the data with the measured data, and converting the data.

As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the first embodiment, a DNA sample obtained by extracting a specific DNA with a restriction enzyme or the like from DNA extracted from cells, blood, or the like. Genetic diagnosis and genetic determination can be performed by knowing the ratio of the presence of normal DNA and abnormal DNA contained therein.

[0062]

As described above, according to the present invention, the following advantageous effects can be obtained.

The genetic diagnostic device according to claim 1 is
The double helix of the DNA sample can be separated in the high temperature part to automatically make a single-stranded DNA sample, and there is no need to perform cumbersome preparations for measurement. In addition, a predetermined constant voltage is applied between the second electrode and the first electrode, and the DNA sample can be electrophoresed. However, since the DNA conjugate for separation is bonded to a polymer compound, the electrophoresis speed is high. DNA
Compared to a sample, it is only a few percent (however, it depends on the type and length of the polymer compound), and can be relatively quasi-fixed. Therefore, the DNA sample passes through the DNA conjugate for separation by electrophoresis. At that time, the migration speed of the normal DNA and the abnormal DNA group can be reduced with respect to the noise DNA by utilizing the difference in hydrogen bonding force with the DNA conjugate for separation. Therefore, noise DNA not affected by the separation DNA conjugate
Can be run at almost the same speed.

As a method for delaying the DNA sample, there is an affinity DNA, but the affinity DNA is generally fixed on the wall surface of a closed flow path such as a capillary tube, and the fixing treatment is difficult. Although the closed channel must be disposable, according to the present invention, it is not necessary to disposable because it is pseudo-fixed,
It becomes extremely easy to adjust the concentration of each of the DNA conjugate for separation and the sample and the mixing ratio of the DNA conjugate for separation and the sample.

The normal DNA and the abnormal DNA are affected by the binding force of the separating DNA conjugate while moving, and the noise DNA which is not affected by the binding is detected by the detection unit first when a voltage is applied. The abnormal DNA starts to migrate next after the noise DNA, and the normal DNA starts to migrate after a further delay, and is finally detected by the detection unit. This can cause a difference in the detection time of the three DNAs. The use of electrophoresis and hydrogen bonding of DNA allows DNA to be separated in a short time of several minutes to several tens of minutes, and can be easily adjusted to an optimal voltage with high resolution simply by applying a predetermined constant voltage. The presence / absence of DNA abnormality can be accurately detected, a small-sized, light-weight, low-running-cost inexpensive apparatus can be obtained, and automation of diagnosis is extremely easy.

Thus, with respect to diseases caused by mutations of various genes such as cancer, diabetes, hypertension, and Alzheimer's disease, it is possible to predict the risk of the onset of the disease and to prevent the disease before the onset or to detect it very early. it can.
In addition, by investigating the genetic diversity of each individual,
By estimating the side effects and the effects of the drug in advance, it is possible to provide the most suitable drug for each individual. In addition, mutations in genes collected from tissues that have undergone mutation and have become cancerous can be quickly determined and used. An individual can be specified, and a genetic diagnostic apparatus can be given definitive reliability for criminal investigation, identification of parent-child relationship, and security of each individual.

The genetic diagnostic device according to claim 2 is
Since one or more closed flow paths are provided, DN
A conjugate and DNA conjugate for fixation, DN
By changing the A-binding control agent, the separation environment between the normal DNA and the abnormal DNA of the DNA sample can be changed for each closed channel.

[0068] The genetic diagnostic device according to claim 3 is
Since the DNA conjugate is a vinylated DNA, it is possible to clearly separate and measure normal DNA and abnormal DNA of the DNA sample.

The gene diagnostic apparatus according to claim 4 is
Since the temperature of the high temperature part is adjusted to 90 ° C or more, DNA
Can be separated by adjusting the temperature.

[0070] The genetic diagnostic apparatus according to claim 5 is
The separation unit adjusts the liquid inside to 20 ° C to 60 ° C and returns to normal D
Since normal DNA and abnormal DNA are separated, normal DNA and abnormal D
The NA can be bound to or detached from the DNA conjugate for separation by adjusting the temperature, the binding force can be adjusted with the temperature, and each can be measured by the detection unit after separation.

The genetic diagnostic device according to claim 6 is
Since the closed channel is a fused silica capillary tube having an inner diameter of 50 to 100 μm, it can transmit 90% or more of ultraviolet rays, and can detect abnormal DNA easily by detecting the amount of passed ultraviolet rays.

The gene diagnostic apparatus according to claim 7 is
Since the closed channel is a combination of a plate having a groove and a plate transmitting ultraviolet rays of 90% or more, the ultraviolet rays can be transmitted by 90% or more, and abnormal DNA can be easily detected by detecting the amount of transmitted ultraviolet rays.

The method for gene diagnosis according to claim 8 is
The double helix of the DNA sample can be separated in the high temperature part to automatically make a single-stranded DNA sample, and there is no need to perform cumbersome preparations for measurement. In addition, a predetermined constant voltage is applied between the second electrode and the first electrode, and the DNA sample can be electrophoresed. However, since the DNA conjugate for separation is bonded to a polymer compound, the electrophoresis speed is high. DNA
Compared to a sample, it is only a few percent (however, it depends on the type and length of the polymer compound), and can be relatively quasi-fixed. Therefore, the DNA sample passes through the DNA conjugate for separation by electrophoresis. At that time, the migration speed of the normal DNA and the abnormal DNA group can be reduced with respect to the noise DNA by utilizing the difference in hydrogen bonding force with the DNA conjugate for separation. Therefore, noise DNA not affected by the separation DNA conjugate
Can be run at almost the same speed.

As a method for delaying the DNA sample, there is an affinity DNA, but the affinity DNA is generally fixed on the wall surface of a closed flow path such as a capillary tube, and the fixing treatment is difficult. Although the closed channel must be disposable, according to the present invention, it is not necessary to disposable because it is pseudo-fixed,
It becomes extremely easy to adjust the concentration of each of the DNA conjugate for separation and the sample and the mixing ratio of the DNA conjugate for separation and the sample. Normal DNA and abnormal DNA move while being subjected to the action of the binding force of the DNA conjugate for separation, and noise DNA that is not affected by this action is detected first by the detection unit when a voltage is applied. The electrophoresis starts next after the noise DNA, and the normal DNA starts electrophoresis after a further delay, and is finally detected by the detection unit.
This can cause a difference in the detection time of the three DNAs. The use of electrophoresis and hydrogen bonding of DNA allows DNA to be separated in a short time of several minutes to several tens of minutes, and can be easily adjusted to an optimal voltage with high resolution simply by applying a predetermined constant voltage. The presence or absence of abnormality in DNA can be accurately detected.

According to this genetic diagnosis method, it is possible to predict the risk of the onset of a disease caused by a gene mutation, to prevent the disease and to detect it very early. In addition, by investigating the diversity of the genes possessed by each individual, it is possible to infer in advance whether there are side effects and effects of the drug,
It is possible to provide the most suitable medicine to each individual. Furthermore, mutation of a gene collected from a cancerous tissue can be quickly determined. Identify individuals and provide definitive credibility in criminal investigations, parent-child relationships, and individual security.

[0076]

[Sequence List] SEQUENCE LISTING <110> Matsushita Electric Industrial Co., Ltd. et al. <120> The apparatus and method for genetic testing <130> 2913040055 <140> <141> <150> JP P2001-047812 <151> 2001-02-23 <160> 3 <170> PatentIn Ver. 2.1 <210> 1 <211> 12 <212> DNA <213> Homo sapiens <400> 1 atcgcgtcta gc 12 <210> 2 <211> 12 <212 > DNA <213> Homo sapiens <400> 2 tagcgcagat cg 12 <210> 3 <211> 12 <212> DNA <213> Homo sapiens <400> 3 atcacgtcta gc 12

[Brief description of the drawings]

FIG. 1 is an external view of a gene diagnosis apparatus according to Embodiment 1 of the present invention.

FIG. 2 is an external view of an upper lid of the genetic diagnosis device according to the first embodiment of the present invention;

FIG. 3 is a device configuration diagram including an electrophoresis unit of the gene diagnosis device according to the first embodiment of the present invention.

FIG. 4 is a main part of a control circuit of the gene diagnosis apparatus according to the first embodiment of the present invention.

FIG. 5 is an introduction state diagram of a DNA conjugate for separation and a DNA sample in a closed channel of the genetic diagnostic apparatus according to Embodiment 1 of the present invention.

FIG. 6 is a conceptual diagram showing the relationship between a DNA sample and a DNA conjugate for separation in a closed channel of the genetic diagnostic device according to the first embodiment of the present invention.

FIG. 7 is a diagram showing the relationship between elapsed time and absorbance.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 Power switch 2 Operation button 3 Display panel 4 Top lid 5 Case 6 Electrophoresis part 7 Support stand 8 Control calculation part 9 Power supply box 9a Power supply part 10 High temperature part 11 Heat insulation part 12 Separation part 13 1st temperature controller 14 2nd temperature regulator 15 detector 15a D ₂ lamp 15b photodiode 15c preamplifier 15d A / D converter 16, 17 electrode 18 buffers 18a, 18b container 18d linear polymer gel 19 capillary tube 20 the suction pump 21 for separation DNA conjugates 23 DNA samples

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 ZNA G01N 33/483 F C12Q 1/68 33/50 P G01N 33/483 27/26 311Z 33 / 50 C12N 15/00 ZNAA G01N 27/26 325A 315K 325E (72) Inventor Hideaki Hashimoto 1006 Kadoma, Kazuma, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Kazuyoshi Mori 1006 Kadoma, Kazuma, Osaka Address: Matsushita Electric Industrial Co., Ltd. (72) Inventor Takeshi Nishida 1006, Ojidoma, Kadoma, Osaka Prefecture Matsushita Electric Industrial Co., Ltd. (72) Inventor: Mizuo Maeda 3-1-1-14 Hakozaki, Higashi-ku, Fukuoka, Fukuoka ) Inventor Yoshiki Katayama 3-4-2, Yokohama, Nishi-ku, Fukuoka City, Fukuoka Prefecture F-term (reference) 2G045 DA12 DA13 FB05 4B024 AA11 CA01 C A09 HA12 HA19 4B029 AA07 AA12 AA21 AA23 BB20 CC03 CC08 FA15 4B063 QA01 QA12 QA18 QA19 QQ02 QQ42 QR32 QR38 QR55 QR82 QS16 QS20 QS28 QS34 QS39 QX01

Claims (8)

[Claims] A first electrode containing a buffer and having a first electrode immersed therein;
A container, a second container containing a buffer solution and immersed in a second electrode, and a linear polymer and DN between the first container and the second container.
Controlling the power supply unit by applying a positive potential to the second electrode and applying a negative potential to the first electrode; A control unit that applies a predetermined constant voltage between the second electrode and the first electrode; and a detection unit that is provided in the closed channel and detects a passing amount of DNA passing through the closed channel. A high-temperature portion that separates the double helix of the DNA sample from the DNA sample;
The normal DNA, abnormal DNA, and noise D
A separation unit for separating the DNA into NA, wherein the DNA sample, a base sequence capable of hydrogen bonding to the single-stranded DNA sample, and a polymer compound are contained in the buffer solution in the closed flow path; A DNA conjugate for separation that binds and separates the DNA sample into normal DNA, abnormal DNA, and noise DNA based on a difference in binding strength is filled, and by applying the constant voltage, DN in the closed flow path is filled.
A is electrophoresed, and the detection unit detects normal DNA and abnormal DN.
A genetic diagnostic apparatus characterized by measuring the amount of A passed. 2. The genetic diagnostic device according to claim 1, wherein one or more closed channels are provided. 3. The genetic diagnostic device according to claim 1, wherein the DNA conjugate is a vinylated DNA. 4. The genetic diagnostic device according to claim 1, wherein the temperature of the high-temperature section is adjusted to 90 ° C. or higher. 5. The genetic diagnostic device according to claim 1, wherein the liquid in the closed flow path is adjusted to 20 ° C. to 60 ° C. to separate normal DNA from abnormal DNA. 6. The genetic diagnostic apparatus according to claim 1, wherein the closed flow path is a fused silica capillary tube having an inner diameter of 50 to 100 μm. 7. The method according to claim 7, wherein the closed flow path is provided with a grooved plate and an ultraviolet ray.
The genetic diagnostic device according to any one of claims 1 to 6, wherein the genetic diagnostic device is a combination with a plate that transmits at least%. 8. A first container containing a buffer solution and a first electrode being immersed therein, and a second container containing a buffer solution and a second electrode being immersed therein. With linear polymer and DN
A high-temperature section for filling the buffer containing the A-binding control agent and communicating with the closed flow path and heating the inside of the closed flow path to a predetermined temperature is provided. In the buffer in the closed flow path, a DNA sample and , The DN
The base sequence capable of hydrogen bonding and the high molecular compound bind to one strand of the A sample, and the DNA sample is converted to a normal D
Separation DN to separate NA, abnormal DNA and noise DNA
A conjugate is introduced. Thereafter, a positive potential is applied to the second electrode and a negative potential is applied to the first electrode, and a predetermined constant voltage is applied between the second electrode and the first electrode. The double helix of the DNA sample is separated at the high temperature part, the buffer solution in the closed channel is subjected to electrophoresis, separated into normal DNA, abnormal DNA and noise DNA, and the ratio of normal DNA to abnormal DNA or abnormal DN
A method for genetic diagnosis, wherein A is detected.