WO2005024424A1 - Dispositif d'examen de genes et procede d'examen l'utilisant - Google Patents

Dispositif d'examen de genes et procede d'examen l'utilisant Download PDF

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Publication number
WO2005024424A1
WO2005024424A1 PCT/JP2003/011020 JP0311020W WO2005024424A1 WO 2005024424 A1 WO2005024424 A1 WO 2005024424A1 JP 0311020 W JP0311020 W JP 0311020W WO 2005024424 A1 WO2005024424 A1 WO 2005024424A1
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WIPO (PCT)
Prior art keywords
dna microarray
information
gene
genetic
nucleic acid
Prior art date
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PCT/JP2003/011020
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English (en)
Japanese (ja)
Inventor
Yuko Saida
Shuzo Mishima
Original Assignee
Olympus Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Olympus Corporation filed Critical Olympus Corporation
Priority to AU2003261819A priority Critical patent/AU2003261819A1/en
Priority to PCT/JP2003/011020 priority patent/WO2005024424A1/fr
Publication of WO2005024424A1 publication Critical patent/WO2005024424A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy

Definitions

  • 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.
  • Genetic testing methods currently used include the steps of extracting nucleic acids from biological samples, amplifying target genes to be tested using nucleic acid amplification methods called PCR, NASBA, etc., and radioisotopes of nucleic acids. And the step of measuring the base sequence of the labeled target gene or the concentration thereof.
  • PCR nucleic acid amplification methods
  • NASBA nucleic acid amplification methods
  • radioisotopes of nucleic acids the step of measuring the base sequence of the labeled target gene or the concentration thereof.
  • capillary electrophoresis apparatuses that can process a large number of samples at a high speed by using a plurality of capillaries of fluorescently labeled nucleic acids have been used.
  • DNA chips are those in which a number of cDNA probes are immobilized on the surface of a glass substrate, and in which a number of oligo probes synthesized in microscopic regions on silicon using the semiconductor manufacturing process. .
  • the base sequence and expression level of DNA in the sample It is a method that can be determined sometimes.
  • the application of DNA chips has made it possible to analyze large amounts of gene expression and multiple mutations.
  • DNA chips can be used to categorize many genes into multiple groups (ie, clustering) or to provide information on gene changes associated with development or differentiation. I have.
  • the genetic information obtained in this way is used as a database that can be easily accessed through the Internet.
  • An object of the present invention is to provide a gene testing apparatus and a method capable of testing a plurality of genes simply and in a short time.
  • the present inventors have found a means for solving the above-mentioned object as a result of earnest research.
  • a genetic testing device using a computer, a temperature controller for adjusting the temperature of the DNA microarray, and a light detector for detecting the optical signal from the DNA microarray.
  • a fluid transport unit for supplying and discharging fluid to and from the DNA microarray
  • a control unit for controlling the entire device in accordance with an application for operating the device
  • a display unit for displaying information
  • a user An input unit for enabling input of information from the beginning, and is controlled by the ablation so that the user can freely set various parameters relating to measurement.
  • FIG. 1 is a configuration diagram showing one embodiment of the present invention.
  • FIG. 2A is a plan view of the slide chip.
  • FIG. 2B is a cross-sectional view of the slide tip taken along line 2B-2B shown in FIG. 2A.
  • FIG. 2C is an enlarged view of the DNA microarray shown in FIG. 2A.
  • FIG. 3 is a flowchart showing the analysis procedure.
  • Figure 4 is a flowchart showing the reaction procedure.
  • Figure 5 is a flowchart showing the analysis method.
  • FIG. 6A shows a nucleic acid probe immobilized on a slide tip for P53 exon 7 mutation analysis.
  • FIG. 6B schematically shows a fixing position of the nucleic acid probe of FIG. 6A.
  • FIG. 6C schematically shows an analysis result obtained by using a sample from a normal subject for the nucleic acid probe of FIG. 6A.
  • FIG. 6D schematically shows an analysis result obtained from a sample from a subject having an abnormality for the nucleic acid probe in FIG. 6A.
  • FIG. 7A shows a nucleic acid probe immobilized on a slide chip for expression level analysis.
  • FIG. 7B schematically shows the fixing position of the nucleic acid probe of FIG. 7A.
  • FIG. 7C schematically shows an analysis result obtained from a sample from a normal subject with respect to the nucleic acid probe of FIG. 7A.
  • FIG. 7D schematically shows an analysis result obtained from a sample from a subject having an abnormality with respect to the nucleic acid probe of FIG. 7A.
  • FIG. 8 is a diagram showing a control block of the genetic test apparatus of FIG.
  • Fig. 9 is a flowchart showing the operation procedure of the genetic test device.
  • an apparatus for easily and quickly detecting the expression level and mutation of a gene in one sample is provided.
  • the basic principle of the detection method carried out in the apparatus of the present invention is that a sample is immobilized using an immobilized nucleic acid probe having a known sequence. It detects nucleic acid strands having a specific sequence contained therein.
  • a single-stranded nucleic acid sample is immobilized on a substrate, and then contacted with a nucleic acid contained in a 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, the target nucleic acid having a sequence complementary to the probe can be detected. is there.
  • a genetic test apparatus 1 using a computer which is the 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 DNA microarray.
  • Temperature co emission controller 9 for controlling the temperature of the DNA microphone lower Rei 2 2 via the part data, further, contained in the genetic testing device 1
  • a computer 10 that is directly or indirectly connected to all devices to be controlled and that controls them collectively.
  • the junction is connected to the DNA microarray 22 in such a manner that the liquid contained in the DNA microarray 22 does not leak to the outside.
  • the other end of the joint section is connected to a pump driver 8 via a tube, and the pump driver 8 is further connected to a reagent holding section containing a desired reagent or the like therein.
  • the pump driver 8 moves fluid into and out of the DMA microarray 22 in accordance with an instruction from the computer 10 in accordance with the analysis program.
  • the heater section is disposed between the slide chip 2 and the stage 4.
  • the heater section is connected to a temperature controller 9, and the temperature is adjusted by the temperature controller 9 in accordance with an instruction from the computer 10.
  • the computer 10 includes components provided in a general computer.
  • the components included in the computer 10 are a CPU (Central Processing Unit) which is a main control unit that controls the components of the computer 10 collectively. ) 11, a memory 12 for storing files such as various programs and image data to be displayed as a basis of control in the CPU 11, and a memory executed by the CPU 11.
  • RAM random access memory 13 for temporarily storing results, etc., based on the program
  • An image processing unit 14 for generating image data according to the instruction of the CPU 11; and an operator such as a keyboard and a mouse for inputting information to the computer 10 by the computer 10. It comprises at least an input unit 15 and a display unit 6 for displaying information according to the instructions of the computer 10.
  • the information captured by the CCD camera 5 is sent to the image processing unit 14 and processed by the image processing unit 14 according to the instruction of the CPU 11 in accordance with the analysis program stored in the memory 12 to obtain the fluorescence. It is quantified as strength.
  • the computer that can be used in the present invention may be any commonly used electronic computer.
  • the microscope that can be used in the present invention may be a commonly used fluorescence microscope, such as an Olympus fluorescence microscope AX-70 or BX-52 TFR. It is not limited. 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.
  • the CCD camera that can be used in the present invention may be a commonly used CCD camera, for example, a general digital camera, for example, a Mega PI us CCD camera of Kodak Co., Ltd. Cameras such as napcfmono, etc., but are not limited to these.
  • the temperature control unit for controlling the temperature of the DNA microarray 22 includes the DNA microarray 22.
  • a heating element for example, an electric heater, an electromagnetic heater, a water bath, an air bath, a Peltier element, etc.
  • a sensor for sensing the temperature there
  • a temperature controller for controlling the heating of the heating element according to the control of the computer and the information from the sensor.
  • the computer may be connected to an integrated digital communication network (hereinafter referred to as ISDN) or to a telephone line or LAN via a modem.
  • ISDN integrated digital communication network
  • modem modem
  • the stage 4 provided in the above-described embodiment can be moved in XY by the XY stage controller 7.
  • the scanning may be performed by the CCD camera 5 itself or by using various scanning mechanisms that change the optical path connecting the CCD camera 5 and the microarray 22.
  • the detector used in the present invention has been described using a CCD camera as an example, but 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. It is. However, the CCD camera is effective because one reaction area as shown by the opening 22 in FIGS. 2A and 2B can be simultaneously detected.
  • FIGS. 2A and 2B Slide Chip with DNA Microarray An embodiment of the DNA microarray 22 used in the present invention is shown in FIGS. 2A and 2B.
  • Figure 2A shows a slide with four DNA microarrays 22 where the reaction takes place.
  • FIG. 3 is a plan view of the chip 2.
  • FIG. 2B is a cross-sectional view of the slide chip 2.
  • the slide chip 2 shown in FIG. 2B includes a filter 19 having a microchannel 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 to form the slide chip 2.
  • openings 21 for defining the DNA microarray 22 are formed in the support plate members located above and below the filter 19 at respective positions facing each other.
  • the filter 19 is two-dimensionally flat between the two support plates 20 over a large area including all four DNA microarrays 22.
  • the support plate 20 is preferably made of a light-shielding member, and more preferably made of a dark black member.
  • the two support plates 20 are joined with the filter 19 interposed therebetween.
  • 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.
  • the “DNA microarray” 22 refers to the portion of the filter 19 exposed from the opening 21 formed in the support plate 20.
  • FIG. 2C shows an enlarged view of the DNA microarray 22 shown in FIG. 2A. Although a substantially circular area is shown in FIG. 2C, the outline of the probe spot 23 is substantially circular, substantially rectangular, or substantially rectangular. May be polygonal.
  • the same probe may be immobilized on a plurality of probe spots 23.
  • the porous body may be subjected to a surface treatment to adjust the frictional resistance to a fluid or the adsorbability of a reagent such as a probe.
  • the size of this slide tip is 0.5 to 20. Ocm XO. 5 to 20. Ocm XO. 01 to 1.0. Ocm, preferably 1.0 to 10.0 cm. 0 to 10 Ocm XO .05 to 0.5 cm. Particularly preferably, it is from 3.0 to 8.0 cmX 3.0 to 8.0 OcmXO.05 to 0.2 cm.
  • One example of the actual slide chip is about 7.5 cm x about 2.5 cm x about 0.1 cm.
  • the microarray has a size of 3.0 mm 2 to 16 cm 2, preferably 12.0 to 400 mm 2, particularly preferably 20.0 to 10.0 mm 2.
  • the circular micro-array on the slide chip actually created is about 6 mm in diameter (about 28.3 mm 2).
  • the DNA spot included in the present DNA microarray has a diameter of 100 to 300 Atm, preferably 120 tm, and 10 to 100 000 per DNA microarray. Preferably, 400 probe spots are provided. In the nucleic acid probe described below, different types of probes can be immobilized for each probe spot.
  • Examples of preferred DNA microarrays for use in the present invention include, for example, those described in Japanese Patent Application Laid-Open No. 2000-5005 ⁇ 5251.
  • the device may be a microfabricated flow-through porous device described in JP-A-9-504804.
  • FIGS. 2A and 2B show, for example, a slide chip 2 having four DNA microarrays 22.
  • the number of DNA microarrays 22 may be four or more or less. Good.
  • a slide chip 2 including four DNA microarrays 22 will be described as one preferred embodiment, but the present invention is not limited to this, and other changes may be made in accordance with the change. It will be apparent to those skilled in the art that the procedure for configuring and executing the apparatus will be changed, and such changes are also included in the scope of the present invention.
  • a different sample may be tested for each DNA microarray, or one sample may be tested in one chip.
  • Four different types of analysis may be performed, for example, mutation of an oncogene, expression analysis, determination of the expression pattern of a drug resistance gene, and an intracellular signal gene, and the like.
  • the size such as the arrangement density and arrangement pattern of the probe spots and the diameter of the probe spots, can be changed as appropriate.
  • 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 a body. For example, a cap that penetrates the flat member vertically and linearly and has both ends open It is also possible to use those having a form in which the rallies are closely paralleled and integrated. Also, for example, the cavities may not be arranged perpendicular to the flat member, but may penetrate linearly at a desired angle, for example, or may be non-linear (ie, curved) ) May be arranged as holes. Further, the angle of the cable 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.
  • the filter ⁇ 9 is described as an example, but the probe spot is provided not only on a porous body such as a filter but also on a surface of a non-porous body such as a slide glass. The case is also applicable. 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.
  • nucleic acid probe generally refers to a polynucleotide or an oligonucleotide consisting of a nucleic acid of about 10 to about 100 nucleotides. Generally, it shows a nucleic acid probe used for detecting a nucleic acid by hybridization.
  • an oligonucleotide corresponding to a tumor suppressor gene or an oligonucleotide corresponding to a disease-related or susceptibility gene.
  • any desired nucleic acid corresponding to a sequence containing a polymorphic site can be used as a probe.
  • a probe for a house-kiving gene such as 8 globulin pectin (for measuring the steady state of the sample) may be used. With the amount of such a housekeeping gene as the standard amount of the gene in the cell, the expression level of the actual oncogene, tumor suppressor gene or drug resistance gene may be measured. By doing so, it is possible to more accurately measure the gene expression level in the cell.
  • target nucleic acid refers to the nucleic acid to be detected contained in a sample.
  • such a slide chip 2 when analysis is performed using the slide chip 2 on which the DNA microarray 22 described above is arranged, such a slide chip 2 includes a plurality of DNA microarrays 22. It is.
  • 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 in a single small DNA microarray. It can be obtained with sensitivity.
  • temperature control and analysis of measured data can be performed for each DNA microarray, so that a large number of reaction results can be obtained quickly with a small device.
  • the temperature may be controlled for each probe spot, or the measurement data may be analyzed. In that case, it is possible to quickly obtain the reaction results of more items.
  • the probe is fixed to the porous body.
  • 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-described embodiment, except that the probe is not fixed.
  • the “probe spot” can also be referred to as the “reaction spot”, but here, the “reaction spot” is included and described as an embodiment of the probe spot.
  • Such an apparatus is also included in the scope of the present invention.
  • the memory 12 includes a program for controlling the apparatus via the CPU 11, information such as a table which is searched by the CPU 11 and provides a basis for control and determination, and information obtained.
  • the image data and the like used when displaying the result are stored.
  • the analysis program is a program for performing a genetic analysis, and may include instructions regarding a work procedure for causing the computer to process the analysis.
  • the reaction progress program may include instructions on the steps of a task to cause a computer to process the reaction to be performed in the DNA microarray 22.
  • the reaction progress program includes the reaction, that is, the selection of reagents necessary for the hybridization, the amount of reagents used, the reaction conditions such as the reaction time and reaction temperature, the number and time of bombing, and the bombing start time.
  • An instruction regarding the liquid transport and stirring conditions may be included in advance, or an instruction instructing the operator to input such information at the start of the analysis may be included.
  • the reaction conditions are stored in the memory 12 as a reaction condition program as a program different from the reaction procedure. May be.
  • 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.
  • the gene correspondence table may be a table specifying associations between the obtained fluorescence intensities, reaction conditions, gene expression levels, expressed genes, and sudden mutations in genes.
  • the slide chip 2 is set on the stage 4 so as to contact a part of the heater, and the slide chip 2 is set on the stage 4 with respect to each DNA microarray 22 of the slide chip 2. Connect the contact section.
  • the CPU! 1 When an instruction to start the analysis is issued by one year old, the CPU! 1 operates according to 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 is instructed so that the unanalyzed DNA microarray 22 having a small identification number among the four DNA microarrays 22 is located in the field of view of the CCD camera 5,
  • the designated XY stage controller 7 is the motor driver 6
  • the XY stage 4 is moved.
  • the number of DNA microarrays in the slide chip 2 to be used, the identification numbers corresponding to each of them, and the coordinates where they are located are stored in the memory 12 as a table in which they are associated. It has been. Therefore, the CPU 11 selects the youngest DNA microarray 22 with the lowest identification number from the unanalyzed DNA microarrays 22 and searches the above-mentioned tapes to obtain the target DNA microarray 22. And read out the coordinates based on the coordinates.
  • the CPU 11 reads from the RAM 13 the identification number of the DNA microarray 22 selected in S3 immediately before and used for the reaction, and based on the read number, the four chips provided in the slide chip 2 are read. Determine whether any unanalyzed DNA remains in the DNA microarray 22. If there is an unanalyzed DNA microarray 22, go to (S3), otherwise go to (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 for a gene correspondence table and reads out the corresponding gene information.
  • the CPU ⁇ 1 causes the image processing unit 14 to create a table of the result of the gene analysis, sends the calculated fluorescence intensity reaction condition and the gene correspondence table to the image processing unit 14, There, a result table is created, and this table is stored in the RAM 13.
  • the CPU 11 instructs the image processing unit 14 to display the image of the table of the gene analysis result stored in the RAM 13.
  • the image processing unit 14 reads necessary information from the RAM, processes the image, and displays it on the display device.
  • the information on the reaction conditions input by the operator in S 2 may be the reaction temperature, the reaction time, the number of reactions, the selection of the reagent, and the like.
  • 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 2 in advance.
  • the results obtained by the analysis can be compared with the information obtained by searching a desired genome database or base sequence database. It is also 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 it with the result obtained by the device, and identifies the result.
  • the CPU 11 reads the reaction progress program and the conditions input by the operator in (S1) and stored in the RAM 13, and further changes the temperature setting and / or the solvent composition, etc. It is determined whether the reaction needs to be performed under the additional reaction conditions described. If necessary, proceed to (S41). If not, proceed to (S5).
  • Examples of analysis that can be performed by the device of the present invention include detection of the presence or absence of a mutation in a subject such as an oncogene, analysis of the expression of a gene in a subject, analysis of the expression of a drug resistance gene, disease-related or Genotyping of susceptibility genes, determination of expression patterns of intracellular signal genes in subjects, etc.Clinical therapeutic and diagnostic analysis, and various gene analyzes in nonclinical basic research It is.
  • sample used for a genetic test in the case of a human, blood, a cultured cell, a living tissue such as a cell or a tissue collected at the time of a biopsy or an operation, or the like can be used.
  • the tissue section was fixed on a slide glass, stained, and a pathological examination was performed. It is possible to use a laser capture system, a microdissection system, obtained from the slide glass obtained by using LCS 200, manufactured by Sai Linpath Co., Ltd. it can.
  • the biological sample obtained in this way was treated with a nucleic acid extraction reagent using the phenol-claw-holm method, microcolumn method, or magnetic particle method. Extract DNA, and / or RNA. Further, the extracted DNA and / or RNA is subjected to gene amplification by, for example, the PCR method, the RT-PCR method, the T7 amplification method, 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.
  • a labeling substance such as FITC, rhodamine, Cy3 and Cy5, etc.
  • FITC FITC
  • rhodamine Cy3 and Cy5
  • the prepared sample obtained manually by the operator 1 by pipetting may be dispensed into the DNA microarray. Thereafter, the analysis may be performed by the above-described device. 7 Analysis method
  • 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.
  • (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.
  • (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 probe is immobilized.
  • (Se) Hybridization is performed on the DNA microarray containing the sample under desired conditions. At this time, it is preferable to agitate the liquid in the DNA microarray by bombing or pitting. After performing hybridization in (S f) and (S e), 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. Measure.
  • the sample in the DNA microarray is maintained as it is, that is, there is no contamination or other contamination such as dilution or impurities. Then, the fluorescence is contained in the labeled nucleic acid bound to the nucleic acid probe.
  • the amount of expressed gene and the presence or absence of a mutated gene are determined to obtain information on the target gene.
  • (S f) to (S g) may be repeated as many times as necessary.
  • the apparatus described above as an embodiment 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. All kinds of reaction systems can be easily achieved in a small space. Therefore, the device of the present invention can be an excellent tool useful for all genetic tests.
  • At least one DNA microarray is small enough to be sufficiently captured by a CCD camera, so that the DNA to be measured is required. There is no need to optically scan multiple separate probe spots in the microarray. 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.
  • the present invention enables a plurality of tests to be continuously performed on the same sample, so that a plurality of test results can be obtained quickly or at any time. It is intended to provide a device capable of performing such operations.
  • each reagent for example, a nucleic acid probe and a nucleic acid primer
  • each reaction condition applied to each probe spot is determined for each reagent or each reaction condition.
  • Analysis software is also provided that analyzes the measured data while comparing it with each of the obtained measured data.
  • the same probe spot is used. It is possible to perform multiple types of reactions simultaneously, sequentially, or sequentially. Therefore, it is easy to prepare the reaction conditions necessary to achieve 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 device according to the invention may be provided with a novel reading means that enables the reading.
  • reading means may be means for performing the following:
  • test item information is stored in a storage means such as a memory circuit by nC Ik ⁇ ⁇
  • the measurement result performed under the monitored control condition of ii) is associated with the collation result obtained in ii), and the measurement result is further processed by data processing means such as a CPU. Performing an arithmetic process for correcting based on the collation.
  • such a reading unit includes a storage unit for storing the inspection item information, the control condition, the measurement result, the measurement data, and the like, and the data stored in the storage unit. It has data processing means such as a CPU for reading and comparing and collating data, and for performing necessary arithmetic processing.
  • a chip containing a porous filter in which a plurality of genes complementary to the target gene to be tested are immobilized on the surface is used.
  • the slide chip containing the porous filter is placed on the reaction part (part of the incubator) placed on the stage of the fluorescence microscope.
  • the fluorescence microscope is configured so that the excitation light shutter, fluorescence cup, stage movement, and the like can be automatically controlled by a host computer.
  • the fluorescence microscope is equipped with a device that captures images such as CCDs, and the timing of photography is controlled by the host computer. The same computer can also control exposure time, image storage, and cooling status.
  • the incubator is provided with a pump for transporting liquid and a heater or Peltier element for temperature control, and these controls can also be performed by the host computer.
  • the reaction solution is moved by a pump to accelerate the hybridization reaction.
  • the solution in the lower part of the incubator is moved, and when the liquid surface of the reaction solution comes in contact with the upper surface of the filter, the excitation light shutter is opened and the fluorescence image of the filter surface is captured by a device such as a CCD camera.
  • the captured image data is stored in a folder specified by the computer, and the measurement results are analyzed by a separately installed analysis program.
  • pumps to promote hybridization heaters to apply a temperature change to the chip, and, if necessary, a pitcher system to change the solution composition are placed around the incubator. In this way, a single inspection system can continuously obtain hybridization patterns at various temperatures and solution states.
  • the sample contains substances labeled with multiple fluorescent dyes. Even in this case, by automatically switching the filter cube, 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 probe immobilized on the chip surface and the amount of the target gene immobilized are analyzed. By using this test device, it is possible to detect the expression level of a gene whose expression level fluctuates greatly in response to canceration of C-myc or the like.
  • the degree of hybridization of the target gene can be measured in real time while changing the solution state of the reaction part and the temperature conditions. .
  • 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.
  • the expression level of the target gene can be accurately measured.
  • the efficiency of hybridization to different probes is forcibly changed by automatically changing the temperature of the microarray and the composition of the reaction solution.
  • SNPs single nucleotide polymorphisms
  • Singl e nucle eot i de polly morph ism single nucleotide polymorphisms
  • the level of expression of drug susceptibility genes such as P450, which affects the sensitivity to the therapeutic agent, and the SNPs that affect the activity can be simultaneously tested.
  • the cause of the disease in this way By simultaneously testing not only for genetic abnormalities but also for those that are sensitive to the drug, it is possible to optimize the type and temperature of the selected therapeutic drug according to the patient's constitution. Also, based on the obtained information on gene abnormalities, it is possible to accurately select genes to be used for gene therapy, which is expected to have a high therapeutic effect in the future.
  • FIGS. 6A to 6D show slide chips for measuring the expression levels of C-mycv estrogen receptor and telomerase. Probes for a plurality of genes including c-myc as shown in FIG. 6A and an eight-skiving gene are immobilized on the slide chip.
  • FIG. 6B schematically shows a fixing position of the nucleic acid probe of FIG. 6A fixed to the probe spot included in the DNA microarray. Each type of nucleic acid probe is fixed to the probe spot for each type.
  • FIG. 6C and FIG. 6D are diagrams schematically showing the analysis results. Figure 6C shows the results obtained from a sample from a normal subject, and Figure 6D 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.
  • 7A to 7D show the comparison of the intensity of the fluorescent spots generated for probes having different nucleotide sequences to detect mutations occurring inside the tumor suppressor gene P53. An example of such a case is shown.
  • the expression level can be detected based on the difference in fluorescence intensity.
  • the above-described gene detection and expression level analysis can be performed continuously for one sample in one DNA microarray.
  • the device and method of the present invention it is possible to determine the stage of canceration, and to accurately predict the degree of malignancy, susceptibility to metastasis, individual differences in drug resistance, and the like.
  • Figure 8 shows the control block of Genetic Testing Equipment 1.
  • the genetic test apparatus 1 includes a control unit 10a, a camera controller 1Ob, and a microscope controller 10c, which correspond to the computer 10. are doing. Further, as described above, the genetic test apparatus 1 includes a CCD camera 5 (preferably of a cooling type), a stage 4, a stage controller 7, a temperature controller 9, and an input unit 15. And a display section 16. Stage 4 includes a motor 4a for driving it.
  • the genetic test apparatus 1 further includes a mirror unit 31, a shut unit 32, and a lighting unit 33 included in a fluorescence observation microscope 3, and a DNA microarray 22.
  • a pump 41 that has an upper heater 51, a lower heater -52, and a cooling fan 53 that constitute a temperature controller for controlling the temperature, and that moves a liquid through the DNA microarray 22.
  • the mirror unit 31 includes a motor 31a for driving it, the shutter unit 32 includes a motor 32a for driving it, and the upper heater 51 measures its temperature.
  • the lower heater 52 includes a resistance thermometer 51b for measuring the temperature.
  • the pump 41 includes a controller 41a for driving and controlling the pump 41. Yes.
  • the mirror unit includes, for example, an excitation filter that transmits only light in the excitation wavelength band from the irradiation light, and an absorption filter that transmits only fluorescence from the target object and blocks the excitation light. For a light of a predetermined wavelength, a dichroic mirror that reflects shorter light and transmits longer wavelength can be integrated.
  • the camera controller 1Ob controls the CCD camera 5, and the microscope controller 10c controls the motor 31a of the mirror unit 31 and the motor 32a of the shutter unit 32 and lighting. Controls unit 3 3.
  • the temperature controller 9 controls the upper heater 51 based on the information from the resistance temperature detector 51a, and controls the lower heater 52 based on the information from the resistance temperature detector 52a.
  • the stage controller 7 controls the motor 4 a of the stage 4.
  • the controller 10a includes a camera controller 10b, a microscope controller 10c, a temperature controller 9, a stage controller 7, and a controller 41 of the pump 41. Control. Further, the control unit 10a causes the display unit 16 to display appropriate messages and information, and acquires information input from the input unit 15.
  • the genetic test device 1 is operated according to a specially designed application.
  • the application includes various programs such as the analysis program and the reaction progress program described above, and appropriately controls each part of the apparatus 1.
  • the control unit 10a Command to display unit 1 Next, necessary operations are displayed on 6 to give instructions to the user, and various information related to measurement is displayed on the display 16.
  • the application uses the input unit 15 and the display unit to input various profiles that are instructions for each unit of the device, such as pump operation instructions, imaging instructions, delay time instructions, fixed stop instructions, temperature change instructions, and cleaning instructions.
  • the entire device is controlled so that the user can change it freely.
  • the operation of the genetic test device 1 is controlled by the application so that the user can freely set various parameters relating to the measurement.
  • the control unit 10a displays a message prompting information input on the display unit 16 and waits for information input from the input unit 15 according to a command from the application.
  • the genetic test apparatus 1 is operated, for example, according to an operation shown in a flowchart shown in FIG.
  • control unit 10a checks the connection of the connected devices such as the microscope controller 10c, the pump 41, the temperature controller and the stage controller 7 ⁇ CCD camera 5, and performs initialization.
  • the pipe is filled with water.
  • a water supply tray containing water is set in the incubator, the incubator is inserted into the device, and the pump 41 is connected to the incubator. This can be done by sucking water from the water supply tray, or by setting the drainage tray in the incubator, inserting the incubator overnight into the device, and using the pump 41. This can be done by sucking water from a water supply tank (not shown) and discharging it to a drain tray.
  • Start temperature control Specify the temperature of the incubator. That is, the target temperature of the heater 51 is set.
  • the profile is an instruction for each unit of the apparatus, and includes a pump operation instruction, a photographing instruction, a delay time instruction, a fixed stop instruction, a temperature change instruction, a cleaning instruction, and the like.
  • a cycle consists of an appropriate combination of these profiles.
  • a sequence consists of the appropriate combination of cycles and specifies which cycles are performed, in what order, and how many times. The sequence determines the order of measurement.
  • the operations 6 to 8 described above that is, setting of conditions and profile / cycle / sequence are performed, for example, by displaying an input screen showing information items to be input on the display unit 16 and inputting information to the user. This is performed by prompting the user to input an appropriate numerical value or the like to each item on the screen from the input unit 15 in response to the prompt.
  • operations 6 to 8 are not limited to such a method, and may be performed by another appropriate method.
  • 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.
  • an auxiliary storage device of a computer such as a hard disk or a floppy disk.
  • this may be performed by reading a data file from the auxiliary storage device and obtaining necessary information.
  • Such a data file can be created, for example, by recording the set values used by a user in a previous measurement.
  • the data file is provided by the manufacturer of the slide chip 2.
  • the side may record and create information suitable for each type of the slide chip 2.
  • the data file may be supplied to the user in a form attached to the slide chip 2, for example.
  • a. Pump operation Set whether to pull (suction) or return (discharge) the pump.
  • the driving amount and the speed can be arbitrarily set.
  • the pump drive amount is often set to be approximately the same as the sample amount.
  • the pump drive amount can be set to the optimum value depending on the amount of sample liquid, viscosity, and tip size of the tip (the tip size may vary depending on the tip type). Since the reaction state differs depending on the moving speed of the sample liquid, the optimal pump drive speed can be set to perform the reaction under the optimal conditions.
  • b. Shooting Set the mirror unit and exposure time used for shooting.
  • the mirror unit can be selected according to the fluorescent dye used to label the specimen, and the exposure time suitable for observing the fluorescence can be set arbitrarily. If the brightness is low, the exposure time can be set longer.
  • Delay time Set the time to stop the operation. By setting the delay time, for example, when driving a pump, the time until the liquid is chipped or rises can be set to an optimum value.
  • Washing This is performed to remove unreacted fluorescently labeled samples and to observe extra fluorescence other than the result of the reaction. In accordance with the instructions of the application, take out a part of the incubator out of the device and instruct the user to drop the cleaning solution. By driving the pump, the washing liquid is moved up and down to wash the chips. Perform the above operations manually. Cleaning may or may not be required.
  • Temperature change Use this when you want to change the temperature during the measurement from the current set temperature. 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.
  • Cycles 2 and 3 above are repeated until the reaction is complete. Also, steps 3 and 4 in cycle 4 are repeated while changing the conditions.
  • the user can measure various parameters under desired conditions and operations using the input unit 15, the display unit 16, and the data file. I can do it.
  • the present invention is not limited to genetic testing depending on the purpose, and can be used for high-throughput various other biological reactions (enzyme reaction, antigen-antibody reaction, metabolic reaction, biological kinetic test, etc.). It should be noted that it can be widely applied to the medical field as a technology to be implemented and monitored in the 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 a case where, for example, an antibody or a tissue is used as a probe.
  • a gene inspection apparatus and method which can easily and simply test a plurality of genes in a short time. Further, by using such an apparatus and method, the degree of the hybridization of the target gene can be measured in real time while changing the state of the solution in the reaction portion 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.

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Abstract

Un dispositif d'examen de gènes utilisant un ordinateur comprend un plateau (4) portant un micro-réseau d'ADN, une unité de commande de température (9, 51, 53) destinée à commander la température du micro-réseau d'ADN, une caméra à CCD (5) destinée à capturer un signal optique du micro-réseau d'ADN, une unité de transport (41) de fluide destinée à amener/extraire un fluide vers le/à partir du micro-réseau d'ADN, une unité de stockage dans laquelle est stockée une application d'exploitation du dispositif, une unité de commande (10a) destinée à commander tout le dispositif selon l'application stockée, une unité d'affichage (16) destinée à afficher des informations, une unité de saisie (15) destinée à permettre à l'utilisateur d'entrer des informations. Le dispositif est commandé par une application de telle sorte que divers paramètres relatifs à la mesure peuvent être changés librement par l'utilisateur.
PCT/JP2003/011020 2003-08-29 2003-08-29 Dispositif d'examen de genes et procede d'examen l'utilisant WO2005024424A1 (fr)

Priority Applications (2)

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AU2003261819A AU2003261819A1 (en) 2003-08-29 2003-08-29 Gene examining device and examining method using same
PCT/JP2003/011020 WO2005024424A1 (fr) 2003-08-29 2003-08-29 Dispositif d'examen de genes et procede d'examen l'utilisant

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002296280A (ja) * 2001-03-30 2002-10-09 Olympus Optical Co Ltd キャピラリアレイを用いた遺伝子発現頻度解析方法
JP2002350350A (ja) * 2001-03-21 2002-12-04 Olympus Optical Co Ltd 生化学的検査方法
JP2003125772A (ja) * 2001-06-20 2003-05-07 Dainakomu:Kk コンピュータを利用して解析対象核酸塩基配列から最適なオリゴ核酸配列の候補を設計するためのコンピュータソフトウエアプログラム、その方法およびそのように設計されたオリゴ核酸配列が搭載されたオリゴ核酸アレイ
JP2003185660A (ja) * 2001-12-20 2003-07-03 Olympus Optical Co Ltd Ｄｎａチップ読み取り装置
JP2003232791A (ja) * 2002-02-12 2003-08-22 Olympus Optical Co Ltd プローブ固相化反応アレイ

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002350350A (ja) * 2001-03-21 2002-12-04 Olympus Optical Co Ltd 生化学的検査方法
JP2002296280A (ja) * 2001-03-30 2002-10-09 Olympus Optical Co Ltd キャピラリアレイを用いた遺伝子発現頻度解析方法
JP2003125772A (ja) * 2001-06-20 2003-05-07 Dainakomu:Kk コンピュータを利用して解析対象核酸塩基配列から最適なオリゴ核酸配列の候補を設計するためのコンピュータソフトウエアプログラム、その方法およびそのように設計されたオリゴ核酸配列が搭載されたオリゴ核酸アレイ
JP2003185660A (ja) * 2001-12-20 2003-07-03 Olympus Optical Co Ltd Ｄｎａチップ読み取り装置
JP2003232791A (ja) * 2002-02-12 2003-08-22 Olympus Optical Co Ltd プローブ固相化反応アレイ

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