JP2004028775A - Genetic testing apparatus and method for detecting using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a genetic testing apparatus which can simply examine a plurality of genes in a short time. <P>SOLUTION: The genetic testing apparatus utilizes a computer. The apparatus comprises a stage 4 for supporting a DNA microarray, temperature regulators 9, 51 and 53 each for regulating a temperature of the DNA microarray, a CCD camera 5 for imaging an optical signal from the DNA microarray, a fluid transporting unit 41 for outputting and inputting a fluid in the DNA microarray, a storage unit for storing an application for operating the apparatus, a controller 10a for controlling an overall apparatus according to the application stored in the storage unit, a display unit 10 for displaying information, and an input unit 15 for enabling the information to be input from a user. A variety of parameters regarding a measurement are controlled by the application so as to be freely set by the user. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an apparatus for detecting the expression level of a gene and the presence or absence of a mutation, and a detection method using the same.
[0002]
[Prior art]
In recent years, gene analysis techniques have been advanced, and gene sequences in many organisms including humans have been revealed. In addition, the causal relationship between the analyzed gene products and diseases has been gradually elucidated. Currently used genetic testing methods include a process of extracting nucleic acid from a biological sample, a process of amplifying a target gene to be tested using a nucleic acid amplification method called PCR, NASBA method, etc. It comprises a step of labeling with a molecule or the like, and a step of measuring the base sequence of the labeled target gene or its concentration. Recently, many capillary electrophoresis apparatuses capable of processing a large number of samples at high speed by using a plurality of capillaries for a fluorescently labeled nucleic acid have been used. As a result, compared to the method using a conventional electrophoresis apparatus or the like, it can be performed in about one third to one quarter of the time. In recent years, a testing method using a DNA chip for simultaneously testing a plurality of genes has been developed. The DNA chip is one in which a large number of cDNA probes are immobilized on the surface of a glass substrate, and one in which a large number of oligo probes are synthesized in a minute area on silicon by applying a semiconductor manufacturing process. Any of these methods is capable of simultaneously determining a plurality of base sequences and expression levels of DNA in a sample. The application of DNA chips has made it possible to analyze a large amount of gene expression and a plurality of mutations. Further, from data obtained using a DNA chip, many genes are classified into a plurality of groups (that is, clustering), and information on gene fluctuations associated with development and differentiation is obtained. The gene information thus obtained is used as a database that can be easily accessed through the Internet.
[0003]
Inspection of gene expression levels and analysis of mutations are performed using an electrophoresis method or a DNA chip method. However, the electrophoresis method has the disadvantage that the time required for the test is long and that the number of tests that can be performed at one time is limited to a small number. The gene analysis method using a DNA chip can perform a large number of tests at once, but requires a long test time, requires a large number of whole test steps, and requires complicated operations. For this reason, there is a drawback that an analysis result with good reproducibility cannot be obtained. In order to overcome these drawbacks, a method using a porous filter as a carrier of the DNA chip, and a method using an electrical A method of forcibly performing the operation by a special force has been developed (Japanese Patent Publication No. 9-504864, Japanese Patent Publication No. 2000-515251, Japanese Patent Publication No. 2001-501301). However, the method using a porous filter has a drawback in that a reaction part for performing liquid movement and temperature control and a measurement part for performing fluorescence detection are separate, and a complicated apparatus configuration is required. In the method of forcibly performing hybridization by an electric force (Japanese Patent Application Laid-Open No. 2001-501301), it is necessary to provide a mechanism for strictly controlling the voltage in the chip. Therefore, these methods have not been able to respond to a demand for a simple and easy gene test in a clinical test or the like.
[0004]
In addition, in a multifactorial disease such as cancer and diabetes, or hypertension, which is caused by a combination of a plurality of genetic abnormalities and environmental factors, the abnormalities of a plurality of genes are analyzed in a multifaceted manner. It is thought that more accurate diagnosis can be made by analyzing external environmental factors. In addition, in actual chemotherapy, in order to avoid side effects of the drug and to improve the therapeutic effect, it is necessary to accurately determine in advance the sensitivity to the administered drug (this is also interpreted as constitution). . Furthermore, in performing advanced gene therapy using the P53 gene or the like, gene information such as the expression level of a cancer gene or a tumor suppressor gene in a patient, the presence or absence of a mutation, or the state of cell information transmission is determined in advance. Must be analyzed accurately.
[0005]
Under the circumstances described above, development of a genetic test system capable of testing a plurality of genes easily and in a short time is now desired.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a gene testing apparatus and a method capable of testing a plurality of genes easily and in a short time.
[0007]
[Means for Solving the Problems]
The present inventors have found a means for solving the above-mentioned object as a result of earnest research. That is, a genetic testing device using a computer, a temperature controller for controlling the temperature of the DNA microarray, a photodetector for detecting an optical signal from the DNA microarray, and a fluid to the DNA microarray. A fluid transport section for supplying and discharging, a control section for controlling the entire apparatus according to an application for operating the apparatus, a display section for displaying information, and an input section for enabling input of information from a user. And is controlled by an application so that a user can freely set various parameters related to measurement.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
1. Overview of the device
According to one aspect of the present invention, there is provided an apparatus for easily and quickly detecting the expression level and mutation of a gene in one sample.
[0009]
The basic principle of the detection method carried out in the apparatus of the present invention is to detect a nucleic acid strand having a specific sequence contained in a sample using an immobilized nucleic acid probe having a known sequence. is there. In this method, a single-stranded nucleic acid sample is immobilized on a substrate, for example, and then contacted with a nucleic acid contained in the sample labeled with a fluorescent substance or the like. If the nucleic acid in the sample has a sequence complementary to the nucleic acid probe, it hybridizes with the nucleic acid probe to form a double strand and is thus fixed on the substrate. Therefore, by detecting the label attached to the probe after removing the unreacted nucleic acid strand by washing, a target nucleic acid having a sequence complementary to the probe can be detected.
[0010]
For example, one embodiment of the device of the present invention will be described below with reference to the configuration diagram shown in FIG. A genetic test apparatus 1 using a computer, which is an apparatus of the present invention, includes a microscope 3 for microscopically observing events in a DNA microarray 22 for reacting a sample therein, and a slide chip 2 having the DNA microarray 22. A stage 4 provided on the microscope 3 to support the microscope, a CCD (charge coupled device) camera 5 for capturing a fluorescence image on the DNA microarray 22 observed by the microscope 3, An XY stage controller 7 for controlling a motor driver 6 connected to the stage 4 to change the field of view, a pump driver 8 for transporting a fluid through a joint connected to the DNA microarray 22, Micro array A temperature controller 9 for controlling the temperature of the DNA microarray 22 via a heater section disposed in contact with the DNA microarray 22, and further directly or indirectly connected to all devices included in the genetic test apparatus 1; A computer 10 for controlling them collectively.
[0011]
Although not shown in FIG. 1, the joint is connected to the DNA microarray 22 in such a manner that the liquid contained in the DNA microarray 22 does not leak outside. Further, the other end of the joint portion is connected to a pump driver 8 via a tube, and the pump driver 8 is further connected to a reagent holding portion containing a desired reagent or the like therein. The pump driver 8 sends and receives fluid to and from the DNA microarray 22 according to instructions from the computer 10 according to the analysis program.
[0012]
Similarly, although not shown in FIG. 1, the heater is disposed between the slide chip 2 and the stage 4. The heater unit is connected to a temperature controller 9, and the temperature is adjusted by the temperature controller 9 according to an instruction from the computer 10.
[0013]
The computer 10 includes components included in a general computer. Although not shown in FIG. 1, for example, the components included in the computer 10 include a CPU (Central Processing Unit) 11 which is a main control unit that controls each unit of the computer 10 as a whole, and a control unit in the CPU 11. A memory 12 for storing files such as various programs and image data to be displayed, and a RAM (random access memory) 13 for temporarily storing the results and the like executed by the CPU 11; An image processing unit 14 for generating image data therein according to instructions of the CPU 11 based on a program or the like; an input unit 15 such as a keyboard and a mouse for an operator, for example, a human, to input information to the computer 10; Information by 10 instructions And a display unit 16 for displaying information.
[0014]
The information captured by the CCD camera 5 is sent to the image processing unit 14, processed by the image processing unit 14 according to the instruction of the CPU 11 according to the analysis program stored in the memory 12, and quantified as fluorescence intensity.
[0015]
The computer that can be used in the present invention may be any commonly used electronic computer.
[0016]
The microscope that can be used in the present invention may be any commonly used fluorescence microscope, and may be, for example, Olympus fluorescence microscope AX-70 or BX-42TFR, but is not limited thereto. In the above description, the case where a microscope is used as the optical system used for detection has been described, but it goes without saying that the optical system applicable to the present invention is not limited to the optical system of the microscope.
[0017]
The CCD camera that can be used in the present invention may be any commonly used CCD camera, for example, a general digital camera such as a MegaPlus CCD camera of Kodak Co., or a camera such as Cool Snap cf mono of Rover Co. Yes, but not limited to this.
[0018]
Here, the temperature control unit for controlling the temperature of the DNA microarray 22 is a heating element (for example, an electric heating device) for directly or indirectly contacting the slide chip 2 having the DNA microarray 22 to transfer heat. Heaters, electromagnetic heaters, water baths, air baths and Peltier elements, etc.), sensors for sensing temperatures therein, and temperature controllers for controlling heating in the heating element according to computer controls and information from the sensors.
[0019]
Furthermore, the computer may be connected to an integrated digital communication network (hereinafter, referred to as ISDN) or to a telephone line or a LAN via a modem.
[0020]
As described above, the XY stage controller 7 can move the XY stage of the stage 4 provided in the above-described embodiment. However, the change of the field of view is not limited to such movement on the stage side. This may be performed by itself or by using various scanning mechanisms that change the optical path connecting the CCD camera 5 and the microarray 22.
[0021]
In the above description, the detector used in the present invention has been described using a CCD camera as an example. However, the detector that can be used is not limited to a CCD camera depending on the purpose, and a photomultiplier or the like can be used. However, the CCD camera is effective because one reaction area as shown by the opening 22 in FIG. 2 can be detected simultaneously.
[0022]
2. Slide chip with DNA microarray
One embodiment of the DNA microarray 22 used in the present invention is shown in FIGS. 2 (A) and (B). FIG. 2A is a plan view of the slide chip 2 including four DNA microarrays 22 that perform a reaction therein. FIG. 2B is a cross-sectional view of the slide chip 2.
[0023]
The slide chip 2 shown in FIG. 2B includes a filter 19 having a microchannel 24 which is a porous body, and a support plate 20 for supporting the filter 19. The support plate 20 is composed of two members, and the two members sandwich the filter 19 and constitute the slide chip 2. According to FIG. 2 (B), openings 21 for defining the DNA microarray 22 are formed at respective positions facing the support plate members located above and below the filter 19.
[0024]
Further, as shown in FIG. 2B, the filter 19 is two-dimensionally and flatly located between the two support plates 20 over a large area including all four DNA microarrays 22. Further, the support plate 20 is preferably made of a member having a light shielding property, and more preferably made of a dark black member. Preferably, the two support plates 20 are joined with the filter 19 interposed therebetween.
[0025]
The material of the filter 19 may be any arbitrary material as long as it does not break within the range of temperature control that can be performed there.
[0026]
The “DNA microarray” 22 refers to a portion of the filter 19 exposed from the opening 21 formed in the support plate 20.
[0027]
Although a substantially circular area is shown in FIGS. 2C and 2D, the contour of the probe spot 23 may be substantially circular, substantially rectangular, or polygonal.
[0028]
If desired, the same probe may be fixed to a plurality of probe spots 23. Further, the porous body may be subjected to a surface treatment to adjust the frictional resistance to a fluid and the adsorbability of a reagent such as a probe.
[0029]
The size of the slide chip is 0.5 to 20.0 cm × 0.5 to 20.0 cm × 0.01 to 1.0 cm, preferably 1.0 to 10.0 cm × 1.0 to 10.0 cm × 0. 0.05 to 0.5 cm. Particularly preferably, the size is 3.0 to 8.0 cm × 3.0 to 8.0 cm × 0.05 to 0.2 cm. One example of an actual prepared slide chip is about 7.5 cm × about 2.5 cm × about 0.1 cm.
[0030]
The present microarray has a size of 3.0 mm2 to 16 cm2, preferably 12.0 to 400 mm2, particularly preferably 20.0 to 100.0 mm2, and the actually formed circular microarray on the slide chip has a diameter of about 6 mm (about 28.10 mm). 3 mm2).
[0031]
The probe spots contained in the present DNA microarray have a diameter of 100 to 300 μm, preferably 120 μm, and each DNA microarray has 10 to 1000, preferably 400 probe spots. In the nucleic acid probe described below, a different type of probe can be immobilized for each probe spot.
[0032]
Examples of preferred DNA microarrays for use in the present invention may be, for example, a device described in JP-T-2000-515251 and a microfabricated flow-through porous device described in JP-A-9-504864.
[0033]
FIG. 2 shows a slide chip 2 provided with, for example, four DNA microarrays 22. However, the number may be four or more or less. In the present specification, a slide chip 2 including four DNA microarrays 22 will be described as one preferred embodiment. However, the present invention is not limited to this. Modifications to the procedure will be apparent to those skilled in the art, and such modifications are also within the scope of the invention.
[0034]
As described above, when the slide chip 2 including the four DNA microarrays 22 is used, different samples may be tested for each DNA microarray, or four different types of one sample may be included in one chip. Analysis, for example, mutation of an oncogene, expression analysis, determination of the expression pattern of a drug resistance gene, and an intracellular signal gene may be performed.
[0035]
The size of the probe spot, such as the arrangement density, the arrangement pattern, and the diameter of the probe spot, can be changed as appropriate.
[0036]
In the above-described embodiment, the appearance of the filter 19 is a flat member having a plane having a two-dimensional spread and a constant thickness in a direction perpendicular to the plane. It is possible to apply For example, it is also possible to use a structure in which capillaries that penetrate vertically and linearly with respect to the flat member and whose both ends are open are closely arranged and integrated. Also, for example, the capillary may not be arranged perpendicular to the flat member, but may penetrate linearly at a desired angle, for example, or as a non-linear (ie, curved) hole. It may be arranged. Further, the angle of the capillary to the flat member may be different for each DNA microarray or each probe spot, and a curve or a straight line may be selected.
[0037]
Further, in the above-described embodiment, the filter 19 is described as an example. However, the present invention is applicable not only to a porous body such as a filter but also to a case where a probe spot is provided on a surface of a non-porous body such as a slide glass. However, it is more preferable to use various porous materials because the effect of promoting the reaction by moving the liquid by the pump is high.
[0038]
As used herein, the term "nucleic acid probe" generally refers to a polynucleotide or oligonucleotide consisting of a nucleic acid of about 10 to about 100 nucleotides, and is generally used for detecting a nucleic acid by hybridization. 1 shows a nucleic acid probe to be used.
[0039]
For example, even if it is an oligonucleotide corresponding to an oncogene such as P53 or c-myc, or a tumor suppressor gene, or an oligonucleotide corresponding to a disease-related or susceptibility gene, it corresponds to a sequence containing a polymorphic site. Any desired nucleic acid can be used as a probe, even if it is an oligonucleotide.
[0040]
Further, in addition to the gene to be analyzed, a probe for a housekeeping gene such as β globulin or actin for measuring the steady state of the sample may be used. With the amount of such a housekeeping gene as the reference gene amount in a cell, the actual expression level of an oncogene, a tumor suppressor gene or a drug resistance gene may be measured. In this way, it is possible to more accurately measure the gene expression level in the cell.
[0041]
The term "target nucleic acid" as used herein refers to the nucleic acid to be detected contained in a sample.
[0042]
According to the aspect of the present invention, when the analysis is performed using the slide chip 2 on which the above-described DNA microarray 22 is arranged, such a slide chip 2 includes a plurality of DNA microarrays 22.
[0043]
For example, if the conditions are changed for each probe spot (for example, the type of the probe to be fixed or the surface treatment is changed), a great deal of information can be obtained with sufficient sensitivity in a single micro DNA microarray. be able to. Further, since temperature control and analysis of measurement data can be performed for each DNA microarray, a multi-item reaction result can be quickly obtained in a small device. Further, the temperature may be controlled for each probe spot, or the measured data may be analyzed. In this case, it is possible to quickly obtain the reaction results of more items.
[0044]
In the above-described embodiment of the present invention, an example in which the probe is fixed to the porous body has been described. However, the probe is not necessarily fixed, and the probe is not necessarily fixed to the porous body. The present invention can be applied. In that case, it is the same as the above-mentioned embodiment except that the probe is not immobilized. In this case, the “probe spot” may be referred to as a “reaction spot”, but here, the “reaction spot” is included and described as one embodiment of the probe spot. Such an apparatus is also included in the scope of the present invention.
[0045]
3. software
The memory 12 is used to display a program for controlling the apparatus via the CPU 11, information such as tables retrieved by the CPU 11 and serving as a basis for control and judgment, and obtained results. Image data and the like are stored.
[0046]
For example, the analysis program is a program for performing a gene analysis, and may include an instruction regarding a work procedure for causing the computer to process the analysis.
[0047]
For example, the reaction progress program may include instructions on a work procedure for causing a computer to process a reaction to be performed in the DNA microarray 22. The reaction progress program includes the reaction, that is, the selection of reagents necessary for hybridization, the amount of reagents to be used, reaction conditions such as reaction time and reaction temperature, the number and time of pumping, liquid transport and stirring conditions at the start of pumping. Etc. may be included in advance, or a command instructing the operator to input such information at the start of analysis may be included. In addition, these reaction conditions may be stored in the memory 12 as a program different from the reaction procedure as a reaction condition program.
[0048]
The above program is an example of a program for controlling the present apparatus, but other necessary programs may be stored in the memory 12.
[0049]
For example, the gene correspondence table may be a table specifying associations between the obtained fluorescence intensity, reaction conditions, gene expression levels, expressed genes, gene mutations, and the like.
[0050]
4. Device action
The operation of the above-described embodiment will be described with reference to the flowchart shown in FIG.
[0051]
(S1) The operator sets the slide chip 2 on the stage 4 so as to be in contact with the heater section, and connects the joint section to each DNA microarray 22 of the slide chip 2.
[0052]
(S2) An operator inputs to the input unit 15, and an instruction to start analysis is issued to the present apparatus. At this time, the operator inputs information relating to the reaction conditions, and the information is stored in the RAM 13.
[0053]
(S3) When an instruction to start analysis is issued by the operator, the CPU 11 sends four DNA microarrays to the XY stage controller 7 in accordance with the coordinates set in advance and stored in the memory 12 and the analysis program stored in the memory 12. The XY stage controller 7 drives the motor driver 6 so that the DNA microarray 22 that has not been analyzed and has a small identification number is within the field of view of the CCD camera 5. Be moved. Here, the number of DNA microarrays in the slide chip 2 to be used, the corresponding identification numbers, and the coordinates where they are located are stored in the memory 12 as a table in which they are associated with each other. Therefore, the CPU 11 reads out the coordinates of the target DNA microarray 22 from the unanalyzed DNA microarrays 22 by selecting the one with the youngest identification number and searching the table to issue an instruction based on the coordinates. .
[0054]
(S4) When the target DNA microarray 22 is placed at a position within the field of view of the CCD camera 5, the CPU 11 issues an instruction according to the reaction progress program stored in the memory 12, and controls the pump driver 8 and the temperature controller 9. To perform a plurality of reactions repeatedly. The CPU 11 issues an instruction to the CCD camera 5 at each end of each reaction according to the reaction progress program stored in the memory 12 to execute the imaging. (S4) will be described later in more detail.
[0055]
(S5) Every time an image is captured by the CCD camera 5 in S4, the CPU 11 transfers the obtained image information from the image processing unit 14 to the RAM 13 and temporarily stores it.
[0056]
(S6) The CPU 11 reads from the RAM 13 the identification number of the DNA microarray 22 selected and used for the reaction in the immediately preceding S3, and, based on the identification number, the four DNA microarrays 22 provided in the slide chip 2 and the unanalyzed ones. It is determined whether or not it remains. If there is an unanalyzed DNA microarray 22, the process proceeds to (S3); otherwise, the process proceeds to (S7).
[0057]
(S7) The CPU 11 reads out the image information stored in the RAM 13 in (S5) based on the analysis program, and calculates the fluorescence intensity from the information. Based on the obtained fluorescence intensity and the reaction conditions when the fluorescence intensity was obtained, the CPU 11 searches the gene correspondence table and reads out the corresponding gene information. From these data, the CPU 11 causes the image processing unit 14 to create a table of the result of the gene analysis, sends the calculated fluorescence intensity reaction conditions and the gene correspondence table to the image processing unit 14, and stores the result table therein. Then, the table is stored in the RAM 13.
[0058]
(S8) The CPU 11 instructs the image processing unit 14 to display the image of the gene analysis result table stored in the RAM 13. Thus, the image processing unit 14 reads necessary information from the RAM, performs image processing, and displays the processed information on the display device.
[0059]
In the above description, the information on the reaction conditions input by the operator in S2 may be the reaction temperature, the reaction time, the number of reactions, the selection of the reagent, and the like. Alternatively, these pieces of information may not be input by the operator in S2, but may be included in a reaction progress program or a reaction condition program stored in the memory 12 in advance.
[0060]
In the above-described procedure, an example in which the analysis is performed for each DNA microarray 22 has been described, but the analysis may be performed simultaneously for all the DNA microarrays 22. In the above description, the case where the DNA microarray 22 to be analyzed is automatically selected in the order of the identification numbers assigned in advance has been described. However, for each analysis, the operator selects the DNA microarray 22 to execute the analysis, and 15 may be input.
[0061]
If the computer is connected to an ISDN, LAN, or telephone line, the result obtained by the analysis can be compared with information obtained by searching a desired genome database or base sequence database. It is possible. This procedure may be set so that the CPU 11 searches the database based on the comparison program stored in the memory 12, compares the result with the result obtained by the device, and identifies the result.
[0062]
5. Reaction in DNA microarray
The reaction in the above (S4) will be further described with reference to FIG.
[0063]
(S41) When the target DNA microarray 22 is placed at a position within the field of view of the CCD camera 5, the CPU 11 instructs the temperature controller 9 to control the heater according to the reaction progress program stored in the memory 12. Thereby, the temperature of the target DNA microarray 22 is maintained at the predetermined first temperature. Further, the CPU 11 issues an instruction to the pump driver 8 in accordance with the reaction progress program, causes the sample to be added to the target DNA microarray 22, and causes the reaction to be performed while stirring by pumping.
[0064]
(S42) After the reaction has been performed for a predetermined reaction time according to the reaction progress program stored in the memory 12, the CPU 11 issues an instruction to the pump driver 8 according to the reaction progress program, and converts the sample into the target DNA. The CCD is temporarily retracted from the microarray 22 and the CCD camera 5 performs imaging.
[0065]
(S43) After the imaging by the CCD camera 5 is completed, the CPU 11 issues an instruction to the pump driver 8 in accordance with the reaction progress program, and causes the DNA microarray 22 to again add the sample primarily evacuated in (S42). At this time, the stirring function is also functioning.
[0066]
(S44) The CPU 11 reads the reaction progress program and the conditions inputted by the operator in (S1) and stored in the RAM 13, and performs the reaction under further reaction conditions in which the temperature setting and / or the solvent composition and the like are changed. Determine if it is necessary. If necessary, proceed to (S41), and if not, proceed to (S5).
[0067]
6. Analysis target and preprocessing
Examples of analyzes that can be performed by the apparatus of the present invention include detection of the presence or absence of a mutation such as an oncogene in a subject, gene expression analysis in a subject, expression analysis of a drug resistance gene, genotype of a disease-related or susceptibility gene in the subject. In clinical therapeutic and diagnostic analysis, such as determination, determination of the expression pattern of an intracellular signal gene in a subject, and various other genetic analyzes in nonclinical basic research.
[0068]
Generally, in the case of a human, blood, a cultured cell, a biological tissue such as a cell or a tissue collected at the time of a biopsy or an operation can be used as a sample used for a genetic test.
[0069]
For example, in the case of a tissue section collected endoscopically, the tissue section is fixed on a slide glass, stained, from the slide glass after performing a pathological examination, a laser capture system that is a micro-dissection system, It is possible to use a sample obtained by collecting only the tissue portion of the cancer lesion portion using Olympus LCS200.
[0070]
Prior to analysis by this apparatus, the biological sample thus obtained is treated with a nucleic acid extraction reagent utilizing a phenol / chloroform method, a microcolumn method, or a magnetic particle method to extract DNA and / or RNA. I do. Further, the extracted DNA and / or RNA is subjected to gene amplification by, for example, PCR, RT-PCR, T7 amplification, or the like. However, in the case of a sample having a large amount of nucleic acid, cDNA may be prepared without performing gene amplification. However, in any case, a reagent is added so that a labeling substance, for example, any of fluorescent labeling substances such as FITC, rhodamine, Cy3 and Cy5 is added to the 5 ′ end of the primer or somewhere in the synthesized nucleotide. It is preferred to optimize the composition.
[0071]
The preparation of the sample used for such an analysis is described in, for example, “DNA Microarray and the Latest PCR Method” (Cell Engineering Separate Volume; Genome Science Series 1, Shujunsha, published on March 16, 2000). Alternatively, it may be carried out by a method generally known per se.
[0072]
After performing the fluorescent labeling described above, the prepared sample obtained manually by the operator using a pipette may be dispensed to the DNA microarray. Thereafter, the analysis may be performed by the above-described device.
[0073]
7. analysis method
As described above, we used a three-dimensional DNA microarray, which can be expected to perform high-efficiency hybridization in a short time, for a reaction unit, a pump for solution control, a temperature control system for temperature control, and a highly sensitive fluorescence. The inventors have invented a genetic test system in which a detection device and an imaging device controlled by a computer are combined.
[0074]
A gene analysis method performed by such a system is also included in the scope of the present invention. The method of the present invention will be described with reference to the flowchart of FIG.
[0075]
(Sa) First, a sample is prepared. Nucleic acids are extracted from tissues or cells collected from a test subject by, for example, the phenol-chloroform method.
[0076]
(Sb) The nucleic acid obtained in (Sa) is amplified by, for example, the PCR method or the NASBA method, and fluorescently labeled. When the amount of nucleic acid is large, only fluorescent labeling is performed.
[0077]
(Sc) The fluorescently labeled nucleic acid obtained in (Sb) is purified by a spin column method, an ethanol precipitation method, or the like.
[0078]
(Sd) The nucleic acid obtained in (Sc) is denatured into a single-stranded labeled nucleic acid. Thereafter, the obtained single-stranded labeled nucleic acid is added to the DNA microarray on which the nucleic acid probes are immobilized.
[0079]
(Se) The DNA microarray containing the sample is subjected to hybridization under desired conditions. At this time, it is preferable to agitate the liquid in the DNA microarray by pumping or pipetting.
[0080]
(Sf) After performing the hybridization in (Se), the liquid in the DNA microarray is retained at the bottom of the array, and the fluorescence intensity contained in the labeled nucleic acid bound to the nucleic acid probe is measured.
[0081]
(Sg) After the measurement of the fluorescence intensity in (Sf) is completed, hybridization is performed again under desired reaction conditions, and the fluorescence intensity is measured as described above. This operation is repeated as many times as necessary under various reaction conditions.
[0082]
(Sh) After the completion of the reaction of (Sg), the sample in the DNA microarray is recovered while maintaining the state as it is, that is, without any contamination such as dilution or impurities. The fluorescence intensity contained in the bound labeled nucleic acid is measured.
[0083]
(Si) Based on the fluorescence intensities obtained in (Sf) and (Sh), the amount of the expressed gene and the presence / absence of a mutant gene are determined to obtain information on the target gene.
[0084]
(Sj) The information on the target gene obtained in (Si) is compared with known gene information disclosed in a database and analysis results obtained from a standard sample.
[0085]
(Sk) Based on the result of the comparison of (Sj), a determination is made as to the gene analysis for the sample from the subject.
[0086]
Here, two reactions were performed continuously using two reaction conditions. However, if necessary, the number of continuous reactions and the number of measurements of the fluorescence intensity can be increased. In that case, the above (Sf) to (Sg) may be repeated as many times as necessary.
[0087]
The apparatus described above as an aspect of the present invention is configured so that liquid dispensing, stirring, temperature control, and measurement can always be performed on a reaction element (that is, a slide chip) placed on a stage. Can be easily achieved in a small space. Therefore, the device of the present invention can be an excellent tool useful for any genetic testing.
[0088]
When a CCD camera is used as a detector according to the present invention, at least one DNA microarray is small enough to be sufficiently captured by the CCD camera. There is no need to optically scan the separate probe spots. This makes it possible to simultaneously and continuously monitor the reaction of each spot in the same DNA microarray, which is very advantageous for simultaneous inspection of multiple items.
[0089]
According to a further aspect of the present invention, the present invention is capable of performing multiple tests on the same sample in succession, so that multiple test results can be obtained quickly or at any time. It provides a possible device.
[0090]
According to the present invention, in such a measurement device, the type of each reagent (for example, a nucleic acid probe and a nucleic acid primer) applied to each probe spot or each reaction condition is changed for each reagent or each reaction condition. Analysis software for analyzing the measured data while checking the obtained measured data is also provided.
[0091]
In particular, in the device according to the present invention, it is possible to simultaneously, sequentially or sequentially execute a plurality of types of reactions at the same probe spot. Therefore, it is possible to easily prepare reaction conditions necessary for achieving such a plurality of kinds of reactions, and to accurately read and process a plurality of data obtained by such a plurality of kinds of reactions. The reading device according to the present invention may be provided with a new reading means.
[0092]
For example, such reading means may be means for performing the following:
i) Test item information obtained by combining position information of a probe spot to be measured in one DNA microarray and control information for the probe spot (for example, a suitable temperature for each reagent, an inspection purpose, etc.) is stored in a memory circuit or the like. Storing in the storage means of
ii) The control actually performed at all spots in the DNA microarray is monitored, the monitored actual control conditions are compared with the test item information of i), and the result of the comparison is stored in a memory circuit or the like. In the storage means of
iii) storing, in a storage means such as a memory circuit, measurement results obtained under the monitored control conditions of ii) as measurement data in association with the verification results obtained in ii); or
iv) The measurement result performed under the monitored control condition in ii) is associated with the collation result obtained in ii), and the collation is performed on the measurement result by data processing means such as a CPU. Performing arithmetic processing that makes corrections based on the
[0093]
Therefore, such reading means reads out and compares each data stored in the storage means with the storage means for storing the test item information, the monitored control conditions, the measurement results, and the data such as the measurement data. And data processing means such as a CPU for performing collation and performing necessary arithmetic processing.
[0094]
In addition, such a novel reading unit itself and an apparatus including such a reading unit are also provided as embodiments of the present invention.
[0095]
According to such a reading means, for all spots in the same CCD visual field, different types of reaction results such that all or some of the reactions overlap in time among a plurality of spots, or at least one spot Reaction results under a plurality of different reaction conditions to be executed can be stored and extracted without being confused, and a desired determination result can be output.
[0096]
As described above, the embodiments of the present invention have been described in detail. However, the above-described detailed description and drawings are intended to illustrate embodiments of the present invention, and therefore, the present invention is not limited thereto. On the contrary, the invention is intended to cover various improved or modified inventions based on various equivalents and presently obvious technical knowledge.
[0097]
【Example】
1. Gene analysis
Gene analysis is performed using the embodiment of the present invention as shown in FIG.
[0098]
As a container for measuring the gene, a chip containing a porous filter in which a plurality of genes having complementarity with the target gene to be tested are immobilized on the surface is used. The slide chip containing the porous filter is set on a reaction part (incubator part) placed on the stage of a fluorescence microscope. The fluorescence microscope is configured so that the excitation light shutter, the fluorescence cube, the stage movement, and the like can be automatically controlled by a host computer. The fluorescence microscope is provided with a device such as a CCD for photographing an image, and the timing of photographing is also controlled by a host computer. In addition, the same computer can also control the exposure time, image storage, cooling state, and the like. Further, the incubator is provided with a pump for transporting a liquid and a heater or a Bertier element for temperature control, and these controls can also be performed by a host computer.
[0099]
After the sample is dropped on the measurement area of the chip, the reaction solution is moved by a pump to accelerate the hybridization reaction. The solution at the lower part of the incubator is moved, and when the liquid surface of the reaction solution comes into contact with the upper surface of the filter, the excitation light shutter is opened and a fluorescent image of the filter surface is photographed by a device such as a CCD camera. The photographed image data is stored in a designated folder of the computer, and the measurement result is analyzed by a separately installed analysis program.
[0100]
Thus, by arranging a pump for promoting hybridization, a heater for applying a temperature change to the chip, and, if necessary, a pipettor system for changing the solution composition around the incubator, this one unit is obtained. Hybridization patterns at various temperatures and solution states can be continuously obtained by the test system. In addition, it can be excited by different wavelengths according to the type of fluorescent label used for measurement, and images of different fluorescent wavelengths can be taken, so even if the sample contains a substance labeled with multiple fluorescent dyes, By automatically switching the filter cubes, for example, the fluorescence intensity ratio of two colors can be easily measured. From the fluorescence intensity of the spot thus obtained, the amount of the target gene that has hybridized with the probe immobilized on the chip surface is analyzed. By using this test apparatus, it is possible to detect the expression level of a gene whose expression level fluctuates greatly in accordance with canceration of C-myc or the like.
[0101]
Further, by analyzing the pattern of the change in the fluorescence intensity of the fluorescent spot caused by the change in the temperature of the incubator, mutations in genes such as K-Ras and P53 which greatly affect canceration can be detected. In this way, two physiologically important gene mutations, that is, the expression level of a gene that controls complex cell functions inside the cell and the mutation that occurs inside the gene, can be converted to the same device in a short time. It becomes possible to analyze by. The sample preparation method used here is the same as that used in the conventional genetic test, and the results obtained by this device are stored in databases such as GenBank published on the Internet. It can be easily compared with genetic information.
[0102]
By using such an apparatus according to the embodiment of the present invention, the degree of hybridization of the target gene can be measured in real time while changing the state of the solution in the reaction part and the temperature conditions. By using such an apparatus, the degree of hybridization of the target gene can be measured in real time while changing the state of the solution in the reaction part and the temperature conditions. By using the present invention, it has become possible to accurately measure the expression level of a target gene. In addition, following the measurement of the expression level, the efficiency of hybridization to different probes is forcibly changed by automatically changing the temperature of the microarray and the solution composition of the reaction solution. , For example, the type of single nucleotide polymorphism (hereinafter, referred to as SNP; Single nucleotide polymorphism) can be detected. By using this system, it is expected that the degree of progress of cancer or the like, the degree of malignancy, the possibility of metastasis, the stage of disease, etc. can be more accurately predicted.
[0103]
At the same time, it is possible to simultaneously examine the expression level of a drug susceptibility gene such as P450 that affects the sensitivity to the therapeutic agent and SNPs that affect the activity. In this way, by simultaneously examining not only the abnormality of the disease-causing gene but also the abnormality of the gene that is sensitive to the drug, the type and temperature of the selected therapeutic agent can be optimized according to the patient's constitution. It becomes possible. Further, based on the obtained information on gene abnormality, it is possible to accurately select a gene to be used for gene therapy, which is expected to have a high therapeutic effect in the future.
[0104]
2. Measurement of expression levels of C-myc, estrogen receptor and telomerase
FIG. 6 shows a slide chip for measuring the expression levels of C-myc, estrogen receptor and telomerase. Probes for a plurality of genes including c-myc as shown in FIG. 6A and a housekeeping gene are immobilized on the slide chip. FIG. 6 (B1) schematically shows the positions where the nucleic acid probes of FIG. 6 (A) are fixed to the probe spots included in the DNA microarray. Each type of nucleic acid probe is fixed to a probe spot for each type. FIGS. 6 (B2) and (B3) are diagrams schematically showing the analysis results. FIG. 6 (B2) shows the results obtained from a sample from a normal subject, and FIG. 6 (B3) shows the results obtained from a sample from an abnormal subject. By judging the presence or absence of fluorescence from such a result, it is possible to detect the gene expressed in the subject.
[0105]
Using such a slide chip, by changing the ambient temperature and solution composition of the chip on which expression analysis was performed, or setting the temperature near the labeled nucleic acid and the probe in the sample to a condition close to the TM (melting temperature) value. As a result, the optimal reaction conditions for each probe are achieved, whereby it is possible to analyze mutations occurring inside these genes.
[0106]
FIG. 7 shows an example in which a mutation occurring inside the gene of the tumor suppressor gene P53 is detected by comparing the intensity of fluorescent spots generated for probes having different nucleotide sequences. The expression level can be detected based on the difference in the fluorescence intensity.
[0107]
The detection of the gene and the analysis of the expression level as described above can be continuously performed for one sample in one DNA microarray.
[0108]
As described above, the apparatus and method of the present invention make it possible to determine the stage of canceration, accurately predict malignancy, susceptibility to metastasis, and individual differences in drug resistance.
[0109]
With the advancement of genetic testing technology and computer science, changes in expression levels, types of mutations, or SNPs as abnormalities of many genes have already been identified to date. Such known information is widely disclosed on the Internet as a database and can be easily obtained. As described above, a clinically useful accurate determination can be made by comparing and evaluating the results of a genetic test obtained by the apparatus and method of the present invention with already known information. For example, accurate metastasis prediction and treatment guidelines standardized by the American Cancer Institute (NCI) can be performed, and the possibility of erroneous judgments resulting from differences in judgment criteria among countries and facilities can be reduced. . In the future, it is expected that important information will be provided in selecting a transgene when performing gene therapy.
[0110]
8. Supplement: Equipment control system and operation procedure
FIG. 8 shows a control block of the genetic test apparatus 1.
[0111]
As shown in FIG. 8, the genetic test apparatus 1 includes a control unit 10a, a camera controller 10b, and a microscope controller 10c, and these correspond to the computer 10. Further, as described above, the genetic test apparatus 1 includes the (preferably cooled) CCD camera 5, the stage 4, the stage controller 7, the temperature controller 9, the input unit 15, and the display unit 16. are doing. The stage 4 includes a motor 4a for driving the stage.
[0112]
The genetic test apparatus 1 further includes a mirror unit 31, a shutter unit 32, and an illumination unit 33 included in the fluorescence observation microscope 3, and an upper part constituting a temperature controller for adjusting the temperature of the DNA microarray 22. It has a heater 51, a lower heater 52, and a cooling fan 53, and has a pump 41 for moving a liquid through the DNA microarray 22.
[0113]
The filter unit 31 includes a motor 31a for driving it, the shutter unit 32 includes a motor 32a for driving it, the upper heater 51 includes a temperature measuring resistor 51a for measuring the temperature, and the lower heater The heater 52 includes a temperature measuring resistor 51b for measuring its temperature. The pump 41 includes a controller 41a for driving and controlling the pump 41. Here, the mirror unit includes, for example, an excitation filter that transmits only light in the excitation wavelength band from the irradiation light, an absorption filter that transmits only fluorescence from the target object, and blocks the excitation light, With respect to light, a dichroic mirror that reflects shorter light and transmits longer wavelengths can be integrated.
[0114]
The camera controller 10b controls the CCD camera 5, and the microscope controller 10c controls the motor 31a of the mirror unit 31, the motor 32a of the shutter unit 32, and the illumination unit 33. The temperature controller 9 controls the upper heater 51 based on information from the resistance temperature detector 51a, and controls the lower heater 52 based on information from the resistance temperature detector 52a. The motor 4a is controlled.
[0115]
The control unit 10a controls the camera controller 10b, the microscope controller 10c, the temperature controller 9, the stage controller 7, and the controller 41a of the pump 41. Further, the control unit 10a displays an appropriate message or information on the display unit 16 or acquires information input from the input unit 15.
[0116]
The genetic test device 1 is operated according to a specially designed application. The application includes various programs such as the above-described analysis program and reaction progress program, and appropriately controls each section of the apparatus 1 and issues a command to the control section 10a during operation of the genetic test apparatus 1 to display the command. The next necessary operation is displayed on the unit 16 to give an instruction to the user, and various information related to measurement is displayed on the display unit 16.
[0117]
In particular, the application uses the input unit 15 and the display unit 16 to input various profiles, which are instructions for each unit of the apparatus, such as a pump operation instruction, a shooting instruction, a delay time instruction, a fixed stop instruction, a temperature change instruction, and a cleaning instruction. And control the entire device so that the user can freely change it. That is, the operation of the genetic test apparatus 1 is controlled by the application so that the user can freely set various parameters related to the measurement. For this purpose, the control unit 10a displays a message prompting input of information on the display unit 16, for example, according to a command from the application, and waits for information input from the input unit 16.
[0118]
The genetic test device 1 is operated, for example, according to the operation shown in the flowchart shown in FIG.
[0119]
1. Turn on the device.
2. Perform user login. At this time, a user name is selected from an existing list, or a user name is newly described.
3. Start the application. At this time, the control unit 10a performs connection confirmation and initialization of connected devices such as the microscope controller 10b, the pump 41, the temperature controller, the stage controller 7, and the CCD camera 5.
[0120]
4. Supply water. That is, the pipe is filled with water. This is performed, for example, by setting a water supply tray containing water in an incubator, inserting the incubator into the device, and sucking water from the water supply tray by the pump 41, or placing a drainage tray in the incubator. It can be carried out by setting, inserting the incubator into the apparatus, sucking water from a water supply tank (not shown) by the pump 41, and discharging the water to a drain tray.
[0121]
5. Start temperature control. Specify the temperature of the incubator. That is, the target temperature of the heater 51 is set.
[0122]
6. Set conditions. It sets the temperature, sets the mirror unit, sets the shooting conditions, and sets the pump operating conditions.
[0123]
7. Set profile and cycle. Here, the profile is an instruction to each part of the apparatus, and includes a pump operation instruction, an imaging instruction, a delay time instruction, a constant stop instruction, a temperature change instruction, a cleaning instruction, and the like. A cycle consists of an appropriate combination of these profiles.
[0124]
8. Set the sequence. A sequence is composed of an appropriate combination of cycles and specifies which cycles are performed, in what order, and how many times. The sequence determines the order of measurement.
9. Set the measurement well, file name, etc. A well to be observed, a folder name for saving an image of the well, a sample name, sample information, and the like are set.
10. The reaction part (incubator part) is discharged out of the apparatus.
11. The cassette containing the DNA microarray to be measured is set in the incubator.
[0125]
12. The incubator part on which the object to be measured is set is inserted into the device.
13. Measure according to the previously set sequence. A graph is displayed by specifying the point where the luminance is to be measured.
14． The incubator part is discharged out of the apparatus, and the cassette whose measurement has been completed is taken out.
15. Exit the application. At this time, the apparatus is initialized, whereby the pump returns to the initial state, the temperature controller is stopped, and the incubator portion is moved to the home position.
[0126]
The operations 6 to 8 described above, that is, setting of conditions, profiles, cycles, and sequences are performed, for example, by displaying an input screen showing information items to be input on the display unit 16 to prompt the user to input information. In response, the user inputs an appropriate numerical value or the like for each item on the screen from the input unit 15.
[0127]
However, operations 6 to 8 are not limited to such a method, and may be performed by another appropriate method. For example, a file (data file) in which necessary information is recorded is stored in advance in an auxiliary storage device of a computer, such as a hard disk or a floppy disk. May be performed by reading a data file from the auxiliary storage device and obtaining necessary information.
[0128]
Such a data file can be created, for example, by recording the set values used by the user in the previous measurement. Alternatively, the data file may be created by the manufacturer of the slide chip 2 by recording information suitable for each type of the slide chip 2, for example. In this case, the data file may be supplied to the user in a form attached to the slide chip 2, for example.
[0129]
In the setting of the profile in the operation 7, for example, the following operations and the like are instructed. This makes it possible to cope with various operations.
[0130]
a. Pump operation: Set whether to pull (suction) or return (discharge) the pump. At this time, the driving amount and the speed can be arbitrarily set. Usually, when reacting a sample, the drive amount of the pump is often set to substantially the same amount as the sample amount. However, when imaging fluorescence, it is not preferable that the sample liquid is present on the DNA chip because the focus position is shifted and the fluorescent substance in the sample liquid becomes noise. Therefore, it is preferable to set a slightly larger amount than the sample liquid at the timing of capturing the fluorescence. This amount depends on the viscosity of the sample liquid and the driving speed of the pump. The drive amount of the pump can be set to an optimum value depending on the amount of the sample liquid, the viscosity, and the well size of the chip (the size of the well may be different depending on the type of the chip). Since the reaction state varies depending on the moving speed of the sample liquid, an optimal driving speed of the pump can be set in order to cause the reaction under the optimum condition.
[0131]
b. Shooting: Set the filter and exposure time used for shooting. A filter is selected according to the fluorescent dye used for labeling the specimen, and an exposure time suitable for observing the fluorescence can be arbitrarily set. When the luminance is low, the exposure time can be set long.
[0132]
c. Delay time: Set the time to stop operation. By setting the delay time, for example, in driving a pump, the time until the liquid is chipped or comes up can be set to an optimum value.
[0133]
d. Pause: The operation is stopped until a restart instruction is issued.
[0134]
e. Washing: This is performed in order to remove the unreacted fluorescently labeled sample and not to observe extra fluorescence other than the result of the reaction. According to the instructions of the application, take out the incubator part from the apparatus and instruct the user to drop the cleaning liquid. By driving the pump, the cleaning liquid is moved up and down to clean the chip. Perform the above operations manually. Washing may or may not be necessary.
[0135]
f. Temperature change: Used to change the temperature from the current set temperature during measurement. Set the changed temperature and the waiting time after reaching the changed temperature. Since there are optimal reaction temperatures and reaction temperature programs for the genes to be detected, optimal temperature conditions can be set.
[0136]
In the setting of the cycle in the operation 7, for example, the following operation is instructed. This makes it possible to cope with various operations.
[0137]
Cycle 1: pre-wash
1. The washing solution is dropped.
2. Make the cleaning liquid flow
3. Aspirate and discard the washing solution.
4. Take a filter image after cleaning.
5. Drop sample.
[0138]
Cycle 2: Status check
1. Aspirate the sample solution under the filter.
2. Shoot.
3. Drain the sample onto the filter.
[0139]
Cycle 3: Pump drive
1. Aspirate the sample solution under the filter.
2. Drain the sample onto the filter.
[0140]
Cycle 4: Reaction result shooting
1. Remove the sample solution with a pipette.
2. The washing solution is dropped.
3. Drive the pump.
4. Shoot.
[0141]
Cycles 2 and 3 above are repeated until the reaction is completed. Steps 3 and 4 in cycle 4 are repeated while changing the conditions.
[0142]
An example of the sequence setting in operation 8 is shown below.
1. Execute cycle 1 once.
2. Execute cycle 2 once.
3. Execute cycle 3 29 times.
4. Execute cycle 2 once.
5. Execute cycle 3 29 times.
6. Execute cycle 2 once.
7. Execute cycle 3 29 times.
8. Execute cycle 2 once.
9. Execute cycle 3 29 times.
10. Execute cycle 2 once.
11. Execute cycle 3 29 times.
12. Execute cycle 2 once.
13. Execute cycle 4 twice.
[0143]
The operation of each member level in cycle 1 to cycle 4 described above is shown below.
[0144]
Cycle 1: pre-wash
1. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
Stop for 2.5 seconds.
3. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
4. Stop for 10 seconds.
5. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
Stop for 6.5 seconds.
7. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
8. Stop for 10 seconds.
9. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
Stop for 10.5 seconds.
11. After taking an image with an exposure time of 3000 ms using the FITC mirror unit, the same image is taken with the Cy3 mirror unit.
12. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
13. Stop for 30 seconds.
[0145]
Cycle 2: Status check
1. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
Stop for 2.5 seconds.
3. After taking an image with an exposure time of 200 ms using a mirror unit for FITC, an image is similarly taken with a mirror unit for Cy3.
4. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
5. Stop for 10 seconds.
[0146]
Cycle 3: Pump drive
1. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
2. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
Stop for 3.5 seconds.
[0147]
Cycle 4: Reaction result shooting
1. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
2. After taking an image with an exposure time of 1000 ms using a mirror unit for FITC, an image is similarly taken with a mirror unit for Cy3.
3. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
4. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
5. After taking an image with an exposure time of 1000 ms using a mirror unit for FITC, an image is similarly taken with a mirror unit for Cy3.
6. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
7. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
[0148]
8. After taking an image with an exposure time of 1000 ms using a mirror unit for FITC, an image is similarly taken with a mirror unit for Cy3.
9. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
10. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
11. After taking an image with an exposure time of 1000 ms using a mirror unit for FITC, an image is similarly taken with a mirror unit for Cy3.
12. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
13. Pull the syringe at 50 μl at a speed of 5 μl / s and stop for 1 second.
14． After taking an image with an exposure time of 1000 ms using a mirror unit for FITC, an image is similarly taken with a mirror unit for Cy3.
15. Return the syringe by 50 μl at a speed of 5 μl / s and stop for 1 second.
[0149]
By operating the genetic test apparatus 1 according to the application as described above, the user can measure various parameters under desired conditions and operations using the input unit 15, the display unit 16, and the data file.
[0150]
The present invention is not limited to a genetic test depending on the purpose, and can perform various other biological reactions (enzyme reaction, antigen-antibody reaction, metabolic reaction, biological kinetic test, etc.) at high throughput. It should be noted that the technology to be implemented and monitored can be widely provided to the medical field. Although the present invention has been described with respect to a genetic test apparatus using a DNA probe, the present invention is applicable not only to a DNA probe but also to, for example, a case where an antibody or a tissue is used as a probe.
[0151]
【The invention's effect】
By using such an apparatus according to an embodiment of the present invention, a gene testing apparatus and method capable of testing a plurality of genes easily and in a short time is provided. Further, with such an apparatus and method, the degree of hybridization of the target gene can be measured in real time while changing the state of the solution in the reaction part and the temperature conditions. Therefore, by using the present invention, the presence / absence of the expression of the target gene and the expression level thereof can be easily, efficiently, and accurately measured.
[Brief description of the drawings]
FIG. 1 is a configuration diagram illustrating one embodiment of the present invention.
FIG. 2 is a diagram showing an example of a slide chip.
FIG. 3 is a flowchart showing an analysis procedure.
FIG. 4 is a flowchart showing a reaction procedure.
FIG. 5 is a flowchart showing an analysis method.
FIG. 6 is a diagram showing an example of a slide chip for P53 exon 7 mutation analysis.
FIG. 7 shows a slide chip for expression level analysis.
FIG. 8 is a diagram showing a control block of the genetic test apparatus of FIG. 1;
FIG. 9 is a flowchart showing an operation procedure of the genetic test apparatus.
[Explanation of symbols]
1. Genetic testing device 2. Slide tip 3. Microscope 4. stage
5. CCD camera 6. Motor driver 7. XY stage controller
8. Pump driver 9. Temperature controller 10. Computer
22. DNA microarray 23. Probe spot

Claims (13)

A genetic testing device using a computer, comprising: a temperature control unit for controlling the temperature of a DNA microarray, a photodetector for detecting an optical signal from the DNA microarray, and a fluid supply / discharge to / from the DNA microarray. A fluid transport unit for controlling the entire device according to an application for operating the device, a display unit for displaying information, and an input unit for enabling input of information from a user. A device that is provided and is controlled by an application so that the user can freely set various parameters related to the measurement. The device according to claim 1, wherein the detector is a CCD camera. A genetic testing device using a computer according to claim 1 or 2, further comprising a storage unit storing an application for operating the device, wherein the application is required next to a display unit. A device that displays operations and gives instructions to the user, and displays various information related to measurement on the display unit. A genetic test apparatus using a computer according to any one of claims 1 to 3, wherein the gene test apparatus is controlled by an application so that a user can freely change various profiles that are instructions for each part of the apparatus. apparatus. 5. The genetic test apparatus using a computer according to claim 4, wherein the profile includes at least one of a pump operation instruction, an imaging instruction, a delay time instruction, a fixed stop instruction, a temperature change instruction, and a cleaning instruction. Equipment. A genetic test apparatus using a computer according to any one of claims 1 to 5, wherein a message prompting input of information is displayed on a display unit, and the application is configured to wait for information input from the input unit. Equipment controlled by. A genetic test apparatus using the computer according to claim 4, wherein the apparatus is controlled by an application such that a cycle configured by combining a plurality of profiles can be set freely. A genetic test apparatus using the computer according to claim 7, wherein the apparatus is controlled by an application so that a sequence configured by combining a plurality of cycles can be set freely. 9. A genetic testing device using a computer according to claim 1, wherein the DNA microarray substrate is a porous material, and a probe spot on which a nucleic acid probe is fixed. Equipment to be equipped. A genetic test method using the device according to any one of claims 1 to 9,
(1) Amplifying nucleic acids extracted from tissues or cells collected from a test subject and adding a fluorescent labeling substance;
(2) adding the labeled nucleic acid obtained in the above (1) to a DNA microarray having a desired nucleic acid probe;
(3) performing a hybridization reaction on the DNA microarray of (2) under desired conditions;
(4) measuring the fluorescence intensity of the DNA microarray obtained in (3);
(5) obtaining the result of a genetic test by determining the amount of expressed gene and / or determining the presence or absence of a mutated gene based on the fluorescence intensity obtained in (4) above;
A method comprising: A genetic test method using the device according to any one of claims 1 to 9,
(1) Amplifying nucleic acids extracted from tissues or cells collected from a test subject and adding a fluorescent labeling substance;
(2) adding the labeled nucleic acid obtained in the above (1) to a DNA microarray having a desired nucleic acid probe;
(3) performing a hybridization reaction on the DNA microarray of (2) under desired conditions;
(4) After the reaction of (3) is completed, the solution contained in the DNA microarray is allowed to remain at the bottom of the array;
(5) measuring the fluorescence intensity of the DNA microarray obtained in (4);
(6) stirring the liquid collected at the bottom of the array in (4) again;
(7) repeating the hybridization reaction under the desired conditions as necessary for the DNA microarray of (6);
(8) measuring the fluorescence intensity of the DNA microarray obtained in (6);
(9) obtaining the result of a genetic test by determining the amount of expressed gene and / or determining the presence or absence of a mutated gene based on the fluorescence intensities obtained in (4) and (8) above;
A method comprising: The method according to claim 10 or 11, further comprising determining the amount of the expressed gene based on the fluorescence intensity obtained in (4) or (8) of claim 10 and / or A method comprising obtaining a result of a genetic test by comparing information obtained by determining the presence or absence of a mutant gene with known gene information disclosed in a database and / or information obtained from a standard sample. . The method according to claim 11, wherein the operations (5) to (8) are repeated two or more times.

Images