JP3804538B2 - Genetic diagnostic apparatus and genetic diagnostic method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a genetic diagnostic apparatus and a genetic diagnostic method that can easily and accurately detect the presence or absence of a genetic abnormality.
[0002]
[Prior art]
All diseases involve genetic factors and environmental factors with various probabilities, but diseases such as congenital metabolic disorders, cancer, diabetes, hypertension, Alzheimer, autoimmune diseases, atopy, obesity, and alcoholism Hereditary factors account for a very large proportion. On the other hand, diseases that have a large contribution of environmental factors include diseases caused by infections and aftereffects of trauma.
[0003]
By the way, due to the rapid development of molecular biology in recent years, genetic elements, that is, gene involvement in various diseases have been elucidated fairly accurately, and attention has been focused on gene-targeted medicine. Yes. At present, what is attracting the most attention is SNPs (snips). This is an abbreviation for single nucleotide polymorphism and is generally translated as “single nucleotide polymorphism”, and is a general term for the difference of one code (one base) in genes between individuals.
[0004]
All life-form genes on earth, including humans (or the genome that means the entire collection of genes), are composed of four common bases, and various proteins are created by the sequence of these bases. Life activities are taking place. 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). Human genes are said to be composed of about 3 to 3.2 billion base sequences, but each individual has a base that is different from other people at a rate of about one in every hundred to 1000 bases. Exists. Usually, this single base change is present at a frequency of 1% or more in the total population of a certain human population, and is called SNPs.
[0005]
Therefore, it is said that there are 3 to 10 million SNPs in all genes (genome), and the search for SNPs is currently continued all over the world. The reason why SNPs are attracting attention is that it is considered that the morbidity rate for many diseases, which are said to be statistically related to the genes of each individual, can be estimated by the classification of SNPs. For example, taking breast cancer as an example, SNPs common to those who are likely to have breast cancer can be identified by comparing SNPs between a group of patients with breast cancer and a normal group. At the time of a health examination, it becomes possible to find a person who has investigated the gene and has the SNPs, that is, a person who is normal but is likely to have breast cancer in the future.
[0006]
With this diagnosis, a person with a constitution that is likely to have breast cancer can be treated very early, and the possibility of survival can be improved even if he / she suffers from cancer. The same can be said for lifestyle-related diseases such as diabetes and hypertension, and it is possible to accurately diet and guide living before the onset of diseases that many people around the world suffer.
[0007]
In addition, SNPs are expected to play an important role regarding drugs used for treatment of diseases. Drugs used during treatment are not equally effective for everyone. In general, it is well known that drugs are effective for a certain proportion of people but not for others at all, and may cause adverse results due to side effects. Incidentally, it is well known that drug side effects are at the top of the causes of death in the United States. The effect of the drug is deeply related to the constitution of the person, and the constitution is said to be distinguishable by the classification of SNPs. That is, the classification based on the analysis of SNPs makes it possible to predict the effect and sensitivity of a certain drug in advance and make an appropriate prescription, and the optimal drug administration and risk of side effects according to the individual patient's constitution. Avoidance is expected. Such medical care is called tailor-made medical care or tailor-made medical care, and future practical use is surely expected.
[0008]
In addition, it is known that cancer is caused by a mutation occurring at a specific site on a gene that 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 from an early stage whether or not a cell is cancerous. SNPs are also effective in identifying criminals in criminal investigations, determining ambiguous parent-child relationships, and identifying whether or not they are individuals. As described above, there are 3 to 10 million SNPs in each individual, and since there are separate SNPs from parents, there are absolutely no humans on the planet who have the same SNPs even if they are parents and siblings. It is said not to. This is the reason why complete identification of individuals is possible.
[0009]
In this manner, observing a mutation that occurs at one base in a specific gene may greatly contribute to various matters including medical treatment. However, at present, the method of observing a 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. is there.
[0010]
The most commonly used method for examining SNPs at present is a method called sequencing (determining the base sequence) in which the base sequence of DNA is directly read from the end. Since a gene is a unit of DNA having base sequence information for forming one kind of protein, SNPs can be elucidated by reading the base sequence from the end. There are several reports on the sequencing method, but the most commonly performed method is dideoxy sequencing (Sanger method) described below. All of these methods, including this method, are based on a technique that can separate and identify the difference in length of one base length by denaturing polyacrylamide gel electrophoresis or capillary electrophoresis with high resolution.
[0011]
The dideoxy sequencing (Sanger method), also called enzymatic sequencing, uses DNA polymerase to synthesize complementary strands of single-stranded template DNA, and then produces four special types of dideoxynucleotides artificially created. The feature is to use. As a sequencing operation, a synthetic base sequence that complements the 3 'end of the single-stranded DNA to be sequenced is used as a primer, and deoxynucleotides added equally from the primer to DNA polymerase are extended by an enzymatic reaction. At this time, four reaction vessels were prepared at the same time, and each did not have a hydroxyl group at the 3 'end of the ATGC four bases, and therefore dideoxynucleotide, a base analog that could not continue the DNA extension reaction any more. Mix a small amount separately. As a result, DNA synthesis was stopped when dideoxynucleotide was added to the end of the extending DNA, and each reaction vessel had various lengths, but the ends were always added base analogs. Stranded DNA is formed. The reaction vessel is reacted with S1 endonuclease to digest all the single-stranded DNA into only double-stranded DNA. If the DNA strands of the four reaction vessels thus obtained are subjected to gel electrophoresis or capillary electrophoresis, and the separated DNA is read in order from the shorter one (moved faster), the base sequence of the DNA complementary to the template strand will be Recognize. At this time, the electrophoresis results are identified by, for example, radioactively labeling the added dideoxynucleotide phosphorus or sulfur or binding a fluorescent chemical substance. In the case of a radioactive label, detection is performed by exposure to a film. In the case of a fluorescent chemical substance, fluorescence is detected by irradiating a laser beam. Recently, a method has been developed in which four types of base analogs 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.
[0012]
In this way, conventionally, the exact base sequence of a gene isolated and purified from a subject is determined by this dideoxy sequencing (Sanger method) or other base sequence determination method, and compared with a normal or standard base sequence. Confirmation of the presence of SNPs and mutations, identification of individuals, etc.
[0013]
[Problems to be solved by the invention]
As described above, genetic diagnosis and identification of individuals by gene using conventional gene sequencing methods are performed by isolating the target gene, then amplifying and purifying it, and using a device for determining the base sequence of the gene. Since it was performed by reading the base sequence of the target gene, the experiment required a huge amount of work, a very long time, and a great running cost. In addition, an automated apparatus for determining a base sequence is very expensive, occupies a large space, and requires a large amount of expensive reagents.
[0014]
Therefore, in order to solve such a conventional problem, the present invention can detect a genetic abnormality of one or more nucleotides in a short time, easily and accurately, and is small, light and inexpensive, and has a very low running cost. An object of the present invention is to provide a genetic diagnosis apparatus capable of automating diagnosis.
[0015]
Furthermore, an object of the present invention is to provide a genetic diagnosis method that can easily and accurately determine a gene abnormality of one or more nucleotides in a short time.
[0016]
[Means for Solving the Problems]
In order to solve the above problems, the genetic diagnosis apparatus of the present invention is a DNA sample by applying a constant voltage. In the DNA conjugate for separation and separation in which DNA complementary to the normal DNA of the DNA sample is bound to a polymer compound, and the DNA sample is separated into normal DNA, abnormal DNA, and noise components by the difference in hydrogen bonding force. When detecting the spectral waveform of the light transmitted through the DNA sample, the detector has a detection unit for detecting a change in the passage amount of the DNA sample passing through the flow channel from the absorbance of the irradiated light. And the control calculation unit detects the spectral waveform in a predetermined wavelength region including the DNA absorption wavelength. Inflection point Due to the presence of Other than DNA It is characterized by determining which of the noise components corresponds.
[0017]
Thereby, a genetic abnormality of one base or more can be detected easily and accurately in a short time, and the diagnosis can be automated with a small size, light weight, low cost, and very low running cost.
[0018]
The gene diagnosis method of the present invention also comprises a DNA sample. In the DNA conjugate for separation and separation in which DNA complementary to the normal DNA of the DNA sample is bound to a polymer compound, and the DNA sample is separated into normal DNA, abnormal DNA, and noise components by the difference in hydrogen bonding force. Electrophoresis of the DNA in the DNA sample and For separation The difference in binding force due to the difference in base sequence from the DNA conjugate causes a difference in migration speed, and the absorbance is measured by separating into normal DNA, abnormal DNA and noise components, and normal DNA is detected from the peak of the absorbance pherogram. When the absorbance is measured, the spectral waveform is detected and the wavelength is in the range of 200 nm to 260 nm. Inflection point Is determined to be a DNA peak, and the remaining cases are Other than DNA It is characterized by determining the peak of the noise component.
[0019]
Thereby, even a single base difference gene abnormality can be detected easily and accurately in a short time, and the diagnosis can be automated at a low cost with a very low running cost.
[0020]
DETAILED DESCRIPTION OF THE INVENTION
The invention described in claim 1 includes a first container containing a first electrode and filled with a conductive medium, a second container containing a second electrode and filled with a conductive medium, A medium having electrical conductivity is filled between the first container and the second container, and a DNA sample and DNA complementary to the normal DNA of the DNA sample are converted into a polymer compound in the medium. Combined For separation of DNA samples into normal DNA, abnormal DNA, and noise components due to differences in hydrogen bonding strength A flow path filled with a DNA conjugate, a power supply unit that applies a positive potential to the second electrode and a negative potential to the first electrode, and a power supply unit that controls the power supply unit to provide a predetermined gap between the second electrode and the first electrode. A control operation unit that applies a constant voltage of DNA sample A genetic diagnosis apparatus having a detection unit for detecting a temporal change in the amount of DNA sample passing through the flow path from the absorbance of irradiated light when electrophoresis is performed, and the detection unit transmits the DNA sample. The spectrum waveform of the light to be detected is detected, and the control calculation unit detects the spectrum waveform in a predetermined wavelength region including the DNA absorption wavelength. Inflection point Due to the presence of Other than DNA It is a genetic diagnosis apparatus characterized by determining which of the noise components corresponds to, applying a predetermined constant voltage between the second electrode and the first electrode, DNA sample Electrophoresis is performed to reduce the migration speed of normal DNA and abnormal DNA using the difference in binding force, and noise components that do not receive the action can be migrated at almost the same speed. That is, normal DNA and abnormal DNA move while For separation The noise component that is affected by the binding force of the DNA conjugate and does not receive this effect is migrated first when a voltage is applied and detected by the detection unit. However, for abnormal DNA, For separation The binding force acts slightly weaker than normal DNA from the DNA conjugate, and the action of the conjugate is not easily shaken, and then the migration starts after the noise component. The maximum binding force acts on the normal DNA, the electrophoresis starts further later, and is finally detected by the detection unit. Thereby, a difference can be caused in the detection time of the three DNAs. Some noise components have the same absorption wavelength as normal DNA and abnormal DNA, but such noise components have a spectrum waveform different from that of DNA, and a spectrum waveform in a predetermined wavelength region of DNA. Seen in Inflection point Therefore, noise components and DNA can be separated, and normal DNA and abnormal DNA can be discriminated. DNA can be separated within a short period of time, such as electrophoresis and DNA hydrogen bonding, and it can be adjusted very easily to an optimum voltage with high resolution simply by applying a predetermined constant voltage. It is possible to detect the presence or absence of abnormalities, and it is possible to provide a small, lightweight, low-cost apparatus with a low running cost, and it is extremely easy to automate diagnosis.
[0021]
The invention described in claim 2 For separation 2. The DNA conjugate separates a DNA sample into normal DNA and abnormal DNA by causing a difference in migration speed between normal DNA and abnormal DNA due to a difference in binding force due to a difference in DNA base sequence. A genetic diagnosis apparatus according to the description, For separation The difference in hydrogen bonding strength with the DNA conjugate can be used to reduce the migration speed of normal DNA and abnormal DNA, respectively, and noise components that do not receive the action can be migrated at almost the same speed. For separation Since the DNA conjugate is bound to a polymer compound, the migration speed is only a few percent compared to a DNA sample (however, it depends on the type and length of the polymer compound), and is relatively pseudo-fixed as seen relatively. Can be in a state. Conjugate fixation is difficult and usually the flow path must be disposable, but it is not necessary because of pseudo-fixation, and it is extremely necessary to adjust the concentration of various conjugates and samples and the mixing ratio of various conjugates and samples. It becomes easy.
[0022]
According to a third aspect of the present invention, the detection unit detects a light emitting unit, a separation unit capable of decomposing light irradiated from the light emitting unit and transmitted through the flow path for each wavelength, and light that has passed through the separation unit. 3. A genetic diagnostic apparatus according to claim 1 or 2, wherein a spectral waveform can be obtained by a separation unit such as a prism or a light-transmitting filter, and normal DNA can be obtained from the spectral waveform. Can be distinguished from abnormal DNA and the amount of passage can be measured. In addition, a light receiving unit capable of receiving each decomposed light is required for the prism.
[0023]
The invention described in claim 4 is the genetic diagnosis apparatus according to claim 3, wherein the separation part decomposes the irradiation light in a wavelength region of 200 nm to 260 nm. A peak at 260 nm which is an absorption wavelength; Inflection point As a result, a local minimum value around 240 nm appears, which is different from normal DNA and abnormal DNA. Inflection point Therefore, it is possible to accurately discriminate noise components having no noise and to perform measurement easily and easily.
[0024]
According to the fifth aspect of the present invention, when the absorbance at the wavelength of 260 nm detected by the detection unit is greater than the absorbance at the wavelength of 240 nm, and the absorbance at the wavelength of 200 nm is greater than the absorbance at 240 nm, the peak is a peak indicating DNA. The genetic diagnosis apparatus according to any one of claims 1 to 4, wherein the determination is simple by simply detecting the absorbance at a wavelength of 200 nm, the absorbance at a wavelength of 240 nm, and the absorbance at a wavelength of 260 nm. In Inflection point Information can be obtained, and DNA and noise components can be distinguished very easily.
[0025]
The invention described in claim 6 is the genetic diagnosis apparatus according to any one of claims 3 to 5, wherein the separation unit is a transmission filter that transmits only a predetermined wavelength region. The range of the target wavelength from the light transmitted from the light can be detected by one light receiving unit.
[0026]
The invention described in claim 7 is the genetic diagnosis apparatus according to claim 6, wherein the transmission filter selects and transmits a plurality of different wavelengths within a predetermined wavelength region. The light absorbance at a plurality of wavelengths can be detected by a single light receiving unit from the light transmitted through the light.
[0027]
The invention described in claim 8 is a DNA sample in which a predetermined constant voltage is applied between the second electrode and the first electrode. In a DNA conjugate for separation in which DNA complementary to normal DNA of the DNA sample is bound to a polymer compound, and the DNA sample is separated into normal DNA, abnormal DNA, and noise components by a difference in hydrogen bonding force Electrophoresis of the DNA in the DNA sample For separation A difference in the binding speed due to the difference in base sequence from the DNA conjugate causes a difference in migration speed, separates into normal DNA, abnormal DNA, and noise components, and measures the absorbance. From the pherogram peak, normal DNA and abnormal DNA Is a genetic diagnosis method for diagnosing the abundance ratio, detecting a spectral waveform when measuring absorbance, and in a wavelength range of 200 nm to 260 nm Inflection point Is determined to be a DNA peak, and the remaining cases are Other than DNA It is a genetic diagnosis method characterized by determining a peak of a noise component, a predetermined constant voltage is applied between the second electrode and the first electrode, For separation The DNA conjugate can be electrophoresed and the migration speed of normal DNA and abnormal DNA can be reduced by utilizing the difference in binding force, and the noise component that is not affected can be migrated at almost the same speed. Normal DNA and abnormal DNA move while this For separation The noise component that is affected by the binding force of the DNA conjugate and does not receive this effect is migrated first when a voltage is applied and detected by the detection unit. However, for abnormal DNA, For separation A weak binding force of one base with the DNA conjugate acts, and the action of the conjugate is not easily shaken, and the migration is started after the noise component. The maximum binding force acts on the normal DNA, the electrophoresis starts further later, and is finally detected by the detection unit. Thereby, a difference can be caused in the detection time of the three DNAs. Some noise components have the same absorption wavelength as normal DNA and abnormal DNA, but such noise components have a spectrum absorption waveform different from that of DNA, and are found in the spectrum waveform of DNA. Inflection point Therefore, noise components and DNA can be separated, and normal DNA and abnormal DNA can be discriminated. DNA can be separated in a short period of time of ten minutes or more using electrophoresis and DNA hydrogen bonding, and it can be adjusted very easily to an optimum voltage with high resolution simply by applying a predetermined constant voltage. It is possible to detect the presence or absence of an abnormality, and it is possible to provide a small, lightweight, low-cost apparatus with a low running cost, and it is extremely easy to automate diagnosis.
[0028]
In the invention described in claim 9, when the absorbance at a wavelength of 260 nm is larger than the absorbance at a wavelength of 240 nm, and the absorbance at a wavelength of 200 nm is larger than the absorbance at 240 nm, Inflection point The genetic diagnosis method according to claim 9, wherein the DNA diagnosis method is characterized in that DNA 200 can be detected very simply by detecting absorbance at a wavelength of 200 nm, absorbance at a wavelength of 240 nm, and absorbance at a wavelength of 260 nm. And a noise component can be discriminated.
[0029]
Hereinafter, a genetic diagnostic apparatus and a genetic diagnostic method according to embodiments of the present invention will be described with reference to the drawings.
[0030]
(Embodiment 1)
FIG. 1 is an external view of a genetic diagnosis apparatus according to an embodiment of the present invention, FIG. 2 is an external view of the genetic diagnosis apparatus according to the embodiment of the present invention, and FIG. FIG. 4 is a block diagram of the control circuit of the genetic diagnosis apparatus according to the embodiment of the present invention. FIG. 5 is a DNA conjugate for separation and a DNA sample in the flow path of the genetic diagnosis apparatus according to the embodiment of the present invention. FIG. 6 is a configuration diagram of the detection unit of the genetic diagnosis apparatus according to the embodiment of the present invention, FIG. 8 is a relationship diagram between elapsed time and absorbance, and FIG. 9 is a spectrum waveform diagram when an absorbance peak occurs at 260 nm. .
[0031]
In FIG. 1, 1 is a power switch of the genetic diagnosis apparatus, 2 is an operation button for performing various operations of the apparatus, 3 is a display panel, 4 is an upper lid, and 5 is a casing of the apparatus. 2, 3, and 4, 6 is an electrophoresis unit for performing electrophoresis, 7 is a support base that supports the electrophoresis unit 6, 8 is a control for performing electrophoresis, a suction pump 20 described later, and detection. It is a control calculation part which consists of a board | substrate which controls the part 15 and calculates the detected data. Reference numeral 9 denotes a power supply box, and 9a denotes a power supply unit provided in the power supply box 9. The power supply unit 9a is most suitable for separating abnormal DNA and normal DNA from a DNA sample 23, which will be described later, because there is an optimum applied voltage value that differs depending on the type, concentration, and processing conditions of DNA in this genetic diagnosis apparatus. A predetermined voltage is applied under the control of the control calculation unit 8 so that electrophoresis can be performed. 10 is a high temperature part for separating the double helix (hereinafter, double strand) of DNA, 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 normal DNA and abnormal DNA And a temperature controller for adjusting the temperatures of the high-temperature unit 10 and the separation unit 12. The separation unit 13 adjusts the temperature of the high-temperature unit 10 and the separation unit 12.
[0032]
Although details of the configuration in the high temperature part 10 and the separation part 12 will be described later, at the start of measurement, as shown in FIG. 5, the separation DNA conjugate 21 is near the position of the separation part 12 and the DNA sample 23 is in the high temperature part 10. Arrange them so that they are close. Then, by performing the arrangement, voltage control, concentration adjustment, temperature control, etc. set in this way, the present gene diagnostic apparatus can convert the DNA sample 23 into normal DNA, abnormal DNA, and noise components while performing electrophoresis. To separate.
[0033]
Among them, the DNA sample 23 is filled and arranged in the high temperature part 10 with double strands in the present embodiment, and is used with the temperature controller 13 (within a predetermined temperature of 90 ° C. or higher and in a range of ± 5 ° C.). It is heated and controlled so that the temperature becomes. By heating to this temperature, the double strands of DNA introduced are automatically separated into single strands. Subsequently, in the separation unit 12, the temperature of the high-temperature unit 11 is set so that the DNA sample 23 can be separated from normal DNA, abnormal DNA, and noise components by the difference in hydrogen bond binding force while the separation DNA conjugate 21 is subjected to a migration action. The temperature is controlled to be kept at a predetermined temperature within a temperature range of at least 10 ° C lower, that is, 15 ° C to 80 ° C, desirably 20 ° C to 60 ° C. In order to separate normal DNA from abnormal DNA, it is preferable to control within a range of ± 1 ° C. of this predetermined temperature suitable for DNA separation. Furthermore, the heat insulating part 11 is originally provided to thermally cut off the space between the separating part 12 and the high temperature part 11 so as to adjust the temperature, so that the temperature of the separating part 12 becomes 20 ° C. to 60 ° C. Adjusted. Normal DNA and abnormal DNA can be separated from noise components while undergoing a migration action due to the difference in hydrogen bond strength at this temperature.
[0034]
14 is provided in front of the high-temperature part 10 with reference to the direction of electrophoresis, and position information is obtained prior to measurement in order to accurately place the separation DNA conjugate 21 near the position of the separation part 12 in FIG. A preliminary detection unit 15 is provided on the downstream side of the separation unit 12 and is a detection unit that measures the amount of DNA sample that is separated and electrophoresed. The preliminary detection unit 14 is used before the start of measurement. The preliminary detection unit 14 is inserted through the capillary tube 19 to detect the existing position of the separation DNA conjugate 21, and the capillary tube 19 is moved by a predetermined distance based on the existing position. The separation DNA conjugate 21 is arranged near the position of the separation portion 12. If only the measurement is performed, the detection unit 15 is sufficient. However, since the separation DNA conjugate 21 needs to be accurately arranged at the position shown in FIG. 5 before the measurement is performed, the preliminary detection unit 14 is used. is there. Thus, since the position is determined based on the information detected by the preliminary detection unit 14, the position can be controlled very accurately.
[0035]
In FIG. 4, 15a is a D2 lamp that irradiates ultraviolet rays, 15b is a photodiode that receives ultraviolet rays, 15c is a preamplifier that amplifies a weak current detected by the photodiode 15b, and 15d is an A / D converter that converts it into a digital quantity. . Reference numeral 24 denotes a transmission filter (a separation unit of the present invention) that transmits only the target wavelength region of transmitted light, and is provided between the D2 lamp 15a and the photodiode 15b. Although details of the transmission filter 24 will be described later, DNA can be detected by removing a noise component from a spectrum waveform of light having wavelengths of 200 nm, 240 nm, and 260 nm. The preliminary detection unit 14 has the same internal configuration as the detection unit 15 and includes a D2 lamp (not shown), a photodiode (not shown), and a transmission filter (not shown). A signal is sent to the control calculation unit 8 via the / D converter 15d. Thereby, the position of the separation DNA conjugate 21 can be detected by the preliminary detection unit 14. By the way, in the present embodiment, the separation unit of the present invention is configured by the transmission filter 24. However, when configured by a prism or the like, the number of the photodiodes 15b is increased so that each of the decomposed lights can be detected. It is good to make it. Reference numeral 16 denotes an electrode that applies a positive potential during electrophoresis (second electrode of the present invention), and 17 denotes an electrode that applies a negative potential during electrophoresis (first electrode of the present invention). Controls the power supply unit 9a and applies a predetermined constant voltage between the electrode 16 and the electrode 17 to cause the most effective electrophoresis.
[0036]
3, 4, and 5, 18 is a buffer solution (electroconductive medium of the present invention) for transporting electric charges and stabilizing the pH of the DNA sample during electrophoresis, and 18 a is a buffer solution 18. 18b is a cathode side container (second container of the present invention) 18b containing a buffer solution 18, 18c is a DNA binding control agent and the following linear polymer 18d. An added buffer solution 18d added to the buffer solution 18 is a linear polymer such as polyacrylamide which enables electrophoresis and separates normal DNA, abnormal DNA and noise components. 19 is a capillary tube (the flow path of the present invention) for electrophoresis of the DNA sample 23 during electrophoresis, and 20 is a reagent such as the DNA sample 23 or linear polymer 18d and a DNA binding control agent injected into the capillary tube 19. This is a suction pump. The electrode 16 is immersed in the buffer solution 18 of the container 18a, and the electrode 17 is immersed in the buffer solution 18 of the container 18b. The buffer 18 in the container 18a and the container 18b is communicated with the added buffer 18c in the capillary tube 19. When a voltage is applied between the electrode 16 and the electrode 17, electrophoresis is induced in the capillary tube 19, and the separation DNA conjugate 21 and the DNA sample 23 are negatively charged. Therefore, separation from the container 18a to the container 18b is performed. The DNA conjugate 21 for use and the DNA sample 23 move.
[0037]
In FIG. 6, the transmission filter 24 transmits only the target wavelength region in the light irradiated from the D2 lamp 15 a and transmitted through the conjugate tube 19. A wavelength transmission filter, 24b is a 240 nm wavelength transmission filter, and 24c is a 260 nm wavelength transmission filter. The transmission filter 24 is rotated during electrophoresis, and the absorbance at 200 nm, 240 nm, and 260 nm is measured. If the transmission filter 24 is composed of three or more wavelength transmission filters, a more detailed spectral waveform can be obtained.
[0038]
Incidentally, FIG. 9 shows a spectrum waveform at each peak of absorbance shown in FIG. “A” in FIG. 9 is a typical spectrum waveform of DNA, and “b” is an example of a spectrum waveform of impurities other than DNA. Normally, when the spectrum waveform near the position of each peak of absorbance shown in FIG. 8 is examined, whether the spectrum waveform is “a” or “b” as shown in FIG. Inflection point The feature of whether or not there will appear. That is, since 260 nm is the DNA absorption wavelength, if there is a minimum value in the region near this spectrum waveform, that is, the region of 200 nm to 260 nm, there is a peak at 260 nm, which indicates the presence of DNA. In this way, it is possible to determine whether or not the absorbance peak component is DNA by selecting the absorption wavelength by the transmission filter 24 and examining the difference in the spectrum waveform.
[0039]
Next, the measurement principle for performing measurement with the genetic diagnosis apparatus of the present invention and the matters to be noted will be described. The DNA sample 23 measured by this genetic diagnostic apparatus is DNA obtained from human cells, blood, or the like. It is necessary to cut out the target portion from the human genomic DNA, which is said to have about 3 billion base pairs, to a target length using 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 diagnostic apparatus, a specific description is omitted.
[0040]
The target DNA extracted by PCR or the like is about 6 to 1000 in terms of the number of bases, but it is probable that the same DNA sequence does not exist in human genomic DNA which is said to have about 3 billion base pairs. The number considered is about 50. However, this means that the total number should be about 50. Therefore, when the DNA is cut out in several times, it may be divided into 6 to 12 pieces. According to the experiments of the present inventors, when separating normal DNA and abnormal DNA having one base difference with this genetic diagnosis apparatus, the result is that the number of bases can be separated most efficiently from 6 to 12. Yes. However, because this abnormal DNA separation is closely related to the concentration of the DNA sample (μM), the concentration of the DNA conjugate for separation (mol%), the measurement temperature, etc., separation does not occur unless the conditions are appropriate. It is necessary to pay attention to whether such a condition can be set. For example, the concentration of the DNA conjugate 21 for separation is suitably about 0.004 to 0.008 (mol%), but the concentration of the DNA sample 23 is suitably 1 to 10 (μM) and is 500 to 600 times higher. Is desirable. Thus, a DNA sample having about 6 to about 1000 base sequences obtained by pretreatment by PCR or the like is necessary for the diagnosis of this gene diagnostic apparatus, and it is desirable to set conditions.
[0041]
Next, the capillary tube 19 will be described. When a gel is placed in the capillary tube 19 and electrophoresis is performed, a liquid flow called electroosmotic flow is generated. Therefore, it is necessary to prevent this phenomenon from occurring in the gene diagnostic apparatus. For this reason, it is preferable to coat the inner wall of the capillary tube 19 with acrylamide or the like. As long as the generation of the electroosmotic flow can be prevented, another coating method may be used. For example, a commercially available coated capillary tube may be used. As the capillary tube 19, a fused silica capillary tube having an inner diameter of 50 to 100 μm can transmit ultraviolet rays of 90% or more, and when DNA is detected using ultraviolet rays as will be described later, the amount of passing ultraviolet rays is easy. It is most suitable because it can be detected. Even if the capillary tube 19 is configured by a combination of a plate with a groove and a plate that transmits 90% or more of ultraviolet rays, it can transmit 90% or more of ultraviolet rays and detect abnormal DNA by detecting the amount of ultraviolet rays passing through. Can be easily performed. When the grooved plate is made a plate that can transmit ultraviolet rays, transmitted light is detected. When the grooved plate is made a plate that does not transmit ultraviolet rays, abnormal DNA is detected by reflected light. When detecting reflected light, it is necessary to form a rectangular cross section in order to make the groove shape a uniform reflecting surface.
[0042]
As the buffer solutions 18 and 18c accommodated in the containers 18a and 18b and the capillary tube 19, it is appropriate to use a Tris-Borate (pH 7.2 to pH 8) buffer solution or the like. Among these, the DNA binding control agent mixed in the added buffer 18c accommodated in the capillary tube 19 includes a binding promoter such as magnesium chloride that promotes the binding of DNA to the DNA conjugate for separation, and the separation of urea or the like that promotes the separation. There are two types of agents. These two types of DNA binding control agents can control various migration speeds for DNA by selecting two types of mixing ratios and substances (for example, other electrolytes as binding promoters). The linear polymer 18d for electrophoresis of the DNA conjugate 21 for separation, the DNA sample 23, and the like is suitable because it has a good compatibility because polyacrylamide is also used for producing the conjugate.
[0043]
Next, the order of filling the capillary tube 19 is as follows. As shown in FIG. 5, first, an addition buffer solution 18c containing a linear polymer 18d and a DNA binding control agent is introduced into the capillary tube 19, and then described in detail below. The separating DNA conjugate 21 is added. At this time, the linear polymer 18d may be mixed and introduced, or after the separation DNA conjugate 21 is introduced, the linear polymer 18d may be introduced. Thereafter, the DNA sample 23 diluted with the addition buffer 18c is introduced and electrophoresed.
[0044]
In the present embodiment, since acrylamide and DNA are polymerized to produce the separation DNA conjugate 21, the weight difference and the structure difference from the DNA are large, and the migration speed (0. 6 cm / min to 0.7 cm / min) is about 1/20 to 1/30 of the optimal migration speed of DNA (13 cm / min to 20 cm / min), and is relatively slow and relatively pseudo. A state that may be said to be fixed is realized. Therefore, before starting the measurement, the preliminary detection unit 14 provided at the entrance of the electrophoresis unit 6 detects the separation DNA conjugate 21 and, based on this positional information, the capillary tube containing the packing. 19 is moved between the high temperature part 10, the heat insulation part 11, and the separation part 12, the separating DNA conjugate 21 is placed in the separation part 12, and the DNA sample 23 is placed in the high temperature part 10. Since the arrangement is obtained by obtaining the position information by the preliminary detection unit 14, the arrangement can be accurately arranged at a predetermined position. By performing electrophoresis from this state, the double strand of the DNA sample 23 is separated into single strands at the high temperature portion 10, and normal DNA and abnormal DNA are separated from the high temperature portion 10 through the heat insulating portion 11 and the separation portion 12. In addition, noise components can be clearly separated.
[0045]
As a mechanism for exchangeably introducing each solution of DNA sample, DNA conjugate for separation, and linear polymer into the capillary tube 19 of the genetic diagnosis apparatus, for example, in addition to the suction pump 20 shown in FIG. It is desirable to equip a column for storing each DNA sample, DNA conjugate for separation, and each solution of linear polymer, and a mechanism for automatically replacing the column and connecting it to the capillary tube 19.
[0046]
Furthermore, the capillary tube 19 is unitized in units of one or a plurality, and this can be attached to the separation unit 12, the high temperature unit 10, and the heat insulation unit 11 as a separation flow path cartridge for replacement. It is preferable to control the temperature inside the part 10 and the separation part 12. In this case, the capillary tube 19 is preliminarily filled with the addition buffer 18c containing the linear polymer 18d and the DNA binding control agent, and the separation DNA conjugate 21 and the DNA sample 23 are filled in the separated state to separate the separation channel cartridge. And the separation channel cartridge is replaced every time measurement is performed. Since the arrangement of the separation DNA conjugate 21 and the DNA sample 23 can be accurately set in advance for each separation channel cartridge, the measurement can be performed easily and accurately. In this separation channel cartridge, the separation DNA conjugate 21 and the DNA sample 23 are separated and filled with a linear polymer 18d interposed therebetween. The linear polymer 18d may be mixed and filled in each of them.
[0047]
Subsequently, the separation DNA conjugate will be described. FIG. 7 is a conceptual diagram showing the relationship between the DNA conjugate for separation and the DNA sample in the flow path of the genetic diagnosis apparatus according to the embodiment of the present invention. In FIG. 7, 21 is a DNA conjugate for separation. There are two types of DNA: double-stranded and single-stranded, but the four bases A, T, C, and G of DNA have the property that A and T, and G and C are easy to hydrogen bond with each other. The double strands of DNA are also paired with AT and GC. Therefore, when the DNA of one strand is aligned with ATCGCGTCTAGC (described in SEQ ID NO: 1), the DNA of the other strand has a base sequence of TAGCGCAGATCG (described in SEQ ID NO: 2). This relationship is called a complementary relationship. As long as this complementary relationship is satisfied, A and T and G and C are bonded by hydrogen bonds to form a double strand. In the present invention, in order to utilize this relationship, as shown in FIG. 7, the DNA portion of the separating DNA conjugate 21 has a base sequence complementary to the normal DNA of the DNA sample. Therefore, the normal DNA base sequence of the DNA sample contains ATCGCGTCTAGC (described in SEQ ID NO: 1), and the abnormal DNA is ATCA. ^* In CGTTAGAGC (described in SEQ ID NO: 3), when the bases of normal DNA and abnormal DNA are different in the part indicated by *, the DNA part sequence of the DNA conjugate for separation 21 (first base sequence of the present invention) Is TAGCGCAGATCG (described in SEQ ID NO: 2), the abnormal DNA is A ^* In FIG. 2, hydrogen bonds do not occur because they are no longer complementary to the separation DNA conjugate 21, and the binding force of the entire hydrogen bond is larger by the binding force of one base than the abnormal DNA in the normal DNA of the DNA sample. As a result, during electrophoresis described later, normal DNA binds to separation DNA conjugate 21 for a longer time with a stronger binding force than abnormal DNA, so normal DNA is delayed relatively than abnormal DNA. This is because not only the binding force during electrophoresis but also the pulling force due to electrophoresis acts, so that a large number of DNAs are in a binding state that repeats binding and detachment intermittently. When this migration speed is viewed on average, the migration speed of normal DNA that binds for a long time is lower than that of abnormal DNA. The DNA sample also contains impurities other than normal DNA and abnormal DNA, such as the remaining single-stranded DNA that is not to be measured among double-stranded DNA, and these impurities will be described later. However, since this noise component hardly reacts and binds to the DNA of the separating DNA conjugate 21, it is separated with the fastest electrophoresis speed.
[0048]
Next, a method for producing and synthesizing such a separation DNA conjugate 21 will be described. In order to synthesize vinylated DNA, the DNA for separating DNA conjugate 21 is cut out by PCR. In the above example, TAGCGCAGATCG is the DNA of the DNA conjugate for separation.
[0049]
Next, the 5 ′ end of the DNA having the desired base sequence is aminated (usually aminated via a hexyl group). The aminated DNA thus obtained is diluted by adding ultrapure water sterilized to 2.6 mM. Next, MOSU (methacryloid oxysuccinimide) is diluted with DMSO (dimethyl sulfoxide) to 71.388 mM. Then, the aminated DNA thus obtained and MOSU are added and adjusted so as to have a ratio of 1:50. Further, a solution adjusted with sodium hydrogen carbonate and sodium hydroxide so as to have a pH of 9 for pH adjustment is added to this adjusted solution in an amount equal to the amount of aminated DNA.
[0050]
The resulting solution is then shaken overnight. Thereafter, the vinylated DNA in the shaken solution is separated from aminated DNA, MSU, and others using HPLC (High Performance Liquid Chromatography). Since vinylated DNA contains an eluent (TEAA; mixed solution of triethylamine-acetic acid and acetonitrile), it is further concentrated under reduced pressure by a centrifugal evaporator having a vacuum drying function.
[0051]
Then, 53 μmol of nitrogen is substituted for 10% AAM (acrylamide) as a polymerization solution. Further, 1.34% of TEMD (N, N, N′N′-tetramethylethylenediamine) which is a polymerization initiator is diluted with sterilized ultrapure water deaerated by ultrasonic waves. This is diluted with sterilized ultrapure water in which 1.34% APS (ammonium persulfate), which is a polymerization initiator, is similarly degassed by ultrasonic waves. Furthermore, the concentrated vinylated DNA is diluted with sterilized ultrapure water. Then, in order to synthesize 100 μl of DNA conjugate, 34 μl of the above AAM, 5 μl each of the above TEMD and APS, and 0.01% mol to 0.05% mol of vinylated DNA with respect to AAM In addition, sterilized ultrapure water is added to 100 μl and left for about 60 minutes to obtain an acrylamideated DNA conjugate.
[0052]
In order to remove unreacted vinylated DNA, the obtained DNA conjugate is sealed inside a dialysis membrane such as cellophane, and the dialysis membrane is rotated in a large amount of ultrapure water. Unreacted DNA is removed. Thereby, a DNA conjugate with high purity is obtained.
[0053]
Subsequently, the operation of the genetic diagnosis apparatus in the present embodiment will be described. First, a capillary tube 19 into which a DNA sample or each solution has been introduced is set in the genetic diagnosis apparatus, and the buffer solution 18 in the containers 18a and 18b is immersed at both ends as shown in FIGS. A predetermined voltage described later is applied between the electrode 16 and the electrode 17 by the power supply unit 9a. The appropriate voltage width is preferably 10 to 20 kilovolts, but may be 100 to 30 kilovolts depending on the state of the electrolyte and electrodes. For example, when the electrolyte concentration is low or the electrode area is large, the voltage is lowered. Then, instead of applying a predetermined voltage from the beginning, if a high voltage of 30 kilovolts or more for preparation is applied before applying a constant voltage and noise components are separated quickly, measurement can be performed quickly.
[0054]
Here, the reason why the predetermined voltage must be applied will be explained. The difference between the binding force of the DNA sample to the separation DNA conjugate 21 and the separation force due to the electrophoretic force is the difference in DNA migration speed and movement. This is because it is necessary to perform electrophoresis by applying a predetermined constant voltage that most effectively separates normal DNA, abnormal DNA, and noise components. This voltage is (1) the condition that the normal DNA that is most difficult to migrate can be electrophoresed; and (2) the resolution of normal DNA and abnormal DNA is reduced when high voltage is applied. This is a voltage that satisfies the conditions (1) and (2) of the condition that the electrophoresis must be performed. Accordingly, an optimum voltage to be applied in advance is examined for each DNA and for each condition, and after initially applying a preparation voltage of about 30 kilovolts or more for a short time, this voltage is applied. Since the DNA in the capillary tube 19 is negatively charged and proceeds to the anode side, the variable power source 9a needs to apply the electrode 16 so as to have a positive potential and the electrode 17 have a negative potential. In addition, in electrophoresis, if the electrophoresis time is too long, the addition buffer solution 18c is dried in the capillary tube 19, so that it is necessary to avoid running for a longer time than necessary.
[0055]
When a voltage is applied, the DNA sample is migrated, but the separation conjugate 21 has a difference of one base in the binding force between the normal DNA and the abnormal DNA of the sample DNA. A difference in moving speed is generated, and a large moving difference is generated between noise components. Similarly, the normal DNA group and the abnormal DNA group have the same binding force to the delay conjugate and migrate in exactly the same manner, so that they move between the noise component and the normal DNA group and the abnormal DNA group. There will be a difference.
[0056]
Next, the high temperature part 10 where the separation is performed, the separation part 12, and the heat insulating part 11 will be described. As described above, in order to release the double strands of DNA at the high temperature portion 10, the temperature controller 13 is used to control the temperature so as to be 90 ° C. or higher (predetermined temperature ± 5 ° C.). Further, in the separation unit 12, although it depends on the state of the DNA sample, a high-temperature part can be separated so that normal DNA, abnormal DNA, or noise components can be separated based on the difference in binding force between the DNA conjugate for separation and the DNA sample. The temperature must be about 10 ° C. lower than the temperature of 11, ie, 15 ° C. to 80 ° C., preferably 20 ° C. to 60 ° C. In the genetic diagnosis apparatus of the embodiment, a silicon rubber heater, a nichrome wire, etc., and a temperature sensor such as a thermocouple, thermal, etc. are arranged at the lower part of the separation unit 10 covering the capillary tube 19, and a predetermined temperature controller 13 is used. The temperature is controlled to be ± 1 ° C or less. However, depending on the environmental temperature, the room temperature may exceed the target predetermined temperature, and it is better to use a component that can be heated and cooled such as Peltier instead of a silicon rubber heater or nichrome wire. Further, the heat insulating part 11 may be made of a heat insulating material such as glass wool, a foaming agent, mica, porous ceramics or the like whose contents are evacuated.
[0057]
Next, how the detection unit detects normal DNA, abnormal DNA, and noise components in which a movement difference has occurred will be described. As shown in FIG. 8, the detection is performed by measuring the absorbance when ultraviolet irradiation is shielded by normal DNA, abnormal DNA, and noise components. In the embodiment, as shown in FIG. 6, the glass part is exposed by a slit of a slit plate in a part of the capillary tube 19, and ultraviolet rays are irradiated from the D2 lamp 15a. Only the light that has passed through is detected by the photodiode 15b, and the absorbance is measured. In addition, when a prism etc. are used as a separation part, it is good to detect with the some photodiode 15b. The control arithmetic unit 8 controls the power source unit 9a to cause the D2 lamp 15a to emit light, the current detected by the photodiode 15b is amplified by the preamplifier 15c, and converted into a light quantity by the A / D converter 15d and converted to the absorbance by the control arithmetic unit 8 And displayed as a ferrogram on the display panel 3. The control calculation unit 8 has a built-in timer (not shown), and can measure the elapsed time from the migration start time. In FIG. 8, the longer elapsed time (faster) (I) is the noise component that does not bind to the DNA conjugate, the middle elapsed time (II) is abnormal DNA, and the shorter elapsed time (III) Normal DNA.
[0058]
By reading the peak height, time and number of peak peaks from this ferrogram, it can be seen that the DNA sample contains abnormal DNA. That is, abnormal DNA exists if the number of peaks is 3 or more. Depending on the applied voltage, DNA concentration, and length of DNA (number of bases), the number of peaks can be limited to abnormal DNA and noise components. In this case, there are only two peaks, One of these becomes abnormal DNA. In determining the amount of each passing normal DNA and abnormal DNA, the heights of the peaks indicating both are compared on the ferrogram. This is because the two peaks to be compared were electrophoresed under the same conditions. This is because it shows the difference in absorbance between two sets having different migration speeds in DNA, in other words, this ratio corresponds to the abundance ratio of abnormal DNA and normal DNA. To determine the abundance of abnormal DNA, standard data obtained from the detected peak waveform of a standard DNA sample may be input in advance to the control calculation base 8, and the measured data and the measured data may be compared and converted.
[0059]
By the way, when abnormal DNA is contained in a DNA sample as described above, three or more peaks appear, and the difference between normal DNA, abnormal DNA, and noise components can be determined by considering the difference in migration speed. In the case of two cases or the case where peaks overlap, it cannot be distinguished from the case of normal DNA alone. Therefore, in the genetic diagnosis method of the present invention, the spectral waveform at each peak is measured to accurately determine whether the main component showing the peak is a noise component composed of DNA or other impurities. . As shown in FIG. 6, in the genetic diagnosis apparatus according to the embodiment of the present invention, the transmission filter 24 transmits the transmitted light only in the target wavelength region, and is provided between the D2 lamp 15a and the photodiode 15b. ing. The wavelength transmission filter 24a is a wavelength transmission filter of 200 nm, 24b is a wavelength transmission filter of 240 nm, 24c is a wavelength transmission filter of 260 nm, and the transmission filter 24 is composed of wavelength transmission filters 24a, 24b, and 24c arranged every 120 °. By rotating the transmission filter 24 during electrophoresis, it becomes possible to measure the absorbance at 200 nm, 240 nm, and 260 nm by the wavelength transmission filters 24a, 24b, and 24c.
[0060]
The reason why the filter for measuring the absorbance at 200 nm is used as the wavelength transmission filter 24 a, the wavelength transmission filter 24 b is 240 nm and the wavelength transmission filter 24 c is 260 nm is based on the characteristics of the spectrum waveform in FIG. 9. That is, 260 nm is the DNA absorption wavelength, and in the wavelength region of 200 nm to 260 nm, as shown in FIG. 9, the absorption spectrum waveform “a” of DNA shows a decreasing tendency near 200 nm and shows a minimum value near 240 nm. And a peak is formed at around 260 nm. On the other hand, the absorption spectrum waveform “b” of impurities other than DNA has a tendency to simply decrease as it advances to around 200 nm, around 240 nm, and around 260 nm. The absolute value of the absorbance varies at 200 nm, 240 nm, and 260 nm, respectively, but around 240 nm and 260 nm. Inflection point The characteristic of the presence of γ is almost unchanged, with the tendency that the absorbance at 200 nm is greater than the absorbance at 240 nm, and the absorbance at 260 nm is greater than the absorbance at 240 nm. In many cases, the absorbance at 260 nm is larger than that at 200 nm, and the absorbance at 260 nm usually shows the largest value in this region. On the other hand, the absorption spectrum waveform “b” of the impurity has only a tendency that the absorbance increases as the wavelength decreases. From this characteristic, selective absorption around 200 nm, 240 nm, and 260 nm by the transmission filter 24 is achieved. By detecting the wavelength, it is possible to determine whether the absorbance peak component is DNA.
[0061]
Therefore, in this embodiment, the absorption wavelength is detected by the photodiode 15b, and if the relationship between the absorbance at each wavelength converted by the control calculation unit 8 is 200 nm> 240 nm and 240 nm <260 nm, it is determined as DNA. If this relationship does not hold, it is determined that the noise component is other than DNA. By adding this judgment to the number of peaks and the ratio of the peaks, it is possible to reliably distinguish the DNA from any DNA even if any impurities are mixed in the DNA sample.
[0062]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the present embodiment, among DNA extracted from cells, blood, etc., a specific DNA is included in a DNA sample extracted with a restriction enzyme or the like. Genetic diagnosis and determination can be made by accurately knowing the abundance ratio of normal DNA and abnormal DNA without being affected by noise components.
[0063]
【The invention's effect】
As described above, according to the genetic diagnosis apparatus of the present invention, the spectral waveform of light transmitted through a DNA sample is detected, and the spectral waveform in a predetermined wavelength region including the DNA absorption wavelength is detected. Inflection point It is possible to determine whether the peak of the passage time of the passing amount indicates a DNA or a noise component due to the presence of, and applying a predetermined constant voltage between the second electrode and the first electrode, DNA sample Electrophoresis is performed to reduce the migration speed of normal DNA and abnormal DNA using the difference in binding force, and noise components that do not receive the action can be migrated at almost the same speed. That is, normal DNA and abnormal DNA are affected by the action of the binding force of the DNA conjugate while moving, and noise components that do not receive this action are migrated first when voltage is applied and detected by the detection unit. . However, a slightly weaker binding force than normal DNA acts on the abnormal DNA, and the action of the conjugate cannot be easily shaken, and the migration is started next after the noise component. The maximum binding force acts on the normal DNA, the electrophoresis starts further later, and is finally detected by the detection unit. Thereby, a difference can be caused in the detection time of the three DNAs. Some noise components have the same absorption wavelength as normal DNA and abnormal DNA, but such noise components have a spectrum absorption waveform different from that of DNA, and the spectrum in a predetermined wavelength region of DNA. Seen in the waveform Inflection point Therefore, noise components and DNA can be separated, and normal DNA and abnormal DNA can be discriminated. DNA can be separated in a short period of time of ten minutes or more using electrophoresis and DNA hydrogen bonding, and it can be adjusted very easily to an optimum voltage with high resolution simply by applying a predetermined constant voltage. It is possible to detect the presence or absence of an abnormality, and it is possible to provide a small, lightweight, low-cost apparatus with a low running cost, and it is extremely easy to automate diagnosis.
[0064]
In addition, since the DNA conjugate causes a difference in the migration speed between normal DNA and abnormal DNA due to the difference in binding force due to the difference in DNA base sequence, the DNA sample is separated into normal DNA and abnormal DNA. By utilizing the difference in bonding strength of hydrogen bonds, the migration speed of normal DNA and abnormal DNA can be reduced, and noise components that do not receive the action can be migrated at almost the same speed. Since the DNA conjugate is bound to a polymer compound, the migration speed is only a few percent compared to a DNA sample (however, it depends on the type and length of the polymer compound), and is relatively pseudo-fixed as seen relatively. Can be in a state. Conjugate fixation is difficult and usually the flow path must be disposable, but it is not necessary because of pseudo-fixation, and it is extremely necessary to adjust the concentration of various conjugates and samples and the mixing ratio of various conjugates and samples. It becomes easy.
[0065]
A spectrum waveform can be obtained by the separation unit, and normal DNA and abnormal DNA can be distinguished from the spectrum waveform, and the amount of passage can be measured.
[0066]
Since the separation part decomposes the irradiation light in the wavelength region of 200 nm to 260 nm, the spectral waveform of DNA has a peak at 260 nm which is the DNA absorption wavelength, Inflection point As a result, a local minimum value around 240 nm appears, which is different from normal DNA and abnormal DNA. Inflection point Therefore, it is possible to accurately discriminate noise components having no noise and to perform measurement easily and easily.
[0067]
When the absorbance at the wavelength of 260 nm detected by the detection unit is greater than the absorbance at the wavelength of 240 nm and the absorbance at the wavelength of 200 nm is greater than the absorbance at 240 nm, the peak is determined to be a peak indicating DNA. Simply detect absorbance, absorbance at 240nm wavelength, and absorbance at 260nm wavelength. Inflection point Information can be obtained, and DNA and noise components can be distinguished very easily.
[0068]
Since the separation unit is a transmission filter that transmits only a predetermined wavelength region, a target wavelength range can be detected by one light receiving unit from light transmitted from the flow path. Furthermore, it is possible to detect the absorbance of a plurality of wavelengths of interest from light transmitted from the inside of the flow path with one light receiving unit.
[0069]
According to the genetic diagnosis method of the present invention, DNA sample Diagnose the presence ratio of normal DNA and abnormal DNA from the peak of absorbance pherogram by causing electrophoresis to cause differences in the migration speed due to the difference in binding force due to the difference in base sequence between DNA and DNA conjugate in DNA sample When detecting the absorbance, the spectral waveform is detected and the wavelength is in the range of 200 nm to 260 nm. Inflection point If there is a DNA peak, it is judged as a DNA peak, and the remaining case is judged as a noise component peak. Therefore, the DNA sample and the DNA conjugate are electrophoresed, and the difference in binding strength is used to determine whether the normal DNA is abnormal or abnormal DNA. The migration speed is decreased, and noise components that are not affected by the migration can be migrated at almost the same speed. Normal DNA and abnormal DNA are affected by the action of the binding force of the DNA conjugate while moving, and noise components that do not receive this action are migrated first when a voltage is applied and detected by the detector. However, a weak binding force for one base with the DNA conjugate acts on the abnormal DNA, and the action of the conjugate is not easily shaken, and the migration is started next after the noise component. The maximum binding force acts on the normal DNA, the electrophoresis starts further later, and is finally detected by the detection unit. Thereby, a difference can be caused in the detection time of the three DNAs. Some noise components have the same absorption wavelength as normal DNA and abnormal DNA, but such noise components have a spectrum absorption waveform different from that of DNA, and are found in the spectrum waveform of DNA. Inflection point Therefore, noise components and DNA can be separated, and normal DNA and abnormal DNA can be discriminated.
[0070]
In this way, DNA can be separated in a short period of time of ten minutes or more because electrophoresis and DNA hydrogen bonding are used, and by simply applying a predetermined constant voltage, it can be adjusted very easily to an optimum voltage with high resolution, The presence or absence of DNA abnormality can be accurately detected, and the device can be made small, lightweight, and low in running cost, and diagnosis is very easy to automate.
[0071]
If the absorbance at a wavelength of 260 nm is greater than the absorbance at a wavelength of 240 nm and the absorbance at a wavelength of 200 nm is greater than the absorbance at 240 nm, Inflection point Therefore, simply detecting the absorbance at a wavelength of 200 nm, the absorbance at a wavelength of 240 nm, and the absorbance at a wavelength of 260 nm Inflection point Information can be obtained, and DNA and noise components can be distinguished very easily.
[0072]
[Sequence Listing]
SEQUENCE LISTING
<110> Matsushita Electric Industrial Co., Ltd. et al.
<120> The apparatus and method for genetic testing
<130> 2913040005
<140>
<141>
<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 genetic diagnosis apparatus according to an embodiment of the present invention.
FIG. 2 is an external view of the open top lid of a genetic diagnosis apparatus according to an embodiment of the present invention.
FIG. 3 is an apparatus configuration diagram of a genetic diagnosis apparatus according to an embodiment of the present invention.
FIG. 4 is a main part diagram of a control circuit of a genetic diagnosis apparatus according to an embodiment of the present invention.
FIG. 5 is an introduction state diagram of a DNA conjugate for separation and a DNA sample in the flow path of the genetic diagnosis apparatus according to the embodiment of the present invention.
FIG. 6 is a configuration diagram of a detection unit of the genetic diagnosis apparatus according to the embodiment of the present invention.
FIG. 7 is a conceptual diagram of the relationship between a DNA conjugate for separation and a DNA sample in the flow path of the genetic diagnosis apparatus according to the embodiment of the present invention.
Fig. 8 Relationship between elapsed time and absorbance
FIG. 9 is a spectrum waveform diagram when an absorbance peak occurs at 260 nm.
[Explanation of symbols]
1 Power switch
2 display section
3 Display panel
4 Top cover
5 Case
6 Electrophoresis part
7 Support stand
8 Control operation part
9 Power box
9a Power supply
10 Separation part
13 Temperature controller
14 Preliminary detection unit
15 detector
15a D2 lamp
15b photodiode
15c preamplifier
15d A / D converter
16, 17 electrodes
18 Buffer
18c loading buffer
18a, 18b container
18d linear polymer
19 Capillary tube
20 Suction pump
21 DNA conjugate for separation
23 DNA samples
24, 24a, 24b, 24c Transmission filter

Claims (9)

A first container containing a first electrode and filled with a conductive medium; a second container containing a second electrode and filled with a conductive medium; the first container and the second container; In addition, a medium having electrical conductivity is filled, and a DNA sample and a DNA complementary to the normal DNA of the DNA sample are bound to the polymer compound, and the difference in hydrogen bonding force A positive potential is applied to the second electrode and a negative potential is applied to the first electrode, and the flow path is filled with a DNA conjugate for separation that separates the DNA sample into normal DNA, abnormal DNA, and noise components. A power supply unit; a control calculation unit for controlling the power supply unit to apply a predetermined constant voltage between the second electrode and the first electrode; and the DNA sample by application of the constant voltage. when it was allowed to electrophoresis A gene diagnostic apparatus comprising a detection unit for detecting a temporal change in the amount of DNA sample passing through the flow path from the absorbance of irradiated light, wherein the detection unit has a spectrum of light transmitted through the DNA sample. A waveform is detected, and the peak of the passage amount of time changes to either DNA or a noise component other than DNA due to the presence of an inflection point of the spectrum waveform in a predetermined wavelength region including the DNA absorption wavelength by the control calculation unit. A genetic diagnostic apparatus characterized by determining whether it corresponds. The separation DNA conjugate is characterized by causing a difference in the migration speed between normal DNA and abnormal DNA due to a difference in binding force due to a difference in DNA base sequence, and separating the DNA sample into normal DNA and abnormal DNA, The gene diagnostic apparatus according to claim 1. The detection unit is provided with a light emitting unit, a separation unit capable of decomposing light emitted from the light emission unit and transmitted through the flow path for each wavelength, and a light receiving unit for detecting light that has passed through the separation unit. The genetic diagnostic apparatus according to claim 1 or 2, wherein The genetic diagnostic apparatus according to claim 3, wherein the separation unit decomposes the irradiation light in a wavelength region of 200 nm to 260 nm. When the absorbance at a wavelength of 260 nm detected by the detection unit is greater than the absorbance at a wavelength of 240 nm and the absorbance at a wavelength of 200 nm is greater than the absorbance at 240 nm, the peak is determined to be a peak indicating DNA. Item 5. The genetic diagnostic apparatus according to any one of Items 1 to 4. The genetic diagnostic apparatus according to claim 3, wherein the separation unit is a transmission filter that transmits only a predetermined wavelength region. The genetic diagnosis apparatus according to claim 6, wherein the transmission filter selectively transmits a plurality of different wavelengths within a predetermined wavelength region. A predetermined constant voltage is applied between the second electrode and the first electrode to bind the DNA sample to DNA having a complementary relationship with normal DNA of the DNA sample to the polymer compound, and the DNA sample is determined by the difference in hydrogen bonding force. were electrophoresed for separation DNA in the conjugate is separated into a normal DNA and abnormal DNA and a noise component, migration by the difference in binding force due to a difference in nucleotide sequence between the DNA of the DNA sample said separating DNA conjugate A genetic diagnosis method for producing a difference in speed, measuring absorbance by separating normal DNA, abnormal DNA, and noise components, and diagnosing the abundance ratio of normal DNA and abnormal DNA from a pherogram peak, detecting the spectrum waveform when measuring, it is determined that the peak of the DNA if there is an inflection point in the range of wavelength of 200Nm～260nm, the remaining field The gene diagnosis method characterized by determining the peak of the noise components other than DNA. 9. The genetic diagnosis method according to claim 8, wherein when the absorbance at a wavelength of 260 nm is greater than the absorbance at a wavelength of 240 nm and the absorbance at a wavelength of 200 nm is greater than the absorbance at 240 nm, it is determined that an inflection point exists.

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