JP2007322439A - Method for using dna microarray - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for discriminating normal DNAs, and a method for using a DNA microarray that uses only the normal probe DNA. <P>SOLUTION: The method for discriminating DNAs scans the probe DNA manufactured on a substrate with a laser beam, compares reflectivities and/or colors of the normally operating probe DNA and the probe DNA manufactured on the substrate, and discriminates normal DNAs. The method for using the DNA micro array discriminates the normal probe DNA based on a management data and uses only the normal probe DNA. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for distinguishing normal DNA and a method for using a DNA microarray that uses only normal probe DNA.

A DNA microarray has thousands of DNA probes immobilized on a substrate such as a slide glass. A sample (target) labeled with a fluorescent molecule or the like is allowed to flow and hybridize with a DNA probe to generate fluorescence emission by hybridization. By measuring the strength, the expression level of the gene contained in the sample is estimated.
As a result, the expression of a large number of genes can be analyzed comprehensively at the same time, so that it is widely used as a standard instrument for research and development in the fields of life science, pharmacy, and agriculture. For example, if mRNA (messenger RNA) is extracted from a cancer tissue that reacts to a certain drug and a cancer tissue that does not react, and the difference in the expression level between them is estimated by a DNA microarray carrying all human genes, the cancer tissue and the drug Specific gene expression is revealed. This can be used to efficiently elucidate the onset mechanism and conduct drug discovery targeting the gene.
In the age of post-genomic sequencing, the need for comprehensive measurement of gene expression has spread to the new bioindustry such as food testing and production quality control as well as clinical testing in so-called tailored medicine and medical fields. The market is expected to expand significantly.

  Two mainstream types are the Affymetrix type, in which oligonucleotides are stacked vertically on a silicon substrate by optical lithography and solid-phase DNA synthesis technology, and the so-called Stanford type, which attaches DNA on a slide glass. is there. The former is expensive because a gene must be selected in advance and an order design and manufacturing request must be made. The latter is advantageous in that the user can freely select a gene to be spotted in the usage environment. In the present invention, probe DNA (referred to as a DNA spot after hybridization with a sample) means the above-mentioned Affymetrix type, Stanford type, or other types of probe DNA.

As shown in FIG. 11, the current DNA microarray reads a DNA spot array 111 arranged in a two-dimensional lattice pattern on a glass substrate 110 by measuring the fluorescence with a laser scanning or image measuring means and imaging it. Yes. The measurement requires two degrees of freedom, which complicates the apparatus, and as a post-processing, a computer image analysis process for correcting the fluorescence signal and reducing noise is necessary. There are devices that use beads instead of slide glasses and devices that use porous media, but the number of genes that can be handled is at most several hundreds, so it is possible to achieve completeness.
However, a system comprising a spotter, a hybridization, and a scanner for analyzing a DNA microarray has many problems in terms of quantification and sensitivity.

If you list current issues,
1. 1. Insensitive fluorescence measurement 3. It takes time to read. 3. Imaging by 2D scanning is necessary. Poor operability 5. Not enough DNA spots placed on one glass substrate (at most 10,000 spots)
6). It was difficult to record the properties of the samples and the experimental conditions at the same time, and numbers were added to the samples and data sheets were used separately.

  An object of the present invention is to provide a method for distinguishing normal DNA and a method for using a DNA microarray that uses only normal probe DNA.

The present invention relates to the following inventions.
1. The probe DNA produced on the substrate is scanned with a laser beam, and the normal and normal probe DNA produced on the substrate is compared with the reflectance and / or color of the probe DNA produced on the substrate. A method for discriminating DNA, characterized by discriminating DNA (based on International Publication No. 28, line 3-7).
2. A plurality of probe DNAs of the same type are prepared on a microarray substrate, the probe DNA is scanned with a laser beam, and the reflectivity and / or color of the probe DNA that operates normally and the probe DNA are determined. Use of a DNA microarray that discriminates normal probe DNA by comparison, discriminates normal probe DNA from management data, discriminates normal probe DNA from the management data, and uses only normal probe DNA Method (based on International Publication No. 28, lines 8-11).
3. Inspect the DNA prepared on the microarray substrate, select the address where the normal DNA exists, record the selected address information on the recordable part on the substrate on which the DNA is placed, and select A method of using a DNA microarray, characterized in that only DNA existing in the address storage section is used (based on International Publication No. 28, lines 11-15).
4). After generating a hybridization reaction with the probe DNA prepared on the microarray substrate, when detecting the fluorescence contained in the generated DNA spot, the reading laser beam for detecting the fluorescence is modulated at a high frequency. And a method for using the DNA microarray (based on International Publication No. 29, line 3-7).

5). The method for using the DNA microarray according to any one of 2 to 4 above, wherein the DNA prepared on the substrate of the microarray is prepared by the following method:
A track on a substrate capable of tracking a light beam is provided with a storage unit and an address for specifying the storage unit, nucleotides having a protecting group are arranged in at least a plurality of the storage units, and the predetermined storage unit is provided. The address indicating the storage unit is identified by reading with a light beam, and the stored nucleotide having the protective group is irradiated with the light beam, and the nucleotide having the protective group stored in the specific storage unit A method for producing a microarray including a step of removing a protecting group (founded in International Publication No. 24, line 12 to page 25, line 13).
6). A microdisk (International Publication No. 5) characterized by determining the quality of a plurality of probe DNAs or proteins of the same type produced on a microdisk and storing at least address information of the probe DNAs or proteins determined to be non-defective (Found on pages 8-10 lines).

According to the present invention, normal DNA prepared on a substrate can be discriminated.
According to the present invention, DNA prepared on a microarray substrate is inspected, an address where the normal DNA is present is selected, and only the DNA existing in the storage section of the selected address is used. it can.
According to the present invention, after causing a hybridization reaction with the probe DNA prepared on the substrate of the microarray, the reading laser beam for detecting the fluorescence contained in the generated DNA spot is modulated at a high frequency. Thus, fading of the phosphor can be prevented.

In the present invention, DNA microarray spots that are currently arranged in a two-dimensional lattice are arranged in a one-dimensional concentric or spiral shape, and at the same time, the glass plate is changed to a disk shape to provide an index for identifying the spot position. Specifically, spotting can be performed accurately on a pre-groove on a glass disk and / or a place where an index is provided, and a DNA spot can be read without distortion by scanning in a one-dimensional direction. A DNA microdisk and an apparatus for spotting probe DNA on the DNA microdisk.
In addition, a recordable zone is provided on the DNA microdisk, and the correspondence between the creation conditions, the measurement results, the address information indicating the positions of the spots spotted by the pregroove and the spotting liquid names is spotted. Record above. As a result, the conventional glass plate and the data stored at the time of creation can be stored together, and "operability and reliability" and security are improved. This is to provide a specific production method for this purpose.

Embodiments that specifically show the best mode for carrying out the present invention will be described below with reference to the drawings.
The schematic configuration of the first embodiment of the present invention will be described below with reference to FIGS. 1, 2, and 3. FIG. In addition, although protein etc. can also be used, hereafter, it demonstrates mainly using DNA.

FIG. 1 is a block diagram showing the configuration of an apparatus for spotting probe DNA onto the DNA microdisk shown in FIG. 2, and FIG. 2 is a drawing showing the DNA microdisk.
In FIG. 1, reference numeral 1 denotes a DNA microdisk having 23 pregrooves. The rotation is controlled by 14 disk motors. Reference numeral 8 denotes a discharge device for discharging the probe DNA onto the substrate. In the embodiment, an ink jet is used. It is also possible to use a tool such as a micropipette or a nib instead of the ink jet. In order to detect the position of the ink jet, a photodetector 10 (having light receiving elements 10a and 10b) placed at a fixed position with respect to the ink jet discharge port is provided. In addition, regarding the shape of the DNA spot that is spotted and fixed on the pregroove, it has been experimentally confirmed that the ratio of the size in the width direction perpendicular to the tangential direction is large in the tangential direction and more than twice. .
The probe DNA solution (spotting solution) is supplied from a tank provided in the ink jet or by an 11 spotting solution supply tube. The spotting solution is a solution obtained by dissolving probe DNA, protein, etc. in water or other medium (alcohol, etc.). This spotting liquid can be disposed on either or both of the convex and concave portions of the pregroove. Reference numeral 2 denotes a laser, and the emitted laser beam is converted into parallel light by an expander 3 or a collimator lens, and is focused on the pregroove 23 on the DNA microdisk 1 by the objective lens 4 through the beam splitter 5. . The light reflected from the pregroove 23 passes through the objective lens 4 and the beam splitter 5, and the farfield image of the pregroove is formed on the photodetector 7 having the two divided cells 7 a and 7 b by the lens 6. 7a and 7b, the relative positions of the beam spot emitted from the objective lens 4 and the 23 pregrooves can be detected.

The light transmitted through the 23 pre-grooves is incident on the photodetector 10 composed of two photodetectors 10a and 10b. The differential signals of the photodetectors 10a and 10b indicate the relative positions of the nine inkjet discharge ports and the pregroove. Reference numeral 12 denotes a traverse unit A, which includes an inkjet 8, a photodetector 10, and 2 lasers, 3 beam expanders, 4 objective lenses, 4a and 4b actuators, 5 beam splitters, 6 lenses, The optical measurement unit and the mechanism unit including the photodetector are integrally configured as a traverse unit A, and are movable and controlled in the radial direction of one DNA microdisk by 13 traverse motors A.
The control unit for moving the position of the objective lens 4 in the radial direction of the DNA microdisk and causing the laser beam emitted from the objective lens to follow the 23 pregroove is called a tracking servo. A control unit that detects the light detected by the photo detector and arranges the liquid ejected from the ink jet on the pre-groove 23 is called a traverse servo.

  In FIG. 2, the DNA microdisk 1 is provided with a center hole 22 for rotating by a disk motor and an uneven pregroove 23. As a method of creating the pregroove, the glass substrate can be selectively etched. Further, the convex portion of the pregroove can be provided by printing. When the pregroove is formed by printing, a DNA spot is disposed on a substrate to which printing ink does not adhere. Of course, it is also possible to use a resin and use a method similar to that for an optical disk such as a CD, and use injection molding. Further, the pregroove can be cut in the tangential direction to form a prepit, which can be used instead of the pregroove.

If necessary, a thin film that does not emit fluorescence when irradiated with a laser beam, such as SiO ₂ or gold, is provided on the surface on which the probe DNA is spotted. Further, a thin film having good adhesion to the probe DNA is formed on a thin film such as SiO ₂ or gold. This latter thin film promotes close contact with the probe DNA to be spotted and is present on the pregroup due to the surface tension of the probe DNA and / or the presence of a groove wall when spotted in the recess, The liquid in which the probe DNA is dissolved evaporates over time, and the probe DNA is finally fixed on the pregroup. As an example of such a thin film, for example, a thin film formed by treatment with an aqueous solution of poly-L-lysine (abbreviated as PLL) can be given as a representative example. However, an aqueous solution of 3-aminopropyltriethoxysilane (abbreviated as APS) can be used. Alternatively, DNA with biotinylated ends can be used as the probe DNA. In this case, avidin is immobilized on the surface of the substrate, so that the DNA is immobilized on the substrate by the ability to form a specific bond between avidin and biotin. Can do. Further, if a thin film is provided only on the surface of the substrate, the substrate may be warped due to changes in the temperature and humidity environment, so it is preferable to provide the thin film symmetrically on both sides of the substrate. Also at this time, it is preferable to provide a thin film so that the laser beam incident from the substrate surface is transmitted through the substrate.
Address information for indicating the position of the pregroove is added to the pregroove. In FIG. 2, the pregroove is formed concentrically or spirally, and as shown in 27, a part of the concentric pregroove is cut to provide a part with and without the pregroove, and address information indicating the position of the pregroove did. In addition, a mark indicating a rotational position called 26 V mark was provided on the outermost peripheral portion or the inner peripheral portion of the DNA microdisk. FIG. 2A is an enlarged view of the address information and the V mark showing a part of FIG. 2 in an enlarged manner. Since the actual pregroove is along the circumference, it has a fan shape, but here it is shown as a straight line for simplicity. The correspondence with FIG. 2 is given the same number.
A structure in which a DNA spot by spotting described below is provided on the thus configured DNA microdisk is also called a DNA microdisk.

Next, the configuration of the V mark will be described in detail. In FIG. 2A, the V mark 26 indicates an angle in the rotation direction of the pregroove. In this embodiment, a prepit string (addresses of the pregroove in the rotation direction angle between the V marks 26 ( 26a, 26b... By providing the V mark in this way, it becomes easy to detect the position of the address information on the disk circumference. For example, if 26a is the starting point of 0 degree, 26b is an index indicating a position of 0.5 degree on the circumference.
In this way, the V mark constitutes an absolute address representing the rotation angle on the disc. For example, pre-pit trains (26a, 26b,...) Representing 4 bits in the radial direction are formed, and the angles are associated with the addresses using them. In FIG. 2-a, the arrow 28 indicates the radial direction of the DNA microdisk, and the arrow 29 indicates the tangential direction. The pre-groove address information 27 indicates address information indicating the radial position of a plurality of pre-grooves arranged in the radial direction. (For example, the outermost circumference is the first pre-groove and a 4-bit address of 2, 3 and 4 is given in the inner circumference direction) When recording address information and angle information, a well-known modulation method such as FM modulation is used. , Phase modulation. The angle information indicated by the V mark is read by the V mark detector 15 shown in FIG. 6, and the position information of the pregroove is read by the emitted light beam of the objective lens 4 that scans the pregroove. When there is a time shift on the time axis during this period, it is of course corrected by the CPU 36 of the spotting device.

In FIG. 2, the first data recording unit 24 is made of ink that can generate a gray mark in order to use spotting liquid for data recording at the time of spotting or at the end of spotting. Then, the information is recorded by changing the position, size, phase, etc. of the spot produced with this ink or the like. Then, when a DNA spot is provided on the substrate, information such as conditions for creating a DNA spot is created and recorded in a row of recording spots modulated by the information.
In particular, it is effective to record the correspondence between the address information indicating the spotting position and the name in the spotting liquid.
As a modulation method that can be used at this time, a method of changing the position of the recording spot according to an information signal composed of a binary signal, or a method of changing the period of the recording spot according to the information signal Etc. can be used. As a material constituting the recording spot, an ink made of an organic material or an inorganic material can be used.
Needless to say, the correspondence between the address information on the DNA microarray substrate indicating the spotting position and the name of the spotting solution can be recorded in another memory and attached to the DNA microarray substrate. At this time, identification information may be provided on the DNA microarray substrate by a barcode or the like, and the relationship with the memory may be recorded in the memory.
In addition to the first recording portion, a second recordable portion 25 is provided on the substrate, and after reading the DNA spot, it is possible to additionally write information data of the read DNA spot.
In the recordable portion, a dye or a metal thin film conventionally used for a recordable optical disk can be formed on the substrate by vapor deposition or sputtering. It is desirable that the second data recording unit is provided on the pregroove and that the pregroove is provided with address information so that it can be easily distinguished from other parts. Of course, it is also possible to record the address information indicating the spotting position in this data recording section and the correspondence between the names in the spotting liquid. In that case, the information acquired during spotting is temporarily stored in another data. It is also possible to store them in a memory and record them together after spotting.

  Next, the operation will be described with reference to the DNA microdisk in FIG. 2, the block diagram of the spotting device control unit in FIG. 3, and the graph showing the change in the amount of reflected light distribution during spotting in FIG.

1 and 3, the laser beam emitted from the objective lens 4 follows the pregroove 23 on the DNA microdisk 1 by the tracking actuator 4a. For this reason, the output difference between the photodetectors 7a and 7b obtained by dividing the seven photodetectors into two is obtained as the output of 30 differential amplifiers 1, and the response of the tracking servo section is optimized by 31 phase compensators 1. The position of the tracking actuator 4a is controlled by the drive amplifier 1 of 32. A part of the light emitted from the objective lens 4 is transmitted through one DNA microdisk and detected by the photodetector 10a and the photodetector 10 comprising 10b. Is output by. The output of 33 optimizes the response of the traverse servo unit by the phase compensator 2 of 34, drives and controls the 13 traverse motors A and controls the position of the 13 traverse units A by the drive amplifier 1 of 35. As a result, the position of the 9 ink jet outlet is controlled so as to always follow the position facing the 23 pre-groove, and the liquid ejected from the ink jet is ejected and arranged on the 23 pre-groove.
A droplet ejected from the ink jet and adhered onto the pre-groove is irradiated with a beam spot emitted from the four objective lenses. Then, the light is reflected by the pregroove and the adhered droplets and detected by the seven photodetectors. FIG. 4 is a graph showing the situation at this time. The horizontal axis of the graph in FIG. 4 indicates the rotational position of the DNA microdisk, and the vertical axis indicates the amount of reflected light detected by the photodetector 7.

FIG. 4 shows that the amount of reflected light is significantly reduced by the droplet when the droplet adheres to the pre-groove. This is indicated by the arrow 45 in FIG.
The point in time when the droplets are ejected and deposited on the pregroove can be detected by the photodetector 7. In the embodiment, the output of the photodetector 7 is detected by an adder 37, and the adder output 38 is supplied to the CPU 36 to determine whether or not a droplet has adhered, and the position of the adhesion. Can be measured. Since the ejection position accuracy of the ink jet is about ± 30 μm, when the pitch in the radial direction of the pregroove (referred to as the track pitch) is set to 30 μm, the output of the photodetector 7 is measured to measure 13 traverse units A The position of the liquid droplets can be controlled so that the droplets are arranged on the pregroove. Further, the positional accuracy of the pregroove in the tangential direction can be adjusted to the optimum position by adjusting the attachment position of the ink jet.

Next, a method for adjusting the attachment position of the ink jet will be specifically described with reference to FIGS. In FIG. 1, a laser beam emitted from a laser is focused on 23 pregrooves through an objective lens. At this time, the spotting liquid is discharged from the 9 ink jet nozzles.
The state at this time is shown in FIG. In FIG. 4, by rotating 1 DNA microdisk, spotting droplets are ejected from 8 inkjets at the spotting liquid ejection time indicated by 43, 44, reflected from the pregroove 23, and detected by 7 through the objective lens. The amount of light returning to the vessel was plotted on the vertical axis 42 of the graph. The horizontal axis shows the time course as DNA microdisk rotation time course 41.
When the droplets adhere on the pregroove, as shown in the reflected light amount decrease period of 45 in FIG. 4, the light amount reflected from the pregroove and returned to the photodetector 7 through the objective lens is about 1/10. I understand that The light quantity distribution reflected from the 23 pre-grooves is measured by the photo detector 7, and the position of the 8 ink-jet is adjusted so that the discharged liquid droplets are arranged at the center of the 23 pre-grooves. Adjustment is performed by repeating ejection from the ink jet several times to adjust to an optimal position.
The output of the light detector 7 measures the light amount distribution by 30 differential amplifiers, the output of the 37 adders is supplied to the CPU 36 to measure the light amount, and the position of the 13 traverse units A is controlled. While doing.
The 14 disk motors rotate one DNA microdisk.
The 4 objective lens can be moved in the vertical direction with respect to the DNA microdisk by the 4b objective lens actuator, and the distance between the 1 DNA microdisk and the 4 objective lens is detected and controlled to be constant. It is also possible to configure a focus control unit (not shown). At this time, the relative position of the 4 objective lens and the 1 DNA microdisk is detected, and the position of the 4 objective lens (perpendicular to the DNA microdisk) is set to 4b so that the detected position is constant. The drive is controlled by

Next, referring to FIG. 5, a tank (including ink jet) holding an ink jet and spotting liquid is placed on the traverse unit B of 12-1, and a plurality of types of probe DNA droplets are placed on the traverse unit B. A method of sequentially ejecting from the ink-jet 51 and arranging in the pregroove on the DNA microdisk will be described.
Here, the ink jet unit 53 provided with a plurality of ink jets was placed on a traverse unit B 12-1 shown in FIG. 5, and the ink jet unit was movable with respect to the traverse unit B by 55 transfer gears. At this time, a plurality of 51 ink jets are provided so that probe DNA liquids having different components can be ejected to the respective ink jets. And the liquid containing probe DNA was comprised from each inkjet so that spotting was possible. In addition, the inkjet unit 53 is shown in the moving direction of the inkjet unit 54 with respect to the traverse unit B of 12-1, so that the inkjet discharge ports 52 provided in the 10 photodetectors and the inkjet unit always maintain the same positional relationship. Move control in the direction.

6 and 7 are configuration diagrams showing another embodiment of the spotting apparatus. FIG. 6 is a configuration diagram viewed from the side, and FIG. 7 is a configuration diagram viewed from the top. 6, since there are many common points with FIG. 1, the same numbers are used as they are in FIG.
First, a rough operation will be described. A droplet of probe DNA is ejected from the ink jet onto the pregroove 23 of one DNA microdisk, and the DNA microdisk is rotated in order to place the droplet in a pregroove shape. At the same time, the rotary table to which a plurality of ink jets are attached is rotated, and the position of the DNA microdisk is displaced in 62 transport directions so that the desired ink jets are located on the pregroove. When the ink jet comes to a designated position on the pre-groove, droplets are ejected from the ink jet and placed on the pre-groove.

Next, a detailed operation will be described.
In FIG. 6, those having a number of 60 or more are newly added to the configuration of FIG.
A disk motor transfer device 61 transfers the disk motor 14 in the transfer direction 62. Of course, one DNA microdisk clamped by 14 disk motors is transferred together with 14 disk motors.
The laser beam emitted from the objective lens 4 is diffracted by the 23 pregrooves, and the far field pattern of the reflected light is received by the photodetector 7. Since the differential signal of the photodetectors 7a and 7b indicates the relative position of the spot and the pregroove formed on the DNA microdisk by the laser beam described above, the tracking of 4a is performed so that the differential signal becomes zero. The actuator and the traverse motor C of 13-2 are controlled so that the beam spot follows the 23 pregrooves. The traverse unit C of 12-2 is different from the traverse unit B of 12-1 shown in FIG. 5, and the ink jet and the photodetector 10 are attached to the multi-ink jet rotary table 71.
The laser beam emitted from the objective lens 4 is diffracted by 23 pregrooves, and the far field pattern is received by 10 photodetectors.
The differential signals of the photodetectors 10a and 10b indicate the relative positions of the spots and pregrooves formed on the DNA microdisk by the laser beam described above. The follow-up control is performed so that the 9 discharge ports are positioned on the 23 pre-grooves.

  That is, the laser beam spot emitted from the objective lens follows the pregroove by the tracking actuator 4a and the traverse motor C 13-2, and the position of the DNA microdisk follows the position of the ink jet discharge port. The timing of discharging the spotting liquid from the ink jet is performed by reading the output of the 14 V mark detector and the address information 27 specifying the location of the pregroove provided in the pregroove from the output of the 7 photodetector. As the V mark detector 15, for example, a photocoupler, an optical head that scans a laser beam in the radial direction along the V mark pit row, or the like can be used. When the photocoupler is used, light is emitted from one side of the substrate, the light transmitted through the substrate is received by the other photodetector, and the V mark is read.

  When the scanning optical head is used, the laser beam is irradiated from the optical head while scanning the V mark, the light transmitted through the substrate is received by another photodetector, and the V mark is read. The scanning direction is the radial direction indicated by 28 in FIG. The scanning method is performed by displacing the position of a lens for irradiating a laser beam. If the position is detected by scanning the V mark, the position information of the pregroove can be read even if the relative speed between the DNA microdisk and the laser beam is close to zero.

Under such control, the inkjet 8 placed on the multi-inkjet rotary table 71 in FIG. 7 accurately ejects and arranges droplets on the pregroove on one DNA microdisk. The rotation of the multi-inkjet rotary table 71 is controlled by the rotary table rotation control device 83 shown in FIG. Although not shown, the control signal for ejecting the droplets from the ink jet is the address information for the CPU 36 in FIG. 8 to specify the output of the V mark detector 15 and the location of the pregroove provided in the pregroove. Is received by the photodetector 7 and the ejection timing of the ink jet is controlled. In the traverse motor C 13-2, the output of the 31 phase compensator 1 is controlled by a drive signal that passes through the 81 phase compensator and the 82 drive amplifier 3.
FIG. 9 shows an ink jet structure using the multi-spotting apparatus of FIG. Reference numeral 91 denotes a tank for storing probe DNA as a spotting solution, and 90 denotes an ink jet A, which is inserted and connected to the tank 91 through a connection hole 93.
In addition, 92 is a connection seal, 94 is a discharge port, 95 is a pressure chamber for receiving the pressure of 96 pressure devices and discharging spotting liquid from the 94 discharge ports.
The ink jet tank is provided with a liquid name display portion 97 such as a bar code for displaying the kind of spotting liquid in the tank.

When spotting to a predetermined position on the pregroove, the bar code 97 is always read, and the information displayed on the display unit 97 is recorded in the memory together with the address information on the pregroove. Pregroove address information can also be read using the adder output 38 of FIG. Of course, the spotting position on the DNA microdisk can be detected from the output of the V mark detector of FIG. The contents of this memory are finally transferred to the recording area on the DNA microdisk (area 24 or 25 in FIG. 2), and what spotting solution is placed at which position on the DNA microdisk where spotting is completed. You can know what is being done.
By configuring as described above, it becomes possible to detect the position of the pregroove on the substrate and spot the probe DNA on the pregroove as a spotting solution, and to probe at a position having predetermined address information on the substrate. Since the DNA can be provided and the pregroove can restrict the probe DNA from spreading on the substrate, the probe DNA can be arranged at a high density. In FIG. 1, the spotting liquid is disposed on the convex portion of the pregroove, but it may be disposed on the concave portion of the pregroove. In this case, the spotted spotting liquid is disposed along the concave portion of the pregroove.
Further, since the address information 27 can be added in advance to the pregroove, the probe DNA can be accurately indicated.

In the above embodiment, when spotting the probe DNA, a laser beam having a wavelength of, for example, 780 nm is irradiated from the laser beam 2 from the substrate side in order to detect the position of the pregroove on the substrate, and the transmitted laser beam is used. Then, the position of the pregroove 23 on the substrate is detected, and positioning is performed as described above. When a thin film is provided on the substrate, a laser having a wavelength that allows the laser beam irradiated from the substrate side to pass through the substrate is selected and used.
When applying a cDNA as a test sample to a DNA probe on a substrate and a hybridization reaction with the probe DNA occurs, when detecting fluorescence contained in the generated DNA spot, for example, wavelengths of about 650 nm, about 530 nm, A laser beam of about 400 nm is irradiated from above the substrate and the reflected light is used. For this reason, layers such as SiO ₂ and gold are provided on the substrate. In the embodiment, a gold layer is provided on a substrate and a SiO ₂ layer is formed thereon. It is also possible to use an inorganic material or an organic material having equivalent properties in place of gold instead of Pt or SiO ₂ .

The function of the gold and SiO ₂ thin film provided on the substrate will be described. In FIG. 10, the horizontal axis 100 indicates the film thickness [nm] of the SiO ₂ film, the vertical axis 101 indicates the ratio of the electric field strength on the substrate surface to the case of only the polycarbonate substrate, and a thin film of gold and SiO ₂ is provided. It shows the comparison value when nothing was provided. Reference numeral 102 denotes characteristics when a laser beam having a wavelength of 563 nm is used, 103 is a laser beam having a wavelength of 652 nm, and 104 is a laser beam having a wavelength of 532 nm.
In FIG. 10, a SiO ₂ thin film is disposed on a gold thin film. The electric field intensity ratio that can be formed on the SiO ₂ film when the thickness of the gold film is 50 nm is calculated by changing the thickness of the SiO ₂ film.
As a result, it can be seen that the intensity ratio of the vertical axis is 5 times, for example, when the film thickness of SiO ₂ is 70 nm. This indicates that, when the film structure described above is used, the amount of reflected light is five times greater when light having a wavelength of 532 nm is irradiated from the surface on which the film is formed.

In the graph of FIG. 10, when the thickness of the gold thin film is 50 nm, it can be seen that by setting the SiO ₂ film thickness to 70 nm, the electric field on the SiO ₂ surface is about five times that without the thin film. By providing a thin film on the surface of the substrate and irradiating laser light, the electric field is increased so that the DNA spot containing the phosphor is irradiated with the laser beam and the reflected light is observed. The effect of increasing the amount of light and improving the S / N of the reflected light is obtained.
When spotting on a substrate, it is important to remove dirt such as fine oil and fat components on the surface of the substrate to be spotted. Therefore, by increasing the output of the laser beam that irradiates the pre-groove immediately before spotting, the temperature of the substrate surface can be raised and the dirt component can be removed.
For this purpose, it is possible to use a laser beam for position detection. However, spotting can be performed after another laser is prepared and the temperature of the substrate surface is temporarily increased prior to spotting.
Although the description has been made using the embodiment in which the substrate is a disc shape, the substrate is not limited to a disc shape but may be applicable to a shape such as a rectangle. Of course, the pregroove can be used not only in a circumferential shape but also in a shape in which straight line segments are assembled.
In addition, in order to detect the position of the inkjet, a photodetector can be directly attached to the inkjet. Of course, a method other than the position detection method using light, for example, magnetic detection can be used. The spotting solution has been described in the embodiment using probe DNA such as cDNA, but the spotting solution can be applied to any liquid as long as it is a protein such as protein.

The substrate of the present invention can also be used as a probe DNA when preparing an oligo DNA using a photochemical reaction.
Here, a probe DNA prepared by using a photochemical reaction using the substrate of the present invention will be briefly described.
As is well known, DNA is a polymer of deoxyribonucleotides (referred to as nucleotides). This nucleotide is a compound in which phosphoric acid and one of four types of bases of adenine (A), guanine (G), cytosine (C), and thymine (T) are bound to deoxyribose. For example, two nucleotides, now one of these is [X], the other is [Y], the 5 ′ carbon atom of deoxyribose in [X] and the 3 ′ carbon of deoxyribose in [Y] Atoms can be connected by forming an ester bond with phosphoric acid in between.
Nucleotides have two ends, but one end is connected to the 5 'carbon and the other end is connected to the 3' carbon, and the bond between the nucleotides is only between the 5 'and 3' carbons. Bonding does not occur between 5 'carbons or 3' carbons.
By repeating the bond, theoretically, an infinite number of nucleotides can be bound in a straight chain. Since each nucleotide contains one of four types of bases A, G, C, and T, the order in which the bases are arranged in the DNA can be specified, and this is the DNA base sequence. Here, for simplicity, these are called A, G, C, and T. The DNA prepared on the substrate is called probe DNA.
When the probe DNA is generated on the DNA microdisk of FIG. 2, a recordable zone is provided on the DNA microdisk, the preparation conditions and measurement results, address information indicating the storage position of the probe DNA positioned by the pregroove or prepit, and The correspondence relationship with the probe DNA is recorded on the DNA microdisk. As a result, the conventional glass plate and the data at the time of creation can be stored together, and operability, reliability and security are improved. This is to provide a specific creation method for this purpose.

Hereinafter, an embodiment that specifically shows the best mode for carrying out the present invention will be described with reference to the drawings.
The schematic configuration of the first embodiment of the present invention will be described below with reference to FIGS. FIG. 11 is an enlarged view of a pregroove of a DNA microdisk and an address portion indicating its position. FIG. 13 is a block diagram showing the configuration of an apparatus for creating probe DNA.
Here, an oligonucleotide made of four nucleotides of the base sequence A-C-G-T is generated in the pregroove 112 having the address 1 (113) on the DNA microdisk 118 shown in FIG. An example in which an oligonucleotide having a G-A-T-C sequence is prepared on a DNA microdisk in the pregroove 114 indicated by address 2 of 115 will be described.

As described above, it is assumed that A is on the 3 ′ end side and T is on the 5 ′ end side. In the chemical synthesis of DNA, starting from the 3 'end, nucleotides are added one by one to the 5' end, and the DNA of the desired base sequence is synthesized by controlling the type of nucleotide added. . For example, the case of starting from A will be described. Another chemical group is bonded to the 5 ′ end of this nucleotide so that it cannot react with the 3 ′ end of another nucleotide. Such chemical groups are referred to herein as protecting groups. This first A nucleotide is covalently bonded at its 3 ′ end to the resin on the substrate. In addition, DNA having a 3 ′ end biotinylated can be used as a probe DNA. In this case, by fixing avidin on the surface of the DNA microdisk 111, DNA can be formed by a specific binding ability between avidin and biotin. Can also be fixed to the substrate.
As a method for fixing the first A nucleotide at its 3 ′ end, there is the following method. First, a photochemically activated substance is provided on the surface of the DNA microdisk.
Alternatively, in order to increase the electric field generated when the surface of the DNA microdisk is irradiated with laser light, for example, a gold layer or SiO ₂ layer is provided, and the above-mentioned photochemically activated substance is provided thereon. You can also.

First, a photochemically active substance on the surface of the DNA microdisk substrate, a monomer (mononucleotide) terminated by a protecting group having the property of leaving an OH-group after dissociation by light irradiation, is provided. An oligonucleotide (polymer having a short chain length) is synthesized in a state where one end is fixed to the substrate surface by polymerization. Next, after selectively irradiating the surface of the substrate with laser light to remove the protecting group to form an OH-group, when a mononucleotide solution is applied by, for example, spin coating, a chemical reaction occurs, and the polynucleotide (DNA) is irradiated with light. Join the part where Examples of the monomer include 3′-O-activated phosphoramidite nucleotides in which 5 ′ hydroxyl is photoprotected with a photosensitive protecting group. The photoprotected mononucleotide and its synthesis method are described in detail in WO1997 / 039151 (Japanese Patent Laid-Open No. 2000-508542). An example of the photosensitive protecting group is an ortho nitrobenzyl group.
That is, when a nucleotide solution is added after selectively irradiating the surface of the substrate with a laser beam, a chemical reaction occurs, so that the nucleotide has an activation layer that binds to the lighted portion. For example, an OH group is generated in a portion irradiated with light and then bonded to a monomer applied by spin coating.

In the present invention, a photochemical reaction is carried out in a designated storage section provided on the substrate with a storage section that can be specified by address information constituted by pregrooves or prepits. Therefore, the address information is read, and the storage unit is selectively irradiated with laser light.
The storage portion uses a concave or convex portion of the pregroove, or a concave pit or convex pit shape that is shaped like a soccer stadium even if it is a plane that can be specified by the prepit. The dimensions are preferably 1 μm or more in width and 1 μm or more in length in relation to the size of the laser beam spot, but are not limited to this and are set to dimensions that can store the probe DNA.
After reading the address information and tracking the laser beam spot to a pregroove or the like as a storage unit, light is selectively irradiated to an arbitrary storage unit on the surface of the DNA microdisk. Therefore, first, a laser beam is applied to address 1 and then an A nucleotide is added. The end of this A nucleotide is protected with a protecting group. A solution of A nucleotide is applied onto the substrate by spin coating. However, the protecting group is a photochemically unstable one. In other words, a deprotection reaction occurs when a laser beam is applied. When this A nucleotide binds to the surface of the pregroove 112 at address 1, a washing solution is applied to the substrate by spin coating to remove unreacted A nucleotide.

  Next, the pregroove 114 at address 2 is irradiated with light to activate it, and a G nucleotide having a protecting group at the terminal is reacted and arranged there. Thus far, a DNA microdisk has been formed in which A is connected to the pregroove 112 at address 1 and G is connected to the pregroove 114 at address 2. Next, when the pregroove 112 at address 1 is irradiated with a laser beam, a protecting group for A nucleotide is removed, and then C nucleotide is added. A pre-groove 114 at address 2 is irradiated with laser light to deprotect G nucleotides and add A nucleotides. Thereafter, if the same process is repeated, a DNA microdisk having A-C-G-T in the pre-groove 112 at address 1 and G-A-T-C oligo DNA in the pre-groove 114 at address 2 is completed. Although 118 DNA microdisks are written linearly for convenience, they are actually disk-shaped, and the pregrooves are formed concentrically or spirally in the 117 tangential direction. A plurality of concentric or spiral pregrooves are aligned on the disk in the radial direction (for example, several thousand or more with a track pitch of about 2 μm to 20 μm).

A method of irradiating this specific pre-groove with laser light will be described with reference to the block diagram of FIG.
FIG. 13 is a block diagram of a probe DNA production apparatus. Reference numeral 118 denotes a DNA microdisk, which is provided with a pre-groove 112 at address 1 and a pre-groove 114 at address 2. In this probe DNA production apparatus, a pregroove is used as a probe DNA storage.
118 DNA microdisks are placed on 131 motors via 132 turntables, and the rotation is controlled by the disk motor 131. A laser 120 is converted into parallel light by a beam expander 121, passes through a beam splitter 122, and is focused on a pregroove 112 at address 1 by an objective lens 123. For this reason, the objective lens 123 is controlled by a focusing actuator 125, a tracking actuator 124, and a traverse motor 130 so as to focus on the pregroove 112 having the address 1 113. For control purposes, the objective lens detects the reflected light from 118 DNA microdisks collected by the objective lens at address 1 at 113 by means of 128 photodetectors through 126 lenses, and reads address 1 at 113. The position is controlled. First, the power of the laser beam is adjusted to a low power so as not to affect even when the protective group is irradiated, and the beam spot follows the pre-groove of address 1 at 112. Then, when the pre-groove 112 at address 1 is detected, the laser power is increased to remove the protective group. That is, the laser power is set to a high output only during a period in which the laser beam scans the pre-groove 112 at address 1.

FIG. 14 is a block diagram of a probe DNA production apparatus. The laser power of 120 lasers is modulated by a laser power modulator 146. The output of the 128 photodetectors is converted into current by the 140 preamplifier, the address 1 information of the pregroove 112 is detected, demodulated by the decoder 141, and sent to the CPU (controller) 142. After the position of the pregroove 112 is read, the laser power suitable for the photochemical reaction performed on the pregroove is calculated by the CPU 142 and output by the irradiation pulse control unit 143. Then, the output laser power, output pulse width, etc. of 120 lasers are controlled via the laser power modulator 146, and the optical output of the laser irradiates the pregroove 112. The laser power output from 120 lasers is controlled in pulse width and pulse peak value so as to be suitable for the photochemical reaction, but it is necessary to change the power depending on the relative speed between the DNA microdisk and the laser beam spot. Therefore, the rotational speed information of 131 disk motors is input from the servo unit 147 to the CPU 142 and used as information for power control. For example, when a large laser power is required for the photochemical reaction, the relative speed can be reduced, and a large light energy (an integrated value of the light energy applied on the pregroove) can be applied to the target pregroove. The servo unit 147 also performs an operation for focusing and tracking the light beam emitted from the 123 objective lens on the pregroove of the DNA microdisk.
A PC 145 is a personal computer for controlling the probe DNA production apparatus, and controls the entire operation of the probe DNA production apparatus via an interface (I / F) 144. For example, the total number of pregrooves that can be specified by the address information on the DNA microdisk can be 1 million or more, and what DNA is generated at which address only by the CPU 142 inside the probe DNA production apparatus. Since it cannot be controlled, control by an external PC is required.

FIG. 15 shows an operation waveform diagram. Reference numeral 141 denotes an example of the read address information. The light output of the laser 120 reads address information with the reproduction light power 148 and irradiates a pregroove having a specific address with laser light having a peak power of 149.
Especially when a photochemical reaction is caused on the pregroove, the integrated value of the light energy is important. By increasing the initial pulse width of the irradiation laser power and then reducing the pulse width, the light provided on the pregroove is reduced. Appropriate reaction energy for the protecting group can be provided.
In this way, by a tracking servo technology that irradiates light on an arbitrary minute part (pre-groove) on the surface of the DNA microdisk and a DNA synthesis reaction using a nucleotide having a protecting group that undergoes a deprotection reaction upon irradiation with light, An “oligo DNA microdisk” can be produced. As for the laser wavelength, about 350 nm has been found to be suitable for deprotection groups. It is also possible to change the action for the photochemical reaction by selecting and using a plurality of laser wavelengths. For example, the monomer reaction can be selectively performed by changing the wavelength by making the properties of the photoprotective group and the monomer highly wavelength dependent. The probe DNA finally produced can be discriminated whether or not the probe DNA has been produced normally by scanning the laser beam. As a determination method, it is possible to perform scanning by scanning the probe DNA and comparing the reflectance DNA, the color, etc. with the probe DNA operating normally.
A plurality of probe DNAs of the same type can be prepared, and normal ones can be discriminated and managed. Finally, the customer knows the probe DNA that is finally normal from the management data, and can use only the probe DNA having the address information. As a management method, address information in which probe DNAs that have been successfully prepared are present can be stored on a DNA microdisk. This is because the DNA microdisk is provided with a recordable zone, and information is stored there.

Finally, using the probe DNA on the substrate prepared as described above, mRNA was analyzed by the same process as that of a known DNA chip, which was hybridized with the sample to be examined and base-bonded. Thereafter, the probe DNA is irradiated with an inspection laser beam and fluorescence is observed. At this time, when the DNA microdisk of the present invention is used, the DNA spot can be scanned one-dimensionally, so that inspection can be performed at high speed. Since the probe DNA is arranged on the pre-groove, the detection S / N of the base-bonded probe DNA (referred to as DNA spot) can be improved. By providing an optical measurement section and a servo section for this purpose, base binding Realization of high-speed measurement, improved operability and low price. The optical measurement unit constituting the control unit is provided independently of the DNA spot reading optical measurement unit, so that the beam spot for detecting the servo error does not deteriorate the phosphor contained in the DNA spot. In addition, the position of the reading light beam of the DNA microarray was accurately controlled by using a servo mechanism, and errors in the focus and tracking directions were eliminated, thereby realizing accurate scanning.
In addition, the reading laser beam can be amplitude-modulated at a high frequency (for example, 100 to 500 MHz) in order to prevent the phosphor from fading due to the reading laser beam and to improve the reading S / N of the DNA spot. By modulating in this way, it has been found that the accumulation of irradiation energy is reduced on the substrate, and fading can be prevented.

Hereinafter, examples specifically showing the best mode for measuring the DNA microdisk of the present invention will be described with reference to the drawings.
Hereinafter, the schematic configuration of an embodiment of the measurement according to the present invention will be illustrated with reference to FIGS.
FIG. 16 is a block diagram showing the configuration of an apparatus for reading the DNA microdisk shown in FIG. 2, and FIG. 2 is a DNA microdisk.
In FIG. 16, reference numeral 1 denotes a DNA microdisk whose rotation is controlled by a disk motor 152. Reference numeral 153 denotes an objective lens which collects the reflected light by condensing the laser beam on the DNA spot formed on the DNA microdisk. The objective lens 153 includes a tracking actuator 154a, a focus element 154a, and a tracking element 154b. The tracking is performed in a direction perpendicular to the substrate of the DNA microdisk (Z direction of 153b) and the laser beam destination follows the DNA spot array. It can be moved in the direction (X direction of 153a).
Reference numeral 155 denotes a fluorescence excitation light source (output light wavelength λ1). In this case, a laser having a wavelength of 650 nm is used. Reference numeral 156 denotes a collimator lens 1 which converts the output light of the laser 155 into parallel light or divergent light having a specified angle, passes through a half mirror 159 and a beam splitter 1 160, and is supplied by an objective lens 153. Focus on one DNA microdisk. The reason for converting to divergent light having a specified angle is to make the size of the beam spot when focused into the same extent as the size of the DNA spot. At this time, the light beam emitted from the light source having the wavelength λ1 may be provided with an aperture limit before entering the objective lens so that the NA of the objective lens is substantially reduced.
This aperture restriction can be provided on the exit side of the 156 collimator lens in FIG. For example, even when an objective lens having an NA of 0.6 is used, the NA is 0.5 for the light of wavelength λ1 and 0.6 for the servo light of wavelength λ3. I did it. By doing so, the servo error detection sensitivity can be increased and the DNA spot reading beam diameter can be increased.

Reference numeral 157 denotes a servo light source (output light wavelength λ3), which passes through a half mirror of 158 collimator lenses 2 and 159, passes through a beam splitter 1 of 160, and is focused on a DNA microdisk of 1 by an objective lens of 153. Tie. The reflected light reflected by the DNA microdisk passes through the objective lens 153, the beam splitter 1 of 160, the beam splitter 2 of 164, the condenser lens 2 of 165, and the 166 servo error detector. .
On the other hand, the output light of the light source for fluorescence excitation 155 (output light wavelength λ1) condensed on one DNA microdisk by the objective lens 153 is used for the phosphor contained in the DNA spot on one DNA microdisk. The excited light having the wavelength λ2 is collected by the objective lens 153, condensed by the condenser lens 161 through the beam splitters 160 and 164, and then excited by the optical filter 162. Only the light wavelength λ2 is selected and directed to the 163 fluorescence reading detector. At this time, the beam splitters 1 and 2 of 160 and 164 pass the light emission wavelength λ2 of the phosphor in the DNA spot excited by the laser of wavelength λ1, and the beam splitter 14 passes light of the output wavelength λ3 of the servo light source. The optical filter 162 is configured to transmit only the wavelength λ2. The output light of the servo light source 167 is converted into parallel light by the collimator lens 2 168.
The spot shape changes when the light is focused by the objective lens 153 by the cylindrical lens 167 before and after the focus. In other words, on the 166 servo error detector, it changes from a perfect circle to an ellipse. This change is measured by a servo error detector obtained by dividing the 166 photodetectors into four. When the beam reflected from the DNA microdisk is projected in a substantially circular state at the center of the detector, it indicates that the objective lens 153 is in focus. At the same time, the output light of the servo light source is generated by the light beams S1, S2, S3 emitted from the objective lens 153 as shown in FIG. 17 on the DNA spot or pregroup on one DNA microdisk by 168 diffraction gratings. Is formed. Of these, the light beams indicated by S1 and S3 are received by D1 and D2 of 166 servo error detectors, and the difference between the outputs becomes a tracking error.

FIG. 17 is a principle diagram showing the relative relationship between the DNA microdisk, the beam spot formed by the reading laser beam and the servo laser beam.
In FIG. 17, reference numeral 171 denotes a pre-group, indicated by P1, P2, and P3. The width of the pre-group is 1-100 μm, the height is 0.1-10 μm convex or concave, and the interval between the pre-groups is set to about 1-150 μm. 173 DNA spots are formed on 171 pregrooves. Reference numeral 172 denotes a servo beam, which shows a state in which the pre-groove P2 of 171 is irradiated.
The servo beam S1 is applied to the servo error detector D1 shown in FIG. 14, S2 is applied to D3, and S3 is applied to D2.
Reference numeral 170 denotes a DNA spot reading beam (wavelength λ1), which irradiates the 173 DNA spot, excites the phosphor contained in the 173 DNA spot, and the excitation light having the wavelength λ2 by the wavelength λ1 is detected by the fluorescence reading shown in FIG. Read by the instrument 163. The servo beam formed from the servo light source (wavelength λ3) irradiates the DNA spot, but is set so as not to have a wavelength that can excite the phosphor of the DNA spot (for example, 780 nm). Absent.

The present invention configured as described above can achieve the following effects.
When a DNA microdisk in which probe DNA is accurately spotted is used as a pregroove on the DNA microdisk, the reading speed can be increased because the reading beam only needs to be scanned in a one-dimensional direction. For this reason, if the DNA spot is scanned only once, the reading is completed, and imaging by the conventional two-dimensional scanning becomes unnecessary.
Furthermore, since a DNA microdisk having a pre-groove provided on the substrate is used, more DNA spots can be provided on the substrate than before, and for example, a DNA microdisk having 100,000 or more DNA spots can be produced. Since a thin film such as gold is provided on the substrate and a thin film such as SiO ₂ is provided thereon, the amount of reflected light increases when a laser is irradiated on the substrate, and a DNA spot on the DNA microdisk is detected. S / N can be increased.
Resin can be used as the substrate material, and the entire system can be constructed at low cost. Even when resin is used as the substrate material, the thin film is provided symmetrically on both sides of the substrate, so that the warpage of the substrate can be suppressed to a minimum even during hybridization.

When spotting, spotting liquids are stored in separate tanks one by one, and the tank is provided with a display for displaying the type and name of the spotting liquid, so that no wrong liquid is spotted during spotting.
Since spotting can be performed by combining a plurality of rotary tables on a single DNA microdisk for spotting at high speed, spotting time per spot can be saved and spotting time is reduced.
When the probe DNA is synthesized by photochemical reaction in the pregroove on the DNA microdisk, the synthesis can be performed by irradiating the pregroove having the address designated with the laser beam spot with the laser beam. Since it is possible only by controlling the laser beam, it is not necessary to use a conventional mask. Also, by controlling the laser beam, a different probe DNA can be produced each time, and a custom DNA chip can be produced easily.

In the present invention, the pregroove that can be specified by the address information is described on the substrate. However, the storage portion can be specified by the address information instead of the pregroove. It is also possible to use a convex part. That is, a storage unit that can be specified by a pre-pit having address information is provided, and a photochemical reaction is performed in the specified storage unit. Therefore, the address information is read, and the storage unit is selectively irradiated with laser light.
The storage portion may be a concave or convex portion of the pregroove, or a concave pit or a convex pit having a shape like a soccer stadium even if it is a plane as long as it can be specified by the prepit. The dimensions are preferably 1 μm or more in width and 1 μm or more in terms of the current laser beam spot size. However, the present invention is not limited to this, and any dimensions can be set as long as the probe DNA can be stored. You may set to such a dimension.
By providing both a servo laser beam and a reading laser beam as a laser beam for irradiating a DNA spot on a DNA microdisk or DNA microarray, the reading laser beam can be accurately positioned on the DNA spot. The phosphor can be excited efficiently and the sensitivity of fluorescence measurement is improved. Of course, even if there are deformations such as warping of the substrate on which the DNA microspot is placed, or if the placement of the DNA spot is misaligned, the servo can detect the exact position of the spot and the reading beam can accurately irradiate the spot. It was. Further, by modulating the reading beam at a high frequency, fading of the phosphor can be prevented, and a reading laser peak power larger than the conventional one can be irradiated.
In particular, when a microdisk substrate is used, the reading speed can be increased because the reading beam only needs to be scanned in a one-dimensional direction. For this reason, if the DNA spot is scanned only once, the reading is completed, and imaging by the conventional two-dimensional scanning becomes unnecessary.

It is a figure which shows schematic structure of the spotting apparatus which is one Example of this invention. It is a figure which shows schematic structure of the DNA microdisk of this invention. It is the address information and V mark enlarged view which expanded a part of DNA microdisk of this invention. It is a figure which shows the control part block diagram of the spotting apparatus which is one Example of this invention. It is a graph which shows the reflected light amount distribution change at the time of spotting. It is the schematic of the spotting apparatus which has the probe DNA discharge port which is a some spotting liquid. It is a side view of the schematic diagram showing a multi-spotting device. It is a top view of the schematic which shows a multi-spotting apparatus. It is a block diagram which shows the control part of a multi-spotting apparatus. It is the schematic which shows the tank of an inkjet and a spotting liquid. Gold provided on the thin gold film provided on DNA micro disk is a graph showing the calculation results of the electric field strength to the on SiO ₂ film. It is a conventional DNA microarray configuration diagram. Enlarged view of DNA microdisk pre-groove Probe DNA generator block diagram Probe DNA production device control block diagram Probe DNA production device waveform diagram Block diagram of reader Layout of read beam and servo beam on DNA microdisk

Explanation of symbols

1 DNA micro disk 2 Laser 3 Beam expander 4 Objective lens 4a Tracking actuator 4b Focusing actuator 5 Beam splitter 6 Lens 7 Photo detector 7a Photo detector A
7b Photodetector B
8 Inkjet 9 Inkjet outlet 10 Photodetector 10a Photodetector A
10b Photodetector B

11 Spotting liquid supply tube 12 Traverse unit A
12-1 Traverse unit B
12-2 Traverse unit C
13 Traverse motor A
13-1 Traverse Motor B
13-2 Traverse motor C
14 Disc motor 15 V mark detector

22 Central hole 23 Pregroove 24 First data recording section 25 Second data recording section 26 V mark 26a V mark pit a row 26b V mark pit b row 27 Pregroove address information 28 Radial arrow 29 Tangential arrow 30 Differential amplifier 1
31 Phase compensator 1
32 Drive amplifier 1
33 Differential Amplifier 2
34 Phase compensator 2
35 Drive amplifier 2
36 CPU
37 Adder

38 Adder output 39 Disk motor controller 41 FIG. 4 graph horizontal axis DNA microdisk rotation time lapse 42 reflected light amount (FIG. 4 graph vertical axis)
43 Spotting liquid discharge time point 44 Spotting liquid discharge time point 45 Reflected light quantity reduction period 51 Inkjet 52 Inkjet discharge port 53 Inkjet unit 54 Inkjet unit moving direction 55 Transfer gear 61 Disk motor transfer device 62 Transfer direction 71 Multi-inkjet rotary table 81 Rotary table rotation control Device 82 Phase compensator 3
83 Drive amplifier 3

90 Inkjet A
91 Tank 92 Connection seal 93 Connection hole 94 Discharge port 95 Pressurization chamber 96 Pressurization device 97 Liquid name display unit 100 FIG. 10 Horizontal axis: SiO ₂ film thickness [nm]
101 FIG. 10 Vertical axis: electric field intensity on substrate surface 102 characteristic at wavelength 563 nm 103 characteristic at wavelength 652 nm 104 characteristic at wavelength 532 nm 110 glass substrate 111 DNA spot array 112 pregroove 113 at address 1 address 1
114 Address 2 pregroove

115 Address 2
116 Radial direction arrow 117 Tangential direction arrow 118 DNA microdisk 120 Laser 121 Beam expander 122 Beam splitter 123 Objective lens 124 Tracking actuator 125 Focusing actuator 126 Lens 128 Photodetector 129 Traverse unit 130 Traverse motor 131 Disc motor 132 Turntable 140 Preamplifier 141 Decoder 142 CPU
143 Irradiation Pulse Control Unit 144 I / F Interface 145 PC Personal Computer

146 Laser power modulator 147 Servo unit 148 Reproducing light power 149 Peak power 152 Disc motor 153 Objective lens 153a X direction 153b Z direction 154 Objective lens actuator 154a Tracking element 154b Focusing element 155 Fluorescence excitation light source (output light wavelength λ1)
156 Collimator lens 1
157 Light source for servo (output light wavelength λ3)
158 Collimator lens 2
159 half mirror

160 Beam splitter 1
161 Condensing lens 1
162 Optical filter 163 Fluorescence reading detector 164 Beam splitter 2
165 Condensing lens 2
166 Servo error detector 167 Cylindrical lens 168 Diffraction grating 170 DNA spot reading beam 171 Pregroove or DNA spot row 172 Servo beam 173 DNA spot 174 Arrow indicating X direction

Claims (5)

The probe DNA produced on the substrate is scanned with a laser beam, and the normal and normal probe DNA produced on the substrate is compared with the reflectance and / or color of the probe DNA produced on the substrate. A method for discriminating DNA, comprising discriminating DNA. A plurality of probe DNAs of the same type are prepared on a microarray substrate, the probe DNA is scanned with a laser beam, and the reflectivity and / or color of the probe DNA that operates normally and the probe DNA are determined. Use of a DNA microarray that discriminates normal probe DNA by comparison, discriminates normal probe DNA from management data, discriminates normal probe DNA from the management data, and uses only normal probe DNA Method. Inspect the DNA prepared on the microarray substrate, select the address where the normal DNA exists, record the selected address information on the recordable part on the substrate on which the DNA is placed, and select A method for using a DNA microarray, wherein only the DNA present in the address storage section is used. After generating a hybridization reaction with the probe DNA prepared on the microarray substrate, when detecting the fluorescence contained in the generated DNA spot, the reading laser beam for detecting the fluorescence is modulated at a high frequency. A method of using a DNA microarray characterized by: The method for using a DNA microarray according to any one of claims 2 to 4, wherein the DNA prepared on the substrate of the microarray is prepared by the following method:
A track on a substrate capable of tracking a light beam is provided with a storage unit and an address for specifying the storage unit, nucleotides having a protecting group are arranged in at least a plurality of the storage units, and the predetermined storage unit is provided. The address indicating the storage unit is identified by reading with a light beam, and the stored nucleotide having the protective group is irradiated with the light beam, and the nucleotide having the protective group stored in the specific storage unit A method for producing a microarray comprising a step of removing a protecting group.

Images