JP2020000060A - Substrate for nucleic acid analysis, flow cell for nucleic acid analysis, and analysis method - Google Patents

Abstract

To arrange a high-density spot pattern, to which positioning can be performed, on a substrate in order to improve throughput of nucleic acid analysis.SOLUTION: A pattern arrangement for nucleic acid analysis and a substrate for nucleic acid analysis have the same hexagonal lattice pattern in which a spot region having a pattern of a plurality of spots on which formed biopolymers on the substrate are adhered is divided into a plurality of blocks, blocks A,AX,AY,AXY adjacent to each other are highly dense and a pattern of spots with different phases from each other.SELECTED DRAWING: Figure 13

Description

  The present invention relates to a nucleic acid analysis substrate, a flow cell, and an analysis method, and more particularly to a spot pattern arrangement for nucleic acid analysis for measuring a biological substance.

  2. Description of the Related Art In recent years, in a nucleic acid analyzer, a method has been proposed in which a flow cell formed of a glass substrate, a silicon substrate, or the like carries a large number of DNA fragments to be analyzed, and the base sequences of these numerous DNA fragments are determined in parallel. In this method, a substrate with a fluorescent dye corresponding to a base is introduced into an analysis region on a flow cell containing a large number of DNA fragments, and the flow cell is irradiated with excitation light to detect fluorescence emitted from each DNA fragment. Identify the base.

  Also, in order to analyze a large amount of DNA fragments, the analysis region is usually divided into a plurality of detection fields, and after each irradiation, the detection field is changed and analysis is performed in all the detection fields. , A new substrate with a fluorescent dye is introduced, and each detection field is analyzed by the same operation as described above. By repeating this, the base sequence can be determined efficiently (see Patent Document 1).

  Substrates used for such analysis are disclosed in Patent Literature 1 and Patent Literature 2. In Patent Document 1, spots to which sample DNA binds are arranged in a grid on a substrate. A silicon wafer is used for the substrate, and is made by a photolithography technique and an etching technique. After forming a hydrophobic HMDS (hexamethyldisilazane) layer on a silicon wafer, a positive resist is applied. After an opening is provided at a predetermined position of the resist by using a photolithography technique, HMDS at the bottom of the opening is removed by using oxygen plasma. Thereafter, the wafer is held in the aminosilane gas phase, and aminosilane is introduced into the bottom of the opening. After applying a resist on the wafer, the substrate is cut out by dicing. After the resist on the cut-out substrate is removed by ultrasonic cleaning using an organic solvent, a cover glass is attached via a polyurethane adhesive to prepare a flow cell for nucleic acid analysis. When aminosilane having high hydrophilicity and capable of immobilizing DNA is used for the DNA ball fixing spot, and hydrophobic HMDS which prevents DNA adsorption in other areas is used, when a solution containing a DNA ball is introduced onto a substrate, the DNA is spontaneously introduced. This allows the ball to be fixed only on the spot.

  In Patent Document 2, aminosilane was coated on a slide glass, activated with a divalent cross-linking reagent 1,4-diphenylen-diisothiocyanate, and then the 5 'end was aminated using a custom spotting apparatus. DNA oligomers are arranged in a lattice. Thereafter, the DNA oligomer is hybridized with the DNA sample to fix the DNA sample in a lattice. As described above, a technique for forming a substrate on which a spot for fixing a DNA fragment sample is arranged in an analysis region of a flow cell has been developed and put into practical use.

  In the above-described analysis, fluorescence emitted from a DNA sample fixed on a spot placed on a substrate is imaged, and a base is specified by image processing. For this purpose, it is necessary to accurately identify individual spots in a luminescence image which is a fluorescence image. In general, even between the fluorescence images obtained by imaging the same detection visual field, the positions where the images are captured are different on the flow cell due to the limit of the control accuracy of the driving device for changing the visual field. For this reason, a certain spot is imaged at a different coordinate position in each light emission image. Therefore, in order to accurately identify each spot, it is necessary to accurately determine the coordinate position of each spot on the flow chip.

  For such a purpose, there is a method of arranging and placing a reference marker for determining a position on the substrate on the substrate. However, in order to correct the deformation of the substrate and the lens distortion, it is necessary to arrange the reference markers at a high density, and their shapes need to be unique to determine the position, and these are detected. There is a problem that the load of image processing is large.

  Patent Document 3 discloses a technique in which a spot pattern itself has a code indicating position information. However, in the analysis according to the present invention, DNA is not always fixed to all spot patterns, and when a spot of a DNA sample is fluorescent in all luminescence images due to a combination of a base type and a fluorescent filter. Not necessarily. For this reason, the code pattern technology disclosed in Patent Document 3 cannot be applied to the above-described analysis substrate.

  As described above, there is a method of detecting the position of each spot of a captured image by image correlation matching with a reference image instead of detecting a reference marker or a code pattern indicating position information in an image. Here, the reference image is an image whose position information, which is the position coordinates of the spot on the image, is known and is generated from the design information of the spot. Alternatively, any one of a plurality of images captured in each detection field of view may be used as a reference image, and spots of other images may be associated with spots on a reference image.

  However, if the spot pattern has a lattice shape, the image cut out for matching looks like the image at any position as the same pattern, and it is difficult to detect the position by image correlation using only this pattern. In view of this, a technique of a spot pattern that enables alignment is disclosed. Patent Literature 4 discloses a method of performing alignment by image correlation using a pattern in which spots are pseudo-randomly deleted from a grid pattern. Patent Literature 5 discloses a technique in which a spot pattern is divided into blocks, and adjacent blocks have different rotation angles, thereby enabling alignment by image correlation.

US Patent Application Publication No. 2009/0270273 US Patent Application Publication No. 2009/0018024 U.S. Pat. No. 6,663,008 U.S. Pat. No. 8,774,494 U.S. Pat. No. 9,566,560

  However, when a pseudo-random deletion pattern shown in Patent Document 4 is used, a DNA pattern is not always fixed to each spot, and a random pattern similar to the pattern of the alignment target image appears around the spot. Therefore, there is a problem that the accuracy of the alignment is lowered.

  Further, in the spot pattern disclosed in Patent Document 5, since the same pattern having various angles appears in an image, there is a problem that detection accuracy is reduced when detecting an angle difference between two images to be aligned. .

  The present invention has been made in view of such circumstances, and provides a nucleic acid analysis substrate, a nucleic acid analysis flow cell, and an analysis method using a high-density spot pattern arrangement that enables alignment based on image correlation. The purpose is to do.

  In order to achieve the above object, according to the present invention, a substrate and a pattern of spots formed on a surface of the substrate and to which a biopolymer is attached are provided, and a region where the pattern of spots is formed is a plurality of blocks. And a first block and a second block adjacent to each other have the same spot pattern and a phase shift for nucleic acid analysis. Provide a substrate.

  In order to achieve the above object, according to the present invention, there is provided a substrate, and a spot pattern formed on the surface of the substrate and to which a biopolymer is attached, wherein a plurality of spot pattern regions are formed. , And one or more specific patterns of spots are formed on the block, and the first block and the second block adjacent to each other have different configurations or sizes of blocks for nucleic acid analysis. Provide a substrate.

  Furthermore, in order to achieve the above object, in the present invention, there is provided an analysis method, wherein a pattern of a spot where a biopolymer is attached to a surface of a substrate is formed, and a region where the pattern of the spot is formed is a plurality of blocks. The first block and the second block adjacent to each other have the same spot pattern and have a phase shift, and the blocks have a phase shift. Alignment processing is performed between one or more target images cut out from one or more positions of the light emission image obtained by capturing the light emission of one light source and one or more template images cut out from one or more positions of the reference image. Provide an analysis method.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to improve the throughput of a nucleic acid analysis by the nucleic acid analysis board | substrate which has arrange | positioned the spot which can identify a position at high density.

FIG. 2 is a diagram illustrating a schematic configuration example of a nucleic acid analyzer according to each embodiment. FIG. 4 is a diagram showing processing steps for decoding a base sequence of DNA according to each example. FIG. 4 is a diagram for explaining the concept of a detection visual field on a flow cell according to each embodiment. It is a figure which shows the concept of the bright spot of four types of fluorescent images in each detection visual field concerning each Example. FIG. 4 is a diagram showing a concept of determining a base sequence according to each example. FIG. 4 is a diagram illustrating a concept of positional displacement between cycles according to each embodiment. FIG. 7 is a diagram illustrating a concept of measuring the amount of displacement at a plurality of locations in an image according to each embodiment. FIG. 3 is a diagram for explaining an example of a configuration of a flow cell for nucleic acid analysis according to each embodiment. FIG. 3 is a diagram for explaining an example of a substrate for nucleic acid analysis according to each example. FIG. 7 is a diagram for explaining the concept of spot arrangement by a grid pattern according to each embodiment. FIG. 4 is a diagram illustrating a first example of a spot pattern of a basic block according to the first embodiment. FIG. 4 is a diagram illustrating an example of a middle block according to the first embodiment. FIG. 4 is a diagram illustrating an example of a spot pattern of a middle block 1 according to the first embodiment. FIG. 4 is a diagram illustrating an example of a spot pattern of a middle block 2 according to the first embodiment. FIG. 4 is a diagram illustrating an example of a spot pattern of a middle block 3 according to the first embodiment. FIG. 4 is a diagram illustrating an example of a spot pattern of a middle block 4 according to the first embodiment. FIG. 3 is a diagram illustrating an example of a large block according to the first embodiment. FIG. 4 is a diagram illustrating a first example in which basic blocks are adjacent to each other according to the first embodiment. FIG. 6 is a diagram illustrating a second example in which basic blocks are adjacent to each other according to the first embodiment. FIG. 6 is a diagram illustrating a third example in which basic blocks are adjacent to each other according to the first embodiment. FIG. 6 is a diagram illustrating a second example of a spot pattern of a basic block according to the first embodiment. FIG. 14 is a diagram illustrating a concept of dividing a spot area into different block shapes according to the second embodiment. FIG. 14 is a diagram illustrating a concept of dividing a spot area into different block shapes according to the second embodiment. FIG. 14 is a diagram illustrating an example of a spot pattern of a middle block according to the second embodiment. FIG. 14 is a diagram illustrating an example of a pattern arrangement of basic blocks according to the second embodiment. FIG. 13 is a diagram for explaining a case where basic blocks are adjacent to each other according to the second embodiment. FIG. 14 is a diagram illustrating an example of a pattern arrangement of basic blocks according to the second embodiment. FIG. 14 is a diagram illustrating an example of a middle block according to the second embodiment. FIG. 14 is a diagram illustrating an example of a large block according to the second embodiment. FIG. 14 is a diagram illustrating an example of a middle block according to the third embodiment. FIG. 14 is a diagram illustrating an example of a spot pattern of a middle block 1 according to a third embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the accompanying drawings, functionally the same elements may be represented by the same numbers. Although the attached drawings show specific embodiments and implementation examples in accordance with the principle of the present invention, these are for understanding of the present invention, and in order to limit the present invention in a limited manner. Not used. That is, it is to be understood that the description in the present specification is merely a typical example, and is not intended to limit the scope of the claims or the application examples in any way.

  Although the various embodiments described below are described in sufficient detail for those skilled in the art to practice the present invention, other implementations and forms are possible, and the scope and spirit of the technical idea of the present invention are not limited. It is necessary to understand that the configuration and structure can be changed and various elements can be replaced without departing from the scope. Therefore, the following description should not be construed as being limited thereto. In addition, the substrate for nucleic acid analysis according to various embodiments targets a DNA fragment for measurement and analysis, but may target RNA, protein, and the like in addition to DNA, and the present invention can be applied to all biologically related substances. It is.

  Hereinafter, various embodiments of the present invention will be sequentially described with reference to the drawings. First, a schematic configuration of a nucleic acid analyzer common to a plurality of embodiments, a decoding process of a base sequence of DNA, a flow cell configuration for nucleic acid analysis, a substrate for nucleic acid analysis The configuration and the like will be described.

(1) Nucleic Acid Analyzer FIG. 1 shows a schematic configuration example of a nucleic acid analyzer using the nucleic acid analysis substrate according to each embodiment. The nucleic acid analyzer 100 includes a flow cell 109, a liquid sending system, a transport system, a temperature control system, an optical system, and a computer 119. The flow cell 109 is provided with a substrate for nucleic acid analysis of each embodiment described later.

  The liquid supply system provides a means for supplying a reagent to the flow cell 109. The liquid sending system reacted with the DNA fragments as the means, a reagent storage unit 114 containing a plurality of reagent containers 113, a nozzle 111 for accessing the reagent containers 113, a pipe 112 for introducing the reagent into the flow cell 109, and a DNA fragment. A waste liquid container 116 for discharging waste liquid such as a reagent and a pipe 115 for introducing waste liquid into the waste liquid container 116 are provided.

  The transport system moves an analysis area 120 of a flow cell 109 described later to a predetermined position. The transfer system includes a stage 117 on which the flow cell 109 is placed, and a drive motor (not shown) for driving the stage. The stage 117 is movable in the X-axis and Y-axis directions orthogonal to each other in the same plane. The stage 117 can be moved in the Z-axis direction orthogonal to the XY plane by a drive motor different from the stage drive motor.

  The temperature control system adjusts the reaction temperature of the DNA fragment. The temperature control system is provided on a stage 117 and includes a temperature control substrate 118 that promotes a reaction between a DNA fragment to be analyzed and a reagent. The temperature control board 118 is realized by, for example, a Peltier device or the like.

  The optical system provides a means for irradiating the analysis region 120 of the flow cell 109 described later with excitation light and detecting fluorescence emitted from the DNA fragment. The optical system includes a light source 107, a condenser lens 110, an excitation filter 104, a dichroic mirror 105, a bandpass filter 103, an objective lens 108, an imaging lens 102, and a two-dimensional sensor 101. . The excitation filter 104, the dichroic mirror 105, and the band-pass filter 103 also called an absorption filter are included as a set in a filter cube 106. The bandpass filter 103 and the excitation filter 104 determine a wavelength region through which fluorescence having a specific wavelength passes.

  The flow of excitation light irradiation in the optical system will be described. Excitation light emitted from the light source 107 is collected by the condenser lens 110 and enters the filter cube 106. The incident excitation light passes through the excitation filter 104 only in a specific wavelength band. The transmitted light is reflected by the dichroic mirror 105 and condensed on the flow cell 109 by the objective lens 108.

  Next, the flow of fluorescence detection in the optical system will be described. The condensed excitation light excites the phosphor that excites in the specific wavelength band among the four types of phosphors incorporated in the DNA fragment fixed on the flow cell 109. Fluorescence emitted from the excited phosphor passes through the dichroic mirror 105, passes only through a specific wavelength band through the bandpass filter 103, and is imaged as a fluorescent spot on the two-dimensional sensor 101 by the imaging lens 02. I do.

  In the present embodiment, it is designed that only one type of phosphor is excited in a specific wavelength band, and it is assumed that four types of bases can be respectively identified by the type of this phosphor, as described later. Also, four sets of filter cubes 106 are prepared according to the wavelength bands of the irradiation light and the detection light so that these four types of phosphors can be sequentially detected, and these can be sequentially switched. The transmission characteristics of the excitation filter 104, the dichroic mirror 105, and the band-pass filter 103 in each filter cube 106 are designed so that each fluorescent substance can be detected with the highest sensitivity.

  The computer 119 includes a processor (CPU), a storage device (various memories such as a ROM and a RAM), an input device (a keyboard, a mouse, and the like), and an output device (a printer, a display, and the like), like an ordinary computer. Prepare. The computer controls the above-described liquid supply system, transport system, temperature control system, and optical system, analyzes the fluorescent image detected and generated by the two-dimensional sensor 101 of the optical system, and analyzes each DNA fragment. It functions as a control processing unit for identifying bases. However, the above-described control of the liquid feeding system, the transport system, the temperature control system, and the optical system, the image analysis, and the base identification do not necessarily need to be controlled and processed by one computer 119. It may be performed by a plurality of computers each functioning as a control unit or a processing unit for the purpose of time reduction or the like.

(2) Decoding Method of DNA Base Sequence A method of decoding a DNA base sequence will be described with reference to FIGS. As described later, it is assumed that reaction spots in which the same DNA fragment is amplified and densely arranged are arranged on the flow cell 109 in advance. Existing techniques such as the method for amplifying a circular DNA template shown in Patent Document 2 may be used for amplifying a DNA fragment.

  FIG. 2 is a diagram showing processing steps for decoding the base sequence of DNA. The entire run for decoding (S21) is performed by repeating the cycle processing (S22) M times. M is the length of the base sequence to be determined and is determined in advance. Each cycle process is a process for specifying the k-th (k = 1 to M) base, and is divided into a chemistry process (S23) and an imaging process (S24) described below.

(A) Chemistry treatment: treatment for extending a base In the chemistry treatment, the following procedures (i) and (ii) are performed.
(I) In the case of a cycle other than the first cycle, the fluorescently labeled nucleotide (described later) in the immediately preceding cycle is removed from the DNA fragment and washed. A reagent for this is introduced into the flow cell 109 via the pipe 112. The waste liquid after washing is discharged to a waste liquid container 116 via a pipe 115.
(Ii) The reagent containing the fluorescent-labeled nucleotide is flowed to the analysis area 120 on the flow cell 109 via the pipe 112. By adjusting the temperature of the flow cell with the temperature control substrate 118, an extension reaction is caused by the DNA polymerase, and a fluorescently labeled nucleotide complementary to the DNA fragment on the reaction spot is incorporated.

  Here, the fluorescence-labeled nucleotide is one in which four types of nucleotides (dCTP, dATP, dGTP, dTsTP) are labeled with four types of fluorophores (FAM, Cy3, Texas Red (TxR), Cy5), respectively. . Each fluorescently labeled nucleotide is denoted as FAM-dCTP, Cy3-dATP, TxR-dGTP, Cy5-dTsTP. Since these nucleotides are incorporated complementarily into the DNA fragment, if the base of the actual DNA fragment is A, dTsTP, if base C, dGTP, if base G, dCTP, if base T, dATP Are taken in respectively. That is, the phosphor FAM corresponds to the base G, Cy3 corresponds to the base T, TxR corresponds to the base C, and Cy5 corresponds to the base A. The 3 'end of each fluorescently labeled nucleotide is blocked so that it does not extend to the next base.

(B) Imaging Processing: Processing for Generating a Fluorescent Image The imaging processing (S24) is performed by repeating the imaging processing (S25) for each detection field described below N times. Here, N is the number of detection fields.

  FIG. 3 is a diagram for explaining the concept of the detection visual field. The detection visual field 121 corresponds to each area when the entire analysis area 120 is divided into N pieces. The size of the detection visual field 121 is the size of the area that can be detected by the two-dimensional sensor 101 by one fluorescence detection, and is determined by the design of the optical system. As will be described later, a fluorescence image corresponding to four types of phosphors is generated for each detection visual field 121.

(B-1) Imaging processing for each detection visual field In the detection visual field imaging processing (S25), the following procedures (i) to (iv) are performed.
(I) The stage 117 is moved so that the detection visual field 121 for performing fluorescence detection comes to a position where the excitation light from the objective lens 108 is irradiated (S26).
(Ii) The filter cube 106 is switched to a set corresponding to the phosphor (FAM) (S27).
(Iii) A fluorescent image is generated by irradiating the two-dimensional sensor 101 with the excitation light.
(Iv) Perform steps (ii) and (iii) for other types of phosphors (Cy3, TxR, Cy5).

  By executing the above-described processing, fluorescence images for four types of phosphors (FAM, Cy3, TxR, Cy5) are generated for each detection visual field. In this fluorescent image, the signal of the fluorescent substance corresponding to the type of base of the DNA fragment fixed to each spot appears on the image as a luminescent spot. That is, the spot detected in the fluorescent image of FAM is base A, the spot detected in the fluorescent image of Cy3 is base C, the spot detected in the fluorescent image of TxR is the base T, and the spot detected in the fluorescent image of Cy5. Is determined to be base G.

  FIG. 4 is a diagram illustrating the concept of spots of four types of fluorescent images in each detection visual field. As shown in FIG. 4A, for example, spots are present at eight positions P1 to P8 in a certain detection visual field in a certain cycle, and each base is A, G, C, T, A, C, T, G. At this time, the fluorescence images for the four types of phosphors (Cy5, Cy3, FAM, TxR) are, as shown in (b) to (e), at positions P1 to P8 according to the corresponding base type. A spot is detected. The positions of P1 to P8 are the same in the four fluorescence images. However, depending on the design of the optical system, there is a difference in the optical path for each wavelength, so it may not be exactly the same. For this reason, the spot positions of the four types of fluorescent images can be made the same by performing the alignment process described later as necessary. However, when there is crosstalk in which the wavelength characteristics of the filters of the respective phosphors overlap each other, spots of a certain base type are observed in two or more fluorescent images. The base type at this time can be identified by the ratio of the signal intensities of the four types of fluorescent images. As described above, the base type of each spot detected in the detection visual field is determined.

(C) Repetition of Cycle Processing By repeating the above-described cycle processing for the number of desired base sequence lengths M, a base sequence of length M can be determined for each spot.

  FIG. 5 is a diagram showing the concept of this base sequence determination. As shown in FIG. 5, when an individual spot (a DNA fragment having the base sequence ACGTATACGT...) Is extended by one base by chemistry treatment in a certain cycle (#N), for example, Cy3-dATP is incorporated. This fluorescently labeled nucleotide is detected as a spot on the fluorescent image of Cy3 in the imaging process. Similarly, in the cycle (# N + 1), it is detected as a spot on the fluorescence image of Cy5. In the cycle (# N + 2), it is detected as a spot on the TxR fluorescent image. In the cycle (# N + 3), it is detected as a spot on the FAM fluorescence image. By the cycle processing from cycle #N to cycle # N + 3, the base sequence at this spot is determined to be TACG.

(3) Alignment of spots As described above, the DNA fragment to be detected is imaged in a state where it is fixed to the spots arranged on the substrate on the flow cell 109, and each spot has four fluorescent images in each cycle. Observed as upper bright spot. As described above, the nucleic acid analyzer 100 repeatedly images the same detection visual field in each cycle. However, in each cycle, the stage 117 is moved to change the detection visual field, and an image is taken. Therefore, for the same detection visual field, a displacement occurs due to the movement of the stage between different cycles. This displacement is caused by a control error of the stage 117.

  FIG. 6 is a diagram illustrating the concept of positional displacement between cycles. As shown in FIG. 6, the imaging position may be shifted due to a stage control error between the Nth cycle in (a) and the (N + 1) th cycle in (b) with respect to a certain detection visual field. For this reason, the DNA fragment positions (P1 to P8) in the luminescence image which is the N-cycle fluorescence image are detected as different positions (P1 'to P8', respectively) on the (N + 1) -th luminescence image. However, these bright spots are all caused by the same DNA fragment. Therefore, in order to determine the base sequence of each spot, it is necessary to correct the positional shift between the spots detected in each luminescence image.

  In order to correct this positional shift, it is necessary to align each light emission image with respect to a common reference image. Here, the reference image is a common image used for a spot position coordinate system. For example, if a spot position is known as design information, a reference image may be created from a known spot position. As an example, an image having a luminance according to a two-dimensional Gaussian distribution of a predefined variance according to the spot size with the spot position (x, y) as the center may be created. Alternatively, a reference image may be created based on one of the captured real images. As an example, the image of each detection field of view in the first cycle may be used as a reference image, and the image of each detection field of view after the second cycle may be aligned with this reference image.

  A known matching technique can be applied to the alignment between the images. As an example, an image obtained by cutting out a part of the reference image is used as a template image t (x, y), and a cross-correlation function m (u, v) with a target image f (x, y) obtained by cutting out a part of the input image is used. Is obtained, and S_1 = (u, v) which gives the maximum value thereof is set as the displacement amount. Here, an example of t (x, y) is an image of 256 pixels × 256 pixels at the center of the reference image. Similarly, an example of f (x, y) is an image of 256 pixels × 256 pixels at the center of the input image. Instead of the cross-correlation function, a normalized cross-correlation taking into account the difference in brightness may be used for calculating the amount of displacement, or a correlation limited to a phase may be used. When detecting the amount of angular deviation between images, the above cross-correlation and phase-only correlation can be applied to an image whose angle direction has been converted to a horizontal direction by performing polar coordinate conversion on the image.

  In addition, this position shift amount may be obtained at a plurality of points according to the degree of image distortion. This concept is shown in FIG. For example, if there is no distortion in the image and it is possible to assume only the same positional deviation for all pixels, that is, only a uniform deviation due to the stage, the positional deviation amount S_1 (u, u, shown on the left side of FIG. v) can be applied.

On the other hand, for example, when the image is distorted and the amount of displacement differs depending on the position in the image (when the flow cell 109 is deformed by heating and the displacement is not uniform), the right side of FIG. As shown in the figure, the amount of displacement is obtained at n points in the image, and the amount of displacement S_1, S_2,... S_n at these points is obtained. To calculate the amount of displacement at each point, an image centered on the position of each point in the reference image and the input image is cut out and used as a template image and a target image. Can be calculated. Then, based on the obtained n displacement amounts, for example, a coefficient of affine transformation or polynomial transformation is obtained by a least squares method, so that the displacement amount of an arbitrary pixel position can be formalized ((FIG. 7) b))
(4) Flow Cell and Nucleic Acid Analysis Substrate (A) Nucleic Acid Analysis Flow Cell FIG. 8 shows a nucleic acid analysis flow cell 109 used in each embodiment. The flow cell 109 has a nucleic acid analysis substrate 801, a hollow sheet 802 having a hollow section 803 with a hollow center, and a cover glass 802. A hollow portion surrounded by the hollow portion 803, the nucleic acid analysis substrate 801 and the cover glass 804 serves as a flow path. The inlet 807 and the outlet 808 serve as a liquid inlet / outlet. In the figure, the holes in the nucleic acid analysis substrate 801 are the inlet 807 and the outlet 808, but in another embodiment, the holes in the cover glass 804 may be the inlet and the outlet. Further, as still another form, holes formed in the side surface of the hollow sheet 802 may serve as an inlet and an outlet.

(B) Substrate for Nucleic Acid Analysis A plurality of spots 806 for immobilizing DNA fragments in the analysis region 805 are formed on the substrate 801 for nucleic acid analysis. For forming the spot on the substrate, a known technique disclosed in Patent Literature 1 or Patent Literature 2 may be used. As an example, as shown in FIG. 9, by providing a blocking film in a region other than the spot, the fixing ratio of the DNA sample to the spot may be increased. In FIG. 9, a spot 902 is formed on a substrate 901, a coating film 904 containing an amino group is formed on the spot 902, and a blocking layer 903 is coated on a region where the spot 902 is not present. By bringing the solution containing the DNA sample into contact with the substrate, the DNA sample can be fixed on the spot at a high density.

  The material used for the substrate 901 is not particularly limited, and inorganic materials such as silicon, glass, quartz, sapphire, ceramic, ferrite, alumina, and diamond; or metal materials such as aluminum, SUS, titanium, and iron; Epoxy resin, melamine resin, unsaturated polyester resin, alkyd resin, polyurethane, thermosetting polyimide, transparent polyimide, polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, ABS resin, AS resin, acrylic resin Polyamide, nylon, polyacetal, polycarbonate, modified polyphenylene ether, polybutylene terephthalate, polyethylene terephthalate, glass fiber reinforced polyethylene terephthalate, cyclic polyolefin, Li polyphenylene sulfide, polysulfone, polyether sulfone, amorphous polyarylate, liquid crystal polymer, polyether ether ketone, it is also possible to use a resin material and the mixed material and glass fiber or carbon fiber reinforced inorganic material of such. Among these materials, when a DNA sample is analyzed by fluorescence or when the temperature is raised or lowered during the analysis, silicon having low autofluorescence, a low coefficient of thermal expansion, and high resistance of the analysis solution is used. Glass, quartz, SUS, titanium and the like are particularly desirable.

  The spot 902 is formed using a spotting device as shown in Patent Document 2 or a known technique such as wrist-off. As a material to be used, a material which can be formed on a substrate through a covalent bond is preferable. Examples of such materials include inorganic materials such as silicon, glass, quartz, sapphire, ceramic, ferrite, and alumina having an oxide film on the substrate surface, and metal materials such as aluminum, SUS, titanium, and iron. Coupling materials are preferred. Also, among the silane coupling materials, those having a highly reactive functional group capable of forming a coating film containing an amino group via a covalent bond are preferable. Examples of such a functional group include a vinyl group and an epoxy group. Ethoxysilane and methoxysilane having a group, styryl group, methacryl group, acrylic group, amino group, ureide group, isocyanate group, isocyanurate group and mercapto group in the molecule.

  When a plurality of types of DNA samples are fixed to one spot 902, fluorescent dyes from the plurality of types of DNA samples are detected, so that bases cannot be correctly identified. On the other hand, if the size of the spot 902 is small, the probability of contact with the DNA sample in the solution decreases, the number of spots on which the DNA sample is not fixed increases, and the throughput of base sequence determination decreases. Therefore, the diameter of the spot 902 is preferably larger than half of the DNA sample so that only one DNA sample can be fixed at a high fixing rate. Such size depends on the size of the DNA sample to be amplified. Generally, the size of a DNA sample is as small as less than 50 nm, but the size of an amplified DNA as described in Patent Documents 1 and 2 is as large as 50 nm or more. Therefore, when such a DNA sample is used, the size of the spot 902 is desirably about 50 nm or more and less than 1000 nm.

  It is desirable that the pitch, which is the distance between the centers of the spots, is slightly larger than at least the size of the DNA sample used. If the pitch is smaller than the size of the DNA sample, it will be fixed over a plurality of spots, and there will be a problem that the base cannot be identified earlier. On the other hand, if the pitch is too large, the density of the DNA sample to be fixed decreases, and the number of DNA samples that can be analyzed at once decreases. Therefore, the pitch is desirably 50 nm or more and less than 1000 nm, like the spot size.

  The blocking layer 903 is preferably a layer that can prevent the DNA sample from adsorbing to the substrate 901, increase the accessibility of the DNA sample to the substrate, and increase the rate of fixing the DNA sample to the spot. Known examples of such a material include a silane coupling material whose terminal is treated with polyethylene glycol or a carboxylic acid.

  The coating film 904 containing an amino group increases the contact ratio between the DNA sample and the functional group on the spot when the DNA sample is fixed, and prevents the DNA sample from peeling off during analysis, so a material having a high amino group density on the surface is used. It is hopeful. As such a coating film, a method of reacting a functional group introduced on the spot with a polyamine having a plurality of amino groups in one molecule is preferable. Examples of such a polyamine include spermidine, putrescine, spermine, etc., and a dendritic polymer is particularly preferable in that it has many amino groups in the molecule. Examples of such dendritic polymers include polyamidoamine dendrimers. Polyamidoamine dendrimers consist of an alkyldiamine core and a tertiary amine branched structure. The core and molecule include ethylenediamine, 1,12-diamidodecane, 1,4-diaminobutane, cystamine, 1,6-diaminohexane and the like. Dendritic molecules can exponentially increase the number of functional groups in the molecule by increasing the number of generations surrounding the core molecule. In the present invention, by using dendritic molecules of the first generation or later, the amino group density on the coating surface can be greatly increased.

  It is preferable that these polyamines form a covalent bond with the functional group on the spot, because the polyamine can prevent peeling at the interface between the spot and the coating film. As a material for forming such a spot, a material having a functional group capable of reacting with an amino group in a molecule is desirable, and a vinyl group, an epoxy group, a methacryl group, an acryl group, an isothiocyanurate group, an isocyanate group, an isocyanurate Examples thereof include silane coupling materials having a group and the like, alkenes, alkynes, and the like. Alternatively, after introducing a reactive functional group on the spot surface, an amine-reactive functional group may be introduced on the spot using a polyvalent crosslinking reagent. Such amine-reactive functional groups include isothiocyanurate group, isocyanate group, isocyanurate group, sulfonyl chloride group, aldehyde group, carbodiimide group, acrylazide group, fluorobenzene group, carbonate group, N-hydroxysuccinimide ester Groups, imide ester groups, epoxy groups, fluorophenyl ester groups, acid anhydrides and the like.

(5) Pattern Arrangement for Nucleic Acid Analysis As described above, as the number of spots on the substrate for nucleic acid analysis increases, the number of DNA fragments whose base sequence can be determined increases, so that the measurement throughput of analysis improves. However, in order to identify the base type of each spot, there are restrictions on the size of the spots and the minimum pitch between the spots. As a pattern for arranging spots at high density, a square lattice pattern 1001 shown in FIG. 10A and a hexagonal lattice pattern 1002 shown in FIG. However, since these grid patterns are periodic, the spot patterns have no uniqueness. That is, as shown in FIG. 10C, a spot pattern image 1003 obtained by cutting out a region at a certain position of a spot pattern and a spot pattern image 1004 at another region are very similar. If the spot patterns do not have uniqueness in this way, the spot pattern image at one position may be erroneously detected as a spot pattern image at another position in the above-described image registration, and thus the position shift amount may be accurately determined. Cannot be calculated.

  Hereinafter, Examples 1-3 of the substrate for nucleic acid analysis using a high-density spot pattern arrangement that enables alignment based on image correlation will be described.

  In the first embodiment, a substrate and a spot pattern to which a biopolymer is attached are formed on the surface of the substrate, an area where the spot pattern is formed is divided into a plurality of blocks, and each block has a specific pattern of spots. Is formed, and the first block and the second block adjacent to each other have the same spot pattern and a phase shift in the nucleic acid analysis substrate and the analysis method using the same. is there. That is, in the substrate for nucleic acid analysis and the pattern arrangement for nucleic acid analysis according to the present embodiment, the spot pattern region in which the DNA fragments are fixed is divided into blocks having a lattice pattern, and adjacent blocks are separated from each other by the phase of the lattice pattern. Are shifted from each other.

  In the first embodiment, a spot pattern to which a biopolymer is attached is formed on the surface of the substrate, and a region where the spot pattern is formed is divided into a plurality of blocks. The first block and the second block that are formed and adjacent to each other have the same spot pattern and a phase shift, and are located at one or more positions of a luminescence image obtained by capturing luminescence from the surface of the substrate. 7 is an embodiment of an analysis method for performing a registration process between one or more target images cut out and one or more template images cut out from one or more positions of a reference image. The template image and the target image include at least spot patterns of four or more blocks adjacent to each other.

  FIG. 11 shows an example of a pattern of four basic blocks whose phases are different from each other by half a phase based on a hexagonal lattice pattern. The figure shows a pattern AX shifted by half a phase to the right with respect to the pattern A, a pattern AY shifted by a half phase downward, and a pattern AXY shifted by a half phase downward and right. Note that, in the figure, in order to visualize the phase shift, grid lines in one phase unit (the ratio of width to length is 2: √3) are shown, but this grid line does not exist on the actual substrate. . The same applies to the following drawings. Hereinafter, an example will be described in which a combination of these four basic blocks A, AX, AY, and AXY forms a highly unique spot pattern that enables image alignment. Note that the phase shift is not limited to the above-described half phase, that is, 0.5, but is preferably 0.5 ± 0.3. This is for the same reason as the above-mentioned range of the size of the pitch and the spot size.

  FIG. 12 shows an example of middle blocks 1, 2, 3, and 4 composed of four basic blocks and formed by four combinations. FIGS. 13 to 16 show the respective spot patterns of the middle blocks 1-4. As shown in FIGS. 13 to 16, the phases of adjacent middle blocks are different, and the distance between spots in contact with the block boundaries is always equal to or greater than the spot pitch in the hexagonal lattice pattern. Further, from these figures, the same applies to the boundary where the middle blocks are in contact with each other.

  FIG. 17 shows a configuration example of a large block in which the middle blocks 1-4 are arranged. The large block 1005 shown in FIG. The arrangement order of the middle blocks 1-4 is generated by pseudo random numbers. The size of the large block is not limited to the 16 × 16 medium block, and may be adjusted to one imaging field of view. By repeating the above-described large block 1005 in the vertical and horizontal directions, the spot area on the substrate is obtained. A spot pattern may be arranged on the spot.

  Note that the arrangement order of the middle blocks may be locally similar, for example, as indicated by a gray portion in FIG. In order to reduce the risk of alignment failure due to such a similar pattern, the size of the template image and the target image for alignment at the time of image alignment should be at least a side larger than the middle block, and at least It is desirable that patterns of four or more basic blocks A, AX, AY, and AXY are included. Further, it is desirable that many middle blocks 1-4 are included in the image. That is, it is desirable that the size of the basic block and the size of the middle block be determined so as to increase the uniqueness as much as possible in accordance with the size of the image cut out when performing image alignment.

  In addition, in the combination of the patterns of the basic blocks and the combination of the middle blocks, it is necessary to consider that the distance between the spots in contact with the block boundaries is longer than the spot interval of the original grid pattern. FIG. 18 shows a spot pattern in the case where the basic block AXY and the basic block AY in FIG. From the figure, the spots on both sides of the block boundary are smaller than the spot pitch in the hexagonal lattice pattern. In this embodiment, in order to avoid such a combination, in the middle blocks 1 to 4 shown in FIG. 12, AXY and AY are arranged diagonally.

  In the middle blocks 1-4 shown in FIG. 12, no matter which middle blocks are in contact with each other, the phases of adjacent blocks are considered to be different. That is, the pattern arrangement is such that the same basic block does not touch.

  However, even when the same basic blocks are adjacent to each other, it is possible to allow a pattern in which the same basic blocks are adjacent to each other by correcting the spot arrangement on the block boundary. FIG. 19 shows a boundary pattern when the same basic blocks are adjacent in the horizontal direction. FIG. 20 shows a boundary pattern when the same basic block is adjacent in the vertical direction. In FIGS. 19 and 20, the gray spot 1006 does not exist on the original basic block, but a gap between the spots occurs on the boundary by making the basic blocks adjacent to each other. In such a case, such a gray spot 1006 may be arranged on the boundary.

  Considering these facts, it is also possible to configure a spot pattern with higher uniqueness by further increasing the number of combinations of the middle blocks 1-4 shown in FIG.

  In FIG. 19, the basic block patterns A, AX, AY, and AXY based on the hexagonal lattice pattern are shown, but other patterns can be used as a base. For example, a spot pattern with high uniqueness can be generated by the same method using basic block patterns A, AX, AY, and AXY based on a square lattice pattern as shown in FIG. The treatment of the spot near the block boundary described above may be defined each time according to the pattern of the basic block.

  As described above, in the pattern arrangement for nucleic acid analysis and the substrate for nucleic acid analysis according to the first embodiment, the spot pattern area on the substrate is basically composed of blocks whose phases are shifted based on a high-density spot pattern. Since the spots are arranged in the basic block pattern as a unit, a high-density spot arrangement and the uniqueness of the spot pattern enabling image alignment can be ensured, so that an improvement in analysis throughput can be realized.

  In the second embodiment, a pattern of a spot on which a biopolymer is attached is formed on a substrate and a surface of the substrate, an area where the pattern of the spot is formed is divided into a plurality of blocks, and each block includes one or more specific areas. This is an embodiment of the substrate for nucleic acid analysis in which the first block and the second block adjacent to each other are formed with spots having the following patterns and the shapes or sizes of the blocks are different. That is, the second embodiment is an embodiment of the spot pattern arrangement for further improving the uniqueness of the pattern as compared with the first embodiment. In the preferred substrate for nucleic acid analysis and the pattern arrangement for nucleic acid analysis according to the present embodiment, the spot pattern region where the DNA fragment is fixed is divided into block regions having one or more lattice patterns, and adjacent blocks are separated from each other. , The grid patterns are different, and the shapes or sizes of the blocks are different. The configuration of the nucleic acid analyzer, the flow cell, the substrate and the like other than the nucleic acid analysis pattern arrangement are as described above, and the description is omitted here.

(1) Pattern Arrangement for Nucleic Acid Analysis In this embodiment, a square lattice pattern 1001 shown in FIG. 10A and a hexagonal lattice pattern 1002 shown in FIG. However, the present invention is not limited to these lattice patterns, and other geometric patterns may be used.

  FIGS. 22 and 23 show examples in which the spot pattern area is divided into different block shapes and different block sizes. FIG. 22 shows an example in which the spot pattern area 2001 is divided into complicated block shapes. FIG. 23 shows an example in which the spot pattern area 2002 is divided into square middle blocks, and each middle block is further divided into four rectangular basic blocks having different shapes. 22 can be expected to have higher pattern uniqueness in the case of FIG. 22 having a complicated block shape, but the design becomes complicated due to consideration of spot patterns having various block shapes. In practice, it has been confirmed that there is no problem in alignment even with the divided pattern of FIG. Hereinafter, an example of the division pattern shown in FIG. 23 will be described. The middle block of the spot pattern area 2002 in FIG. 23 is not necessarily limited to a square, but may be a rectangle according to the spot pattern.

  It is assumed that the rectangular basic block has a spot pattern of either a square lattice ((a) in FIG. 10) or a hexagonal lattice ((b) in FIG. 10). In this embodiment, in order to make adjacent blocks have different lattice patterns, four basic blocks in each square in FIG. 23 are configured so that diagonal lines have the same lattice pattern. FIG. 24 shows an example of this pattern configuration. In the figure, in the middle block 2003, the upper left and lower right basic blocks are a hexagonal lattice pattern, and the upper right and lower left basic blocks are a square lattice pattern. However, in FIG. 24, since the spot pattern at the block boundary is not considered, unnecessary voids are formed near the boundary, and there is much room for improvement from the viewpoint of increasing the density of spots. Further, depending on the spot pattern, the distance between the spots over the boundary may be reduced, and the accuracy of base identification may decrease.

  Therefore, it is desirable that the spot pattern in each block is separated from the block boundary by at least half of the minimum pitch D of the spot. Further, in order to reduce unnecessary voids, spots across the boundaries may be added according to the combination of the block boundaries.

  FIG. 25 shows two grid patterns separated from the block boundary by at least half the minimum pitch D of the spots. FIGS. 7A and 7B show a square lattice pattern 2004 and a hexagonal lattice pattern 2005, respectively. In these grid patterns, as shown in (c) of the figure, the center of the spot is arranged in a spot area separated from the reference block boundary 2006 by 以上 or more of the minimum pitch D.

  FIG. 26 shows a combination in which the basic block of the square lattice and the basic block of the hexagonal lattice shown in FIG. 25 are in contact with each other. FIG. 14A shows a case where a square lattice block 2004 and a hexagonal lattice block 2005 are vertically in contact with each other. (B) of the same figure is a case where these touch right and left. (C) of the figure shows a case where the upper right and lower left square lattice basic blocks are in contact with each other. FIG. 6D shows a case where the upper left and lower right hexagonal lattice basic blocks are in contact with each other. In any case, the distance between the spots across the block boundary does not become smaller than the minimum pitch D. In addition, in the cases (a), (b), and (c) of the same figure, there is no extra space near the block boundary. However, in (d) of the figure, the spot positions shown in gray are voids. Therefore, in the combination of the patterns shown in (d) of the drawing, the number of spots may be increased by adding gray spots 2007 that extend over the block boundaries. In addition, although there are cases in which the upper and lower sides are reversed in (a) of FIG. 7 and cases where the left and right are reversed in (b) of FIG. Also, whether or not a gap is generated in the combination shown in FIG. 26 depends on the block shape, pattern, and the size of the spot area (distance from the block boundary). May be defined.

  FIG. 27 shows an example of the spot pattern of the middle block 2008 formed by the basic blocks in consideration of the block boundaries shown in FIGS. 25 and 26 described above. The gap is smaller than in FIG. 24, and the number of spots is increased. By changing the vertical and horizontal sizes of the four basic blocks as shown in FIG. 27 in various ways, a highly unique spot pattern can be realized.

  An example of configuring such a spot pattern will be described with reference to FIGS. In (a) of FIG. 28, the middle block is divided into 2: 3 in the horizontal direction, the left side is divided into 4: 1, and the basic blocks are divided into 4: 1, and the right side is divided into 2: 3, respectively. Blocks C and D are used. In (b) of FIG. 28, the middle block is divided into 1: 4 in the horizontal direction, the left side is divided into 3: 2 in the vertical direction, and the basic blocks are respectively E and F, and the right side is divided into 1: 4 to form the basic blocks. Blocks G and H are used. In FIG. 28C, a middle block 1-8 is defined by a combination of 1 to 8 by rearranging the basic blocks A, B, C, and D. Similarly, by rearranging the basic blocks E, F, G, and H, a middle block 9-16 is defined by a combination of 9 to 16. In each of the middle blocks 1-16, as described in the example of FIG. 24, a hexagonal lattice pattern is arranged in the upper left and lower right basic blocks, and a square lattice pattern is arranged in the upper right and lower left basic blocks. ing. As described above, 16 types of block patterns which are divided so that the adjacent blocks have different lattice shapes and block shapes are created.

  FIG. 29 shows that a certain area 2009 in the spot area of the nucleic acid analysis substrate is a large block composed of 16 × 16 middle blocks, and pseudo random numbers from 1 to 16 are generated to arrange the middle blocks 1-16. This is an example determined. Thereby, since each block pattern is arranged irregularly, a spot pattern with high uniqueness can be configured.

  The size of the large block in FIG. 29 is not limited to the 16 × 16 middle block, and may be adjusted to one imaging field of view. By repeating the large block in the vertical and horizontal directions, A spot pattern may be arranged in a spot area on the substrate. Although 16 types of block patterns are created in the example shown in FIG. 28, a combination pattern having more shapes may be created. Also, in FIG. 29, the same numerical value is continuously observed in the vertical and horizontal directions. In such a place, there is a risk that the accuracy of the alignment in the continuous direction may be reduced. Therefore, if necessary, the numerical values may be replaced so that the same numerical value does not continue.

  Also, at least four or more basic blocks are included in the cut-out image for calculating the image correlation, that is, in the template image and the alignment target image, by making these cut-out image sizes larger than the middle block. Is desirable. Further, it is desirable that many middle blocks are included in the image. As a result, uniqueness is increased in the cut-out image due to the difference between the lattice pattern and its size or shape. That is, as in the first embodiment, it is desirable that the size of the block is determined so as to increase the uniqueness as much as possible according to the size of the image cut out when performing image alignment.

  In this embodiment, a unique spot pattern can be generated by combining the same patterns. However, since the pattern is the same except at the block boundary, the similarity as the pattern is high, and there is a risk that the accuracy of the positioning is reduced. For this reason, when the same pattern is used, it is desirable that the block size is made smaller than the image size cut out at the time of aligning the images so that many block boundaries are included.

  As described above, in the pattern arrangement for nucleic acid analysis and the substrate for nucleic acid analysis according to Example 2, the spot pattern area on the substrate is divided into blocks having different shapes and sizes based on one or more spot patterns. Since the spots are arranged in a pattern in which is a basic unit, compared to the first embodiment, the uniqueness of the spot pattern is further ensured, so that the accuracy of image alignment is improved and the accuracy of base identification can be improved. become.

  The third embodiment is an example of a spot pattern arrangement for further increasing the density and the uniqueness of the pattern by combining the configurations of the first and second embodiments. That is, a substrate and a pattern of spots formed on the surface of the substrate and to which a biopolymer is attached are provided. The area where the spot pattern is formed is divided into a plurality of blocks, and each block has a specific pattern. The first block and the second block adjacent to each other have the same spot pattern, are out of phase, and have different block shapes or sizes. This is an example.

  In the pattern arrangement for nucleic acid analysis according to the present embodiment, and the substrate for nucleic acid analysis, and in the spot pattern arrangement, the spot pattern area where the DNA fragment is fixed is divided into block areas having one or more lattice patterns, and Adjacent blocks have a pattern based on one lattice pattern and different phases from each other, and have different shapes or sizes of blocks. The configuration of the nucleic acid analyzer, the flow cell, the substrate, and the like other than the arrangement of the pattern for nucleic acid analysis are the same as those of the first and second embodiments, and thus the description thereof is omitted.

(1) Pattern arrangement for nucleic acid analysis In the hexagonal lattice pattern 2005 and the square lattice pattern 2004 used in Example 2, when spots are arranged at the same pitch, the former can arrange spots with higher density. .

  Therefore, in this embodiment, basic blocks having different shapes including four patterns A, AX, AY, and AXY having different phases based on the hexagonal lattice pattern shown in FIG. 11 are formed. Then, similarly to the second embodiment, the middle block is configured by a combination of basic blocks having different shapes. FIG. 30 shows an example of the middle block 1-16. In FIG. 28, as shown in FIG. 28, the upper left basic block is defined as pattern A, the upper right basic block is defined as pattern AXY, the lower left basic block is defined as pattern AY, and the lower right basic block is defined as AX. . By fixing the pattern position in the middle block in this way, even when the middle blocks are adjacent to each other, unnecessary gaps are prevented from being generated at the block boundaries, and the distance between spots across the block boundaries is reduced by the hexagonal lattice pattern. Can be avoided. However, the definition of the basic block in the middle block is not limited to that shown in FIG. 30, and it is also possible to increase the types of the middle block by a combination of patterns and shapes of the basic blocks.

  The basic block in this embodiment is different from the basic block in the first embodiment, but can be created by arranging the basic blocks shown in FIG. As described with reference to FIGS. 19 and 20, a pattern in which a spot of a gap is newly added to a place where a gap occurs may be defined as a basic block again.

  FIG. 31 shows an example of the spot pattern of the middle block 1 in FIG. In the spot pattern shown in FIG. 31, the blocks indicated by thick frames are the basic blocks having different shapes in the present embodiment. The method of forming a large block using the 16 types of middle blocks 1-16 generated as described above is the same as in the second embodiment, and a description thereof will be omitted.

  As described above, in the pattern arrangement for nucleic acid analysis and the substrate for nucleic acid analysis according to Example 3, the spot pattern area on the substrate is further different in shape and size based on one high-density spot pattern. Since the spots are arranged in a pattern using blocks as a basic unit, the uniqueness of the spot pattern is further secured as compared with the first embodiment, the accuracy of image alignment is improved, and the accuracy of base identification can be improved. . In addition, since the spot density is higher than that in Example 2, it is possible to realize an improvement in the throughput of base sequence analysis.

  Using the nucleic acid analysis flow cell or nucleic acid analyzer of the present invention described above, various nucleic acid reactions can be detected, and nucleic acid analysis such as DNA sequencing can be performed. The present invention is not limited to the embodiments described above, and includes various modifications. For example, the above-described embodiments have been described in detail for a better understanding of the present invention, and are not necessarily limited to those having all the configurations described. Although the nucleic acid analysis substrate of each of the above-described embodiments is used for measuring and analyzing DNA fragments, it may be used for other bio-related substances such as RNA and protein in addition to DNA.

  Furthermore, although each of the above-described configurations, functions, computers, and the like has been described mainly with respect to an example in which a program that realizes some or all of them is created, a part or all of the hardware is designed by, for example, designing an integrated circuit. Needless to say, it may be realized by hardware. That is, all or a part of the functions of the processing unit may be realized by an integrated circuit such as an ASIC (Application Specific Integrated Circuit) or an FPGA (Field Programmable Gate Array) instead of a program.

  In the present specification described above, various inventions are described in addition to the inventions described in the claims. Some of them are listed below.

<List 1>
An analysis method for analyzing spots on a substrate,
A pattern of spots to which biopolymers are attached is formed on the surface of the substrate,
The area where the spot pattern is formed is divided into a plurality of blocks,
In the block, spots of a specific pattern are formed,
The first block and the second block adjacent to each other have different shapes or sizes of the blocks,
A light emission image obtained by capturing light emission from the surface of the substrate is input,
Performing alignment processing between one or more target images cut out from one or more positions of the light-emitting image and one or more template images cut out from one or more positions of the reference image,
An analysis method characterized by that:

<List 2>
The analysis method of column 1, wherein
The reference image is created based on position information of the spot pattern.
An analysis method characterized by that:

<List 3>
The analysis method of column 1, wherein
The reference image is any one of the plurality of light emission images,
An analysis method characterized by that:

<List 4>
The analysis method of column 1, wherein
The template image and the target image include at least spot patterns of four or more blocks adjacent to each other.
An analysis method characterized by that:

<List 5>
The analysis method of column 1, wherein
Spots of a specific pattern are formed on the block,
In the first block and the second block adjacent to each other, the pattern of the spot is the same, and there is a phase shift.
An analysis method characterized by that:

1-16, 2003, 2008 Middle block 100 Nucleic acid analyzer 101 Two-dimensional sensor 102 Imaging lens 103 Band pass filter 104 Excitation filter 105 Dichroic mirror 106 Filter cube 107 Light source 108 Objective lens 109 Flow cell 112, 115 Piping 113 Reagent container 114 Reagent Storage unit 116 Waste liquid container 117 Stage 118 Temperature control board 119 Computer 120 Analysis area 121 Detection field 801, 901 Substrate 802 Hollow sheet 803 Hollow section 804 Cover glass 805 Analysis area 806, 902 Spot 807 Inlet 808 Outlet 903 Blocking layer 904 coating film 1001, 2004 square lattice pattern 1002, 2005 hexagonal lattice pattern 1003, 1004 spot pattern image 100 Large block 1006,2007 spot 2001, 2002 spot pattern area 2006 reference block boundary 2009 area

Claims (17)

Board and
Formed on the surface of the substrate, a pattern of spots to which biopolymers are attached,
The area where the pattern of spots is formed is divided into a plurality of blocks, spots of a specific pattern are formed in the blocks, and a first block and a second block adjacent to each other are formed of the spots. The pattern is the same and there is a phase shift,
A substrate for nucleic acid analysis, characterized in that: The nucleic acid analysis substrate according to claim 1, wherein
The first block and the second block adjacent to each other have different shapes or sizes of blocks.
A substrate for nucleic acid analysis, characterized in that: The nucleic acid analysis substrate according to claim 1, wherein
The specific pattern is a hexagonal lattice pattern,
A substrate for nucleic acid analysis, characterized in that: The substrate for nucleic acid analysis according to claim 1, wherein
In a region where the spots are not formed on the surface of the substrate, there is a blocking layer to which the biopolymer cannot adhere,
A substrate for nucleic acid analysis, characterized in that: The substrate for nucleic acid analysis according to claim 1, wherein
The diameter of the spot, and the pitch of the spot is 50 to 1000 nanometers;
A substrate for nucleic acid analysis, characterized in that: A substrate for nucleic acid analysis according to any one of claims 1 to 5, a hollow sheet, and a second substrate bonded together.
A flow cell for nucleic acid analysis, characterized in that: Board and
Formed on the surface of the substrate, a pattern of spots to which biopolymers are attached,
The area where the spot pattern is formed is divided into a plurality of blocks,
One or more specific patterns of spots are formed on the block,
The first block and the second block adjacent to each other have different shapes or sizes of blocks.
A substrate for nucleic acid analysis, characterized in that: The substrate for nucleic acid analysis according to claim 7, wherein
A pattern is different between the first block and the second block adjacent to each other.
A substrate for nucleic acid analysis, characterized in that: The substrate for nucleic acid analysis according to claim 7, wherein
One of the one or more particular patterns is a hexagonal lattice pattern,
A substrate for nucleic acid analysis, characterized in that: The substrate for nucleic acid analysis according to claim 7, wherein
In a region where the spots are not formed on the surface of the substrate, there is a blocking layer to which the biopolymer cannot adhere,
A substrate for nucleic acid analysis, characterized in that: The substrate for nucleic acid analysis according to claim 7, wherein
The diameter of the spot, and the pitch of the spot is 50 to 1000 nanometers;
A substrate for nucleic acid analysis, characterized in that: The nucleic acid analysis substrate according to any one of claims 7 to 11, a hollow sheet, and a second substrate bonded together.
A flow cell for nucleic acid analysis, characterized in that: An analysis method,
A pattern of spots where biopolymers adhere to the surface of the substrate is formed,
The area where the spot pattern is formed is divided into a plurality of blocks,
A spot of a specific pattern is formed on the block,
In the first block and the second block adjacent to each other, the spot pattern is the same, and there is a phase shift,
Between one or more target images cut out from one or more positions of a light emission image obtained by capturing light emission from the surface of the substrate and one or more template images cut out from one or more positions of the reference image Perform alignment processing,
An analysis method characterized by that: The analysis method according to claim 13, wherein
The reference image is created based on position information of the spot pattern,
An analysis method characterized by that: The analysis method according to claim 13, wherein
The reference image is any one of the plurality of light-emitting images,
An analysis method characterized by that: The analysis method according to claim 13, wherein
The template image and the target image include at least the spot patterns of four or more blocks adjacent to each other,
An analysis method characterized by that: The analysis method according to claim 13, wherein
The first block and the second block adjacent to each other have different shapes or sizes of blocks.
An analysis method characterized by that:

Images