JP7091164B2 - Nucleic acid analysis substrate, nucleic acid analysis flow cell, and analysis method - Google Patents

Description

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

In recent years, in a nucleic acid analysis apparatus, a method has been proposed in which a large number of DNA fragments to be analyzed are supported on a flow cell made of a glass substrate, a silicon substrate, or the like, and the base sequences of these many 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.

In addition, in order to analyze a large amount of DNA fragments, the analysis area is usually divided into a plurality of detection fields, and the detection field is changed for each irradiation, and analysis is performed in all the detection fields, and then the polymerase extension reaction is performed. A new fluorescent dyed substrate is introduced using the same procedure as described above, and each detection field of view is analyzed. By repeating this, the base sequence can be efficiently determined (see Patent Document 1).

The substrate used for such analysis is disclosed in Patent Document 1 and Patent Document 2. In Patent Document 1, spots to which sample DNA binds are arranged in a grid on the substrate. A silicon wafer is used for the substrate, and it is made by photolithography technology and etching technology. After forming a hydrophobic HMDS (hexamethyldisilazane) layer on a silicon wafer, a positive resist is applied. After the openings are provided at predetermined positions on the resist using photolithography techniques, oxygen plasma is used to remove the HMDS at the bottom of the openings. After that, the wafer is held in the gas phase of aminosilane, and aminosilane is introduced into the bottom of the opening. After applying resist on the wafer, dicing is performed to cut out the substrate. After removing the resist on the cut-out substrate 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. By using aminosilane, which is highly hydrophilic and can fix DNA, for the DNA ball fixing spot, and by using hydrophobic HMDS, which prevents DNA adsorption in other regions, when a solution containing DNA balls is introduced onto the substrate, DNA naturally occurs. It makes it possible to fix the ball 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 device. The DNA oligomers are arranged in a grid pattern. After that, the DNA oligomer and the DNA sample are hybridized to fix the DNA sample in a lattice pattern. 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 analysis, the fluorescence emitted from the DNA sample immobilized on the spot arranged on the substrate is imaged, and the base is specified by image processing. For this purpose, it is necessary to accurately identify individual spots in the emission image, which is a fluorescence image. Generally, even in fluorescent imaging in which the same detection field of view is imaged, the imaging position differs on the flow cell due to the limit of the control accuracy of the driving device for changing the field of view. Therefore, a spot is imaged at different coordinate positions in the individual emission images. 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 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 high density, and their shapes need to be unique in order to determine the position, and these are detected. Therefore, there is a problem that the load of image processing is large.

Further, Patent Document 3 discloses a technique in which the spot pattern itself has a code indicating position information. However, in the analysis according to the present invention, the DNA is not always fixed in all the spot patterns, and the spots of the DNA sample fluoresce in all the luminescent images depending on the combination of the base type and the fluorescence filter. Is not always. Therefore, the code pattern technique disclosed in Patent Document 3 cannot be applied to the substrate for the above analysis.

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

However, when the spot pattern is in a grid pattern, the image cut out for matching looks like the same pattern at any position, so it is difficult to detect the position by image correlation only with this pattern. Therefore, a spot pattern technique that enables alignment is disclosed. Patent Document 4 discloses a method of performing alignment by image correlation by using a pattern in which spots are deleted in a pseudo-random manner with respect to a grid pattern. Patent Document 5 discloses a technique that enables alignment by image correlation by dividing a spot pattern into blocks so that adjacent blocks have different rotation angles.

US Patent Application Publication No. 2009/0270273 US Patent Application Publication No. 2009/0018024 US Pat. No. 6,663,008 US Pat. No. 8,774494 US Pat. No. 9,566,560

However, when the pseudo-random defect pattern shown in Patent Document 4 is used, the DNA fragment is not always fixed at each spot, so that a random pattern similar to the pattern of the image to be aligned appears in the surroundings. It is easy, and there is a problem that the accuracy of alignment is lowered.

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

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

In order to achieve the above object, the present invention includes a substrate and a spot pattern on the surface of the substrate to which the biopolymer adheres, and the region where the spot pattern is formed is a plurality of blocks. A specific pattern of spots is formed in the blocks, and the first block and the second block adjacent to each other have the same spot pattern and are out of phase for nucleic acid analysis. Provides a substrate.

Further, in order to achieve the above object, the present invention includes a substrate and a spot pattern on the surface of the substrate to which the biopolymer adheres, and there are a plurality of regions where the spot pattern is formed. For nucleic acid analysis, the blocks are divided into blocks of one or more specific patterns, and the first block and the second block adjacent to each other have different shapes or sizes of blocks. Provides a substrate.

Further, in order to achieve the above object, in the present invention, in the analysis method, a spot pattern on which the biopolymer adheres is formed on the surface of the substrate, and a region in which the spot pattern is formed is a plurality of blocks. The blocks are divided into spots of a specific pattern, and the first block and the second block adjacent to each other have the same spot pattern and are out of phase, and are separated from the surface of the substrate. Alignment processing is performed between one or more target images cut out from one or more positions of the light emission image captured by the light emission of the above and one or more template images cut out from one or more positions of the reference image. Provides an analysis method.

According to the present invention, it is possible to improve the throughput of nucleic acid analysis by using a substrate for nucleic acid analysis in which spots whose positions can be identified are arranged at a high density.

It is a figure which shows the schematic structure example of the nucleic acid analyzer which concerns on each Example. It is a figure which shows the processing process for deciphering the base sequence of DNA which concerns on each Example. It is a figure for demonstrating the concept of the detection field of view on the flow cell which concerns on each Example. It is a figure which shows the concept of the bright spot of four kinds of fluorescent images in each detection field of view which concerns on each Example. It is a figure which shows the concept of determination of a base sequence which concerns on each Example. It is a figure which shows the concept of the position shift between cycles which concerns on each Example. It is a figure which shows the concept of measuring the misalignment amount of a plurality of places in an image which concerns on each Example. It is a figure for demonstrating an example of the flow cell structure for nucleic acid analysis which concerns on each Example. It is a figure for demonstrating an example of the substrate for nucleic acid analysis which concerns on each Example. It is a figure for demonstrating the concept of spot arrangement by a grid pattern which concerns on each Example. It is a figure which shows the 1st example of the spot pattern of the basic block which concerns on Example 1. FIG. It is a figure which shows the example of the middle block which concerns on Example 1. FIG. It is a figure which shows the example of the spot pattern of the middle block 1 which concerns on Example 1. FIG. It is a figure which shows the example of the spot pattern of the middle block 2 which concerns on Example 1. FIG. It is a figure which shows the example of the spot pattern of the middle block 3 which concerns on Example 1. FIG. It is a figure which shows the example of the spot pattern of the middle block 4 which concerns on Example 1. FIG. It is a figure which shows the example of the large block which concerns on Example 1. FIG. It is a figure which shows the 1st example which the basic block is adjacent which concerns on Example 1. FIG. It is a figure which shows the 2nd example which the basic block is adjacent which concerns on Example 1. FIG. 3 is a diagram showing a third example in which basic blocks are adjacent to each other according to the first embodiment. It is a figure which shows the 2nd example of the spot pattern of the basic block which concerns on Example 1. FIG. It is a figure which shows the concept which divides a spot area into a different block shape which concerns on Example 2. FIG. It is a figure which shows the concept which divides a spot area into a different block shape which concerns on Example 2. FIG. It is a figure which shows an example of the spot pattern of the middle block which concerns on Example 2. FIG. It is a figure which shows an example of the pattern arrangement of the basic block which concerns on Example 2. FIG. It is a figure for demonstrating the case which the basic block is adjacent which concerns on Example 2. FIG. It is a figure which shows an example of the pattern arrangement of the basic block which concerns on Example 2. FIG. It is a figure which shows the example of the middle block which concerns on Example 2. FIG. It is a figure which shows the example of the large block which concerns on Example 2. FIG. It is a figure which shows the example of the middle block which concerns on Example 3. FIG. It is a figure which shows the example of the spot pattern of the middle block 1 which concerns on Example 3. FIG.

Hereinafter, examples of the present invention will be described with reference to the accompanying drawings. In the attached drawings, functionally the same elements may be displayed with the same number. The accompanying drawings show specific examples and implementation examples based on the principles of the present invention, but these are for the purpose of understanding the present invention, and in order to never interpret the present invention in a limited manner. Not used. That is, it should be understood that the description of the present specification is merely a typical example and does not limit the scope of claims or application examples in any sense.

In the various examples described below, the description is given in sufficient detail for those skilled in the art to carry out the present invention, but other implementations and embodiments are also possible, and the scope and spirit of the technical idea of the present invention can be understood. It is necessary to understand that it is possible to change the structure / structure and replace various elements without deviation. Therefore, the following description should not be construed as limited to this. Further, although the nucleic acid analysis substrate according to various examples targets DNA fragments for measurement and analysis, it may also target RNA, protein, etc. in addition to DNA, and the present invention can be applied to all biological-related substances. 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 DNA base sequence decoding process, a nucleic acid analysis flow cell configuration, and a nucleic acid analysis substrate will be described in sequence. The configuration and the like will be described.

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

The liquid delivery system provides a means for supplying the reagent to the flow cell 109. The liquid feeding system reacted with the reagent storage unit 114 accommodating a plurality of reagent containers 113, the nozzle 111 accessing the reagent container 113, the pipe 112 for introducing the reagent into the flow cell 109, and the DNA fragment as the means. It is provided with a waste liquid container 116 for discarding waste liquid such as reagents, and a pipe 115 for introducing the waste liquid into the waste liquid container 116.

The transport system moves the analysis region 120 of the flow cell 109, which will be described later, to a predetermined position. The transport 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 each of the orthogonal X-axis and Y-axis directions in the same plane. The stage 117 can also 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 regulates the reaction temperature of the DNA fragment. The temperature control system is installed on the stage 117 and includes a temperature control substrate 118 that promotes the reaction between the DNA fragment to be analyzed and the reagent. The temperature control substrate 118 is realized by, for example, a Pelche element or the like.

The optical system provides a means for irradiating the analysis region 120 of the flow cell 109, which will be described later, with excitation light to detect the 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 bandpass filter 103, also referred to as an absorption filter, are included as a set in the filter cube 106. The bandpass filter 103 and the excitation filter 104 determine a wavelength region through which fluorescence having a specific wavelength is passed.

The flow of irradiation of excitation light in an optical system will be described. The excitation light emitted from the light source 107 is collected by the condenser lens 110 and incident on the filter cube 106. The incident excitation light is transmitted only in a specific wavelength band by the excitation filter 104. 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. Of the four types of phosphors incorporated into the DNA fragment immobilized on the flow cell 109, the condensed excitation light excites the phosphor that excites in the specific wavelength band. The fluorescence emitted from the excited phosphor is transmitted through the dichroic mirror 105, only a specific wavelength band is transmitted by the bandpass filter 103, and is imaged as a fluorescence spot on the two-dimensional sensor 101 by the imaging lens 02. do.

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

The computer 119 includes a processor (CPU), a storage device (various memories such as ROM and RAM), an input device (keyboard, mouse, etc.), and an output device (printer, display, etc.), as in a normal computer. Be prepared. The computer controls the above-mentioned liquid feeding system, transport system, temperature control system, and optical system, and also analyzes the fluorescent image detected and generated by the two-dimensional sensor 101 of the optical system to analyze individual DNA fragments. It functions as a control processing unit that identifies the base of the optical system. However, the control, image analysis, and base identification of the above-mentioned liquid feeding system, transport system, temperature control system, and optical system do not necessarily have to be controlled and processed by one computer 119, and the processing load may be dispersed or processed. It may be performed by a plurality of computers each functioning as a control unit or a processing unit for the purpose of reducing time.

(2) Decoding Method of DNA Base Sequence A method of decoding a DNA base sequence will be described with reference to FIGS. 2 to 4. As will be described later, it is assumed that the reaction spots in which the same DNA fragment is amplified and densely arranged are arranged in advance on the flow cell 109 at a high density. For amplification of the DNA fragment, an existing technique such as a method for amplifying a circular DNA template shown in Patent Document 2 may be used as an example.

FIG. 2 is a diagram showing a processing step for decoding a base sequence of DNA. The entire run (S21) for decoding is performed by repeating the cycle process (S22) M times. M is the length of the base sequence to be obtained, and is predetermined. Each cycle process is a process for identifying the k (k = 1 to M) th 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) If the cycle is 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 onto the flow cell 109 via the pipe 112. The waste liquid after cleaning is discharged to the waste liquid container 116 via the pipe 115.
(Ii) Reagents containing fluorescently labeled nucleotides are flowed through the pipe 112 into the analytical region 120 on the flow cell 109. By adjusting the temperature of the flow cell with the temperature control substrate 118, the DNA polymerase causes an extension reaction, and the fluorescently labeled nucleotide complementary to the DNA fragment on the reaction spot is taken up.

Here, the fluorescently labeled nucleotide is one in which four types of nucleotides (dCTP, dATP, dGTP, dTsTP) are labeled with four types of phosphors (FAM, Cy3, Texas Red (TxR), Cy5), respectively. .. Each fluorescently labeled nucleotide is designated as FAM-dCTP, Cy3-dATP, TxR-dGTP, Cy5-dTsTP. Since these nucleotides are complementarily incorporated into the DNA fragment, dTsTP is used when the base of the actual DNA fragment is A, dGTP is used when the base C is used, dCTP is used as the base G, and dATP is used when the base T is used. 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 detected fields of view.

FIG. 3 is a diagram for explaining the concept of the detection field of view. The detection field of view 121 corresponds to an individual region when the entire analysis region 120 is divided into N pieces. The size of the detection field of view 121 is the size of a region 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, fluorescent images corresponding to the four types of phosphors are generated for each detection field of view 121.

(B-1) Imaging processing for each detected field of view In the detected field of view imaging process (S25), the following procedures (i) to (iv) are performed.
(I) The detection field of view 121 for performing fluorescence detection moves the stage 117 so as to come 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 with excitation light and exposing the two-dimensional sensor 101.
(Iv) Perform procedures (ii) and (iii) for other types of phosphors (Cy3, TxR, Cy5).

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

FIG. 4 is a diagram showing the concept of spots of four types of fluorescent images in each detection field of view. As shown in FIG. 4A, for example, there are spots at eight positions P1 to P8 in a certain detection field of view in a certain cycle, and each base is A, G, C, T, A, C, T, Let it be G. At this time, the fluorescent images for the four types of phosphors (Cy5, Cy3, FAM, TxR) are displayed at the positions P1 to P8 at the positions from P1 to P8, depending on the corresponding base type, as shown in (b) to (e). Spots are detected. The positions of P1 to P8 are the same in the four fluorescent images. However, depending on the design of the optical system, there may be differences in the optical path for each wavelength, so they may not be exactly the same. Therefore, 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. From the above, the base type of each spot detected in the detection field of view is determined.

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

FIG. 5 is a diagram showing the concept of determining this base sequence. As shown in FIG. 5, when each spot (DNA fragment having the base sequence ACGTTACGT ...) is stretched by one base by a certain cycle (#N) chemistry treatment, for example, Cy3-dATP is taken up. 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 fluorescent image of Cy5. In the cycle (# N + 2), it is detected as a spot on the fluorescence image of TxR. In the cycle (# N + 3), it is detected as a spot on the fluorescent image of FAM. By the cycle processing from the above cycle #N to cycle # N + 3, the base sequence at this spot is determined to be TACG.

(3) Spot alignment As described above, the DNA fragment to be detected is imaged in a state of being fixed to the spot placed on the substrate on the flow cell 109, and each spot is an image of four fluorescent images in each cycle. Observed as the bright spot above. As described above, the nucleic acid analyzer 100 repeatedly images the same detection field of view in each cycle. However, in each cycle, the stage 117 is moved to change the detection field of view for imaging. Therefore, for the same detection field of view, the position shift due to the movement of the stage occurs between different cycles. This misalignment is due to a control error in the stage 117.

FIG. 6 is a diagram showing the concept of misalignment between cycles. As shown in FIG. 6, there is a possibility that the imaging position is deviated between the Nth cycle (a) and the (N + 1) th cycle of (b) with respect to a certain detection field of view due to a stage control error. Therefore, the DNA fragment positions (P1 to P8) in the luminescence image which is the fluorescence image of the N cycle are detected as different positions (P1'to P8', respectively) on the luminescence image of the (N + 1) cycle. However, these bright spots are all due to the same DNA fragment. Therefore, in order to determine the base sequence of each spot, it is necessary to correct the positional deviation between the spots detected in each luminescent image.

In order to correct this misalignment, it is necessary to align each emission image with respect to a common reference image. Here, the reference image is a common image used for the position coordinate system of the spot. For example, if the spot position is known as design information, a reference image may be created from the known spot position. As an example, an image of luminance according to a two-dimensional Gaussian distribution of a predefined dispersion according to the spot size may be created centered on the spot position (x, y). Alternatively, a reference image may be created based on any of the captured actual 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 images of each detection field of view in the second and subsequent cycles may be aligned with this reference image.

Known matching techniques can be applied to the alignment between images. As an example, the image obtained by cutting out a part of the reference image is used as the template image t (x, y), and the cross-correlation function m (u, v) with the target image f (x, y) obtained by cutting out a part of the input image. Is obtained, and S_1 = (u, v), which gives the maximum value of this, is set as the amount of misalignment. Here, as an example of t (x, y), an image of 256 pixels × 256 pixels at the center of the reference image can be mentioned. Similarly, as an example of f (x, y), an image of 256 pixels × 256 pixels at the center of the input image can be mentioned. In the calculation of the amount of misalignment, instead of the cross-correlation function, a normalized cross-correlation considering the difference in brightness may be used, or a correlation limited to the phase may be used. When detecting the amount of angular deviation between images, the above-mentioned mutual correlation or phase-limited correlation can be applied to an image in which the angular direction is converted to the horizontal direction by polar coordinate conversion of the image.

Further, the amount of this positional deviation may be obtained at a plurality of points according to the degree of distortion of the image. This concept is shown in FIG. For example, if there is no distortion in the image and the same positional deviation for all pixels, that is, only uniform displacement due to the stage can be assumed, the displacement amount S_1 (u,) shown on the left side of FIG. 7A is shown. v) can be applied.

On the other hand, for example, when the image is distorted and the amount of misalignment differs depending on the position in the image (when the flow cell 109 is deformed by heating and the misalignment is not uniform), the right side of FIG. 7A is shown. As shown, the amount of misalignment is obtained from n plurality of points in the image, and the amounts of misalignment S_1, S_1, ... S_n at these plurality of points are obtained. To calculate the amount of misalignment at each point, an image centered on the position of each point in the reference image and input image is cut out, and each is used as a template image and a target image, and the amount of misalignment that maximizes the correlation as described above. Should be calculated. Then, based on the obtained n misalignments, for example, the coefficients of the affine transformation and the polynomial transformation can be obtained by the least squares method to formulate the misalignment of any pixel position ((FIG. 7). b) See)
(4) Flow cell and substrate for nucleic acid analysis (A) Flow cell for nucleic acid analysis FIG. 8 shows the flow cell 109 for nucleic acid analysis used in each example. The flow cell 109 is bonded with a nucleic acid analysis substrate 801, a hollow sheet 802 having a hollowed out portion 803 with a hollowed out central portion, and a cover glass 802. The 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 injection port 807 and the discharge port 808, but in another embodiment, the holes in the cover glass 804 may be the injection port and the discharge port. Further, as yet another form, the holes formed in the side surface of the hollow sheet 802 may serve as an injection port and a discharge port.

(B) Nucleic Acid Analysis Substrate The nucleic acid analysis substrate 801 is formed with a plurality of spots 806 for fixing DNA fragments in the analysis region 805. For the formation of spots on the substrate, known techniques shown in Patent Document 1 and Patent Document 2 may be used. Further, as an example, as shown in FIG. 9, a blocking film may be provided in a region other than the spot to increase the fixation ratio of the DNA sample to the spot. In FIG. 9, a spot 902 is formed on the substrate 901, a coating film 904 containing an amino group is formed on the spot 902, and the blocking layer 903 is coated on the region without the spot 902. By contacting this substrate with a solution containing the DNA sample, 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 is an inorganic material such as silicon, glass, quartz, sapphire, ceramic, ferrite, alumina, diamond, a metal material such as aluminum, SUS, titanium, iron, or a phenol resin. 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, fiberglass reinforced polyethylene terephthalate, cyclic polyolefin, polyphenylene sulphide, polysulfone, polyether sulfone, amorphous polyarylate, liquid crystal polymer, polyether ether Resin materials such as ketones, mixed materials thereof, glass fiber and carbon fiber reinforced inorganic materials can also be used. Among these materials, silicon, which has low self-fluorescence, low coefficient of thermal expansion, and high resistance to analytical solutions, is used when analyzing DNA samples by fluorescence or when the temperature is raised or lowered during analysis. Glass, quartz, SUS, titanium, etc. are particularly desirable.

The spot 902 is formed by using a spotting device as shown in Patent Document 2 or a known technique such as wrist-off. As the material used, a material that can be formed on a substrate via a covalent bond is preferable. Such materials include inorganic materials such as silicon, glass, quartz, sapphire, ceramics, ferrite, and alumina, which have an oxide film on the substrate surface, and metal materials such as aluminum, SUS, titanium, and iron, especially silane. Coupling material is desirable. Further, 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, and such functional groups include vinyl groups and epoxys. Examples thereof include ethoxysilane and methoxysilane having a group, a styryl group, a methacrylic group, an acrylic group, an amino group, a ureido group, an isocyanate group, an isocyanurate group and a mercapto group in the molecule.

When a plurality of types of DNA samples are fixed in one spot 902, fluorescent dyes from the plurality of types of DNA samples are detected, so that the base cannot be correctly identified. On the other hand, if the size of the spot 902 is small, the contact probability with the DNA sample in the solution decreases, the number of spots where the DNA sample is not fixed increases, and the throughput for base sequence determination decreases. Therefore, the diameter of the spot 902 should be larger than half of the DNA sample so that only one DNA sample can be fixed at a high fixation 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 amplified DNA as shown in Patent Document 1 and Patent Document 2 is as large as 50 nm or more. Therefore, when such a DNA sample is used, the size of the spot 902 is preferably about 50 nm or more and less than 1000 nm in diameter.

It is desirable that the pitch, which is the distance between the centers of the spots, is at least slightly larger than the size of the DNA sample used. If the pitch is smaller than the size of the DNA sample, it will be fixed across a plurality of spots, which causes a problem that the above base identification cannot be performed. On the other hand, if the pitch is too large, the density of the fixed DNA samples will decrease, and there will be a problem that the number of DNA samples that can be analyzed at one time will decrease. Therefore, it is desirable that the pitch is 50 nm or more and less than 1000 nm as in the spot size.

The blocking layer 903 is preferably one that prevents the DNA sample from adsorbing to the substrate 901 and enhances the accessibility of the DNA sample to the substrate to increase the fixation rate of the DNA sample to the spot. Known examples of such materials include silane coupling materials whose ends are treated with polyethylene glycol or carboxylic acid.

The coating film 904 containing an amino group uses a material having a high surface amino group density in order to increase the contact rate between the DNA sample and the functional group on the spot when fixing the DNA sample and to prevent the DNA sample from peeling off during analysis. That's what I want. As such a coating film, a method of reacting a functional group introduced on a spot with polyamines having a plurality of amino groups in one molecule is desirable. Examples of such polyamines include spermidine, putrescine, and spermine, but dendritic polymers are particularly preferable because they have many amino groups present in the molecule. Examples of such a dendritic polymer include polyamide amine dendrimers. Polyamide amine dendrimers consist of an alkyldiamine core and a branched structure of tertiary amines. Cores and molecules include ethylenediamine, 1,12-diamidedecane, 1,4-diaminobutane, cystamine, 1,6-diaminohexane and the like. The dendritic molecule can exponentially increase the functional groups in the molecule by increasing the generations surrounding the core molecule. In the present invention, the amino group density of the coating surface can be greatly increased by using the dendritic molecules of the first generation or later.

It is desirable that these polyamines form a covalent bond with the functional group on the spot in that they 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 the molecule is desirable, and a vinyl group, an epoxy group, a methacryl group, an acrylic group, an isothiocyanurate group, an isocyanate group, and an isocyanurate. Examples thereof include silane coupling materials having a group, alkenes, and alkins. Further, after introducing the reactive functional group on the surface of the spot, the amine-reactive functional group may be introduced on the spot by using a multivalent cross-linking reagent. Examples of such an amine-reactive functional group include an isothiocyanurate group, an isocyanate group, an isocyanurate group, a sulfonyl chloride group, an aldehyde group, a carbodiimide group, an acrylic azide group, a ruolobenzene group, a carbonate group, and an N-hydroxysuccinimide ester. Examples include a group, an imide ester group, an epoxy group, a fluorophenyl ester group, an acid anhydride and the like.

(5) Arrangement of pattern for nucleic acid analysis As described above, the larger the number of spots on the substrate for nucleic acid analysis, the larger the number of DNA fragments whose base sequence can be determined, so that the measurement throughput of analysis is improved. However, in order to identify the base type of each spot, there are restrictions on the size of the spot and the minimum pitch between the spots. Examples of the pattern in which the spots are arranged at high density include the square grid pattern 1001 shown in FIG. 10 (a) and the hexagonal grid pattern 1002 shown in FIG. 10 (b). However, since these grid patterns are periodic, the spot patterns are not unique. That is, as shown in FIG. 10 (c), the spot pattern image 1003 obtained by cutting out a region at a certain position of the spot pattern and the spot pattern image 1004 at a region at another position are very similar. If the spot pattern is not unique in this way, the spot pattern image at one position may be erroneously detected as the spot pattern image at another position in the above-mentioned image alignment, so the amount of misalignment is accurate. Cannot be calculated.

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

In the first embodiment, a pattern of spots to which a biopolymer adheres is formed on the substrate and the surface of the substrate, the region where the pattern of the spots is formed is divided into a plurality of blocks, and the spots having a specific pattern are formed on the blocks. In the first block and the second block adjacent to each other, a nucleic acid analysis substrate having the same spot pattern and a phase shift, and an example of an analysis method using the same are used. be. That is, in the nucleic acid analysis substrate and the nucleic acid analysis pattern sequence according to this embodiment, the spot pattern region in which the DNA fragment is fixed is divided into blocks having a lattice pattern, and the adjacent blocks are the phases of the lattice pattern. Are out of alignment with each other.

Further, in the first embodiment, a spot pattern on which the biopolymer adheres is formed on the surface of the substrate, the region where the spot pattern is formed is divided into a plurality of blocks, and the blocks have spots of a specific pattern. In the first block and the second block that are formed and adjacent to each other, the spot pattern is the same and there is a phase shift, and the emission from the surface of the substrate is taken from one or more positions of the image. This is an example of an analysis method in which alignment processing is performed 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. Then, the template image and the target image include a pattern of spots of at least four or more blocks adjacent to each other.

FIG. 11 shows an example of a pattern of four basic blocks having half-phase differences based on a hexagonal lattice pattern. In the figure, the pattern AX which is half-phase shifted to the right, the pattern AY which is half-phase shifted downward, and the pattern AXY which is half-phase shifted downward and to the right are shown with reference to the pattern A. In the figure, in order to visualize the phase shift, a grid line of one phase unit (horizontal to vertical ratio is 2: √3) is shown, but this grid line does not exist on the actual substrate. .. The same applies to the following figures. Hereinafter, an example of constructing a highly unique spot pattern that enables image alignment by combining these four basic blocks A, AX, AY, and AXY will be described. The phase shift is not limited to the above-mentioned half phase, that is, 0.5, and a shift of 0.5 ± 0.3 is preferable. This is for the same reason as the possible range of the pitch and spot size sizes described above.

FIG. 12 shows an example of medium blocks 1, 2, 3, and 4 consisting of four basic blocks and four combinations. 13 to 16 show each spot pattern of the middle blocks 1-4. As shown in FIGS. 13 to 16, the phases of adjacent middle blocks are different from each other, and the distance between the spots in contact with the block boundary is always equal to or larger than the spot pitch in the hexagonal grid 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 these medium blocks 1-4 are arranged. The large block 1005 shown in the figure is composed of 16 × 16 medium blocks. 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 large block 1005 in the vertical direction and the horizontal direction, the spot area on the substrate can be obtained. A spot pattern may be placed in.

In addition, as shown in FIG. 17, for example, the gray part may be locally similar in the order of the middle blocks. In order to reduce the risk of misalignment due to such similar patterns, the size of the template image and the target image for alignment when aligning the image should be at least on the side larger than the middle block, and at least in the image that could be cut out. It is desirable that four or more basic blocks A, AX, AY, and AXY patterns are included. Furthermore, it is desirable that the image contains many medium blocks 1-4. That is, it is desirable that the size of the basic block and the size of the medium block are determined so as to be as unique as possible according to the image size cut out when the image is aligned.

In addition, in the combination of the patterns of the basic blocks and the combination of the middle blocks, it is necessary to consider so that the distance between the spots in contact with the block boundary is equal to or larger than the spot spacing of the original grid pattern. FIG. 18 shows a spot pattern when the basic block AXY and the basic block AY of FIG. 11 are in contact with each other in the vertical direction. From the figure, the spots on both sides of the block boundary are smaller than the spot pitch in the hexagonal grid pattern. In this embodiment, in order to avoid such a combination, AXY and AY are arranged diagonally in the middle blocks 1 to 4 shown in FIG.

In the middle blocks 1-4 shown in FIG. 12, it is considered that the phases of the adjacent blocks are different regardless of which middle blocks are in contact with each other. That is, the pattern is arranged so that the same basic blocks do not touch each other.

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 modifying the spot arrangement on the block boundary. FIG. 19 shows a boundary pattern when the same basic blocks are adjacent to each other in the horizontal direction. FIG. 20 shows a boundary pattern when the same basic blocks are vertically adjacent to each other. In FIGS. 19 and 20, the gray spot 1006 does not exist on the original basic block, but by making the basic blocks adjacent to each other, a spot gap is generated on the boundary. In such a case, such a gray spot 1006 may be placed on the boundary.

Taking these things into consideration, it is possible to construct a more unique spot pattern by further increasing the number of combinations of the middle blocks 1-4 shown in FIG.

Further, in FIG. 19, the basic block patterns A, AX, AY, and AXY based on the hexagonal lattice pattern are shown, but other patterns can also be used as the base. For example, the basic block patterns A, AX, AY, and AXY based on the square grid pattern as shown in FIG. 21 can also generate a highly unique spot pattern by the same method. The treatment of spots 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 Example 1, the spot pattern region on the substrate is basically a block whose phase is shifted based on the high-density spot pattern. Since the spots are arranged in the basic block pattern as a unit, high-density spot arrangement and uniqueness of the spot pattern that enables image alignment are ensured, so that the analysis throughput can be improved.

In the second embodiment, a pattern of spots to which a biopolymer adheres is formed on the substrate and the surface of the substrate, the region where the pattern of spots is formed is divided into a plurality of blocks, and one or more specific blocks are specified. This is an example of a nucleic acid analysis substrate having a structure in which spots of the above pattern are formed and the first block and the second block adjacent to each other have different shapes or sizes of the blocks. That is, Example 2 is an example of spot pattern arrangement for further enhancing the uniqueness of the pattern as compared with Example 1. In the suitable nucleic acid analysis substrate and nucleic acid analysis pattern arrangement according to this embodiment, the spot pattern region in which the DNA fragment is fixed is divided into block regions having one or more lattice patterns, and adjacent blocks thereof are separated from each other. , The grid pattern is different, and the shape or size of the block is different. The configurations of the nucleic acid analyzer, the flow cell, the substrate, and the like other than the pattern arrangement for nucleic acid analysis are as described above, and the description thereof will be omitted here.

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

22 and 23 are examples of dividing the spot pattern region into different block shapes and different block sizes. FIG. 22 shows an example in which the spot pattern region 2001 is divided into a complicated block shape. FIG. 23 shows an example in which the spot pattern region 2002 is divided into square middle blocks, and the individual middle blocks are further divided into four rectangular basic blocks having different shapes. It can be expected that the uniqueness of the pattern is higher in FIG. 22 where the block shape is complicated, but the design becomes complicated because the spot patterns of the block shapes of various shapes are taken into consideration. In practice, it has been confirmed that there is no problem in alignment even with the division pattern shown in FIG. 23. 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, and may be a rectangle depending on the spot pattern.

It is assumed that the rectangular basic block has a spot pattern of either a square grid ((a) in FIG. 10) or a hexagonal grid ((b) in FIG. 10). In this embodiment, in order to make adjacent blocks have different grid patterns, the four basic blocks in each square of FIG. 23 are configured so that the diagonal lines have the same grid pattern. An example of this pattern configuration is shown in FIG. In the figure, in the middle block 2003, the upper left and lower right basic blocks are used as a hexagonal grid pattern, and the upper right and lower left basic blocks are used as a square grid pattern. However, since the spot pattern at the block boundary is not taken into consideration in FIG. 24, unnecessary voids are formed near the boundary, and there is a lot of room for improvement from the viewpoint of increasing the density of spots. Further, depending on the spot pattern, the distance between the spots straddling the boundary becomes small, which may cause a problem that the accuracy of base identification is lowered.

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 that straddle the boundaries may be added depending on the combination of the block boundaries.

FIG. 25 shows two grid patterns separated from the block boundary by more than half of the minimum pitch D of the spot. In the figure, (a) and (b) show a square grid pattern 2004 and a hexagonal grid pattern 2005, respectively. As shown in (c) of the figure, these grid patterns are arranged so that the center of the spot is in the spot region separated from the reference block boundary 2006 by 1/2 or more of the minimum pitch D.

FIG. 26 shows a combination in which the basic block of the square grid and the basic block of the hexagonal grid shown in FIG. 25 are in contact with each other. (A) in the figure is a case where the square grid block 2004 and the hexagonal grid block 2005 are in contact with each other vertically. (B) in the figure is a case where these are in contact with each other on the left and right. (C) in the figure is the case where the upper right and the lower left square basic block are in contact. The figure (d) is a case where the upper left and the lower right basic blocks of the hexagonal grid are in contact. In any case, the distance between the spots across the block boundaries will never be less than the minimum pitch D. In the cases (a), (b), and (c) in the figure, there is no extra space near the block boundary. However, in (d) of the figure, the spot position shown in gray is a void. Therefore, in the combination of the patterns (d) in the figure, the number of spots may be increased by adding gray spots 2007 that straddle the block boundaries. In (a) of the figure, there are cases where the top and bottom are reversed, and in (b) of the figure, there are cases where the left and right are reversed, but since the cases in the figure have the same characteristics, the description is omitted. Further, whether or not voids are generated in the combination shown in FIG. 26 depends on the block shape, the 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 composed of the basic blocks in consideration of the block boundaries shown in FIGS. 25 and 26 described above. The number of voids is reduced and the number of spots is increased as compared with FIG. 24. By variously changing the vertical and horizontal sizes of the four basic blocks as shown in FIG. 27, a highly unique spot pattern can be realized.

An example of constructing such a spot pattern is shown with reference to FIGS. 28 and 29. In FIG. 28 (a), the middle block is divided into 2: 3 in the horizontal direction, the left side is divided into 4: 1 in the vertical direction to form basic blocks A and B, respectively, and the right side is divided into 2: 3 to be basic. Blocks C and D. In FIG. 28 (b), the middle block is divided into 1: 4 in the horizontal direction, the left side is divided into 3: 2 in the vertical direction to form basic blocks E and F, respectively, and the right side is divided into 1: 4 to be basic. Blocks G and H. In (c) of FIG. 28, the middle blocks 1-8 by the combination of 1 to 8 are defined by such rearrangement of the basic blocks A, B, C, and D. Similarly, the middle blocks 9-16 are defined by the combination of 9 to 16 by rearranging the basic blocks E, F, G, and H. In each of the middle blocks 1-16, as described in the example of FIG. 24, a hexagonal grid pattern is arranged in the upper left and lower right basic blocks, and a square grid pattern is arranged in the upper right and lower left basic blocks, respectively. ing. As described above, 16 types of grid patterns and block patterns divided so that the block shapes are different are created between adjacent blocks.

In FIG. 29, a certain region 2009 in the spot region of the nucleic acid analysis substrate is a large block composed of 16 × 16 medium blocks, and pseudo-random numbers from 1 to 16 are generated to arrange the medium blocks 1-16. This is a determined example. As a result, each block pattern is arranged irregularly, so that a highly unique spot pattern can be configured.

The size of the large block in FIG. 29 is not limited to the medium block of 16 × 16, but may be adjusted to one imaging field of view, or the above large block may be repeated in the vertical direction and the horizontal direction, respectively. A spot pattern may be arranged in a spot area on the substrate. Further, in the example shown in FIG. 28, 16 types of block patterns are created, but a combination pattern composed of more shapes may be created. Further, in FIG. 29, in the same figure, there are places where the same numerical values are continuous in the vertical direction and the horizontal direction. In such a place, there is a risk that the accuracy of alignment in continuous directions will be reduced. Therefore, if necessary, the numerical values may be exchanged so that the same numerical values are not continuous.

In addition, the cutout image for which the image correlation is calculated, that is, the template image and the alignment target image must contain at least four or more basic blocks by making the cutout image size larger than the medium block. Is desirable. Furthermore, it is desirable that the image contains many medium blocks. As a result, in the cut-out image, the uniqueness is enhanced by the difference in the grid pattern and its size and shape. That is, as in the first embodiment, it is desirable that the size of the block is determined so as to be as unique as possible according to the image size cut out when the image is aligned.

In this embodiment, it is possible to generate a unique spot pattern by combining the same patterns. However, since the patterns are the same except for the block boundaries, there is a risk that the similarity as a pattern will be high and the alignment accuracy will be reduced. Therefore, when the same pattern is used, it is desirable to include a large number of block boundaries by making the block size smaller than the image size cut out when the image is aligned.

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 regions on the substrate are further different in shape and size based on one or more spot patterns. Since the spots are arranged in a pattern with the above as the basic unit, the uniqueness of the spot pattern is further secured as compared with Example 1, so that the accuracy of image alignment can be improved and the accuracy of base identification can be improved. become.

Example 3 is an example of spot pattern arrangement for further increasing the density and uniqueness of the pattern by combining the configurations of Example 1 and Example 2. That is, the substrate is provided with a pattern of spots on the surface of the substrate to which the biopolymer adheres, and the region where the pattern of spots is formed is divided into a plurality of blocks, and the blocks have 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. It is an embodiment.

In the nucleic acid analysis pattern arrangement and the nucleic acid analysis substrate according to the present embodiment, and in the spot pattern arrangement thereof, the spot pattern region in which the DNA fragment is fixed is divided into block regions having one or more lattice patterns, and the spot pattern region thereof is divided. Adjacent blocks have patterns having different phases from each other based on one grid pattern, and the shapes or sizes of the blocks are different from each other. Since the configurations of the nucleic acid analyzer, the flow cell, the substrate, and the like other than the pattern arrangement for nucleic acid analysis are the same as those of Examples 1 and 2, the description thereof is omitted here.

(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 at a higher density. ..

Therefore, in this embodiment, four patterns A, AX, AY, and AXY having different phases based on the hexagonal lattice pattern shown in FIG. 11 are included, and basic blocks having different shapes are formed. Then, as in the second embodiment, the middle block is configured by combining basic blocks having different shapes. FIG. 30 shows an example of the middle block 1-16. In the figure, among the 16 types of middle blocks 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, it is possible to prevent unnecessary voids from being generated at the block boundary, and the distance between the spots straddling the block boundary is a hexagonal grid pattern. It is possible to avoid becoming less than the pitch of. However, the definition of the basic block in the middle block is not limited to FIG. 30, and it is possible to increase the types of the middle block by combining the patterns and shapes of the basic blocks.

Although the basic block in the present embodiment is different from the shape of the basic block in the first embodiment, it can be created by arranging the basic blocks shown in FIG. Then, as described in FIGS. 19 and 20, a pattern in which a spot of a gap is newly added may be defined as a basic block again in a place where a gap is generated.

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 block shown by the thick frame is the basic block having a different shape in this embodiment. Since the method of constructing a large block using the 16 types of medium blocks 1-16 generated as described above is the same as that of the second embodiment, the 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 region 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 with the block as the 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. .. Further, since the spot density is improved as compared with Example 2, the analysis throughput of the base sequence can be improved.

Using the nucleic acid analysis flow cell of the present invention or the nucleic acid analyzer described above, various nucleic acid reactions can be detected and nucleic acids such as DNA sequences can be analyzed. The present invention is not limited to the above-mentioned examples, and includes various modifications. For example, the above-mentioned examples have been described in detail for a better understanding of the present invention, and are not necessarily limited to those having all the configurations of the description. The nucleic acid analysis substrate of each of the above-mentioned examples targets DNA fragments for measurement and analysis, but may target other biological substances such as RNA and protein in addition to DNA.

Further, although each of the above-mentioned configurations, functions, computers, etc. has been described mainly with an example of creating a program that realizes a part or all of them, it is hard to design a part or all of them by, for example, an integrated circuit. Needless to say, it can be realized with hardware. That is, the functions of all or part of the processing unit may be realized by, for example, an integrated circuit such as an ASIC (Application Specific Integrated Circuit) or an FPGA (Field Programmable Gate Array) instead of the program.

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

<List 1>
It is an analysis method that analyzes spots on the substrate.
A pattern of spots to which biopolymers adhere is formed on the surface of the substrate.
The area where the spot pattern is formed is divided into a plurality of blocks.
A specific pattern of spots is formed on the block.
The first block and the second block adjacent to each other differ in the shape or size of the blocks.
An luminescence image obtained by capturing the luminescence from the surface of the substrate is used as an input.
Alignment processing is performed between one or more target images cut out from one or more positions of the luminescent 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 in Listing 1
The reference image is created based on the position information of the pattern of the spot.
An analysis method characterized by that.

<List 3>
The analysis method in Listing 1
The reference image is one of a plurality of the emission images.
An analysis method characterized by that.

<List 4>
The analysis method in Listing 1
The template image and the target image include patterns of spots of at least four or more blocks adjacent to each other.
An analysis method characterized by that.

<List 5>
The analysis method in Listing 1
A specific pattern of spots 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.
An analysis method characterized by that.

1-16, 2003, 2008 Medium block 100 Nucleic acid analyzer 101 Two-dimensional sensor 102 Imaging lens 103 Band pass filter 104 Excitation filter 105 Dycroic 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 Board 802 Hollow sheet 803 Hollow part 804 Cover glass 805 Analysis area 806, 902 Spot 807 Injection port 808 Outlet 903 Blocking layer 904 Coating film 1001, 2004 Square lattice pattern 1002, 2005 Hexagonal lattice pattern 1003, 1004 Spot pattern image 1005 Large block 1006, 2007 Spot 2001, 2002 Spot pattern area 2006 Reference block boundary 2009 area

Claims (16)

With the board
A pattern of spots to which a biopolymer adheres, which is formed on the surface of the substrate, is provided.
The area where the spot pattern is formed is divided into a plurality of blocks.
Spots of a specific pattern are formed in the blocks, and the first block and the second block adjacent to each other have the same spot pattern and are out of phase .
At least one target image cut out from one or more positions of a light emission image captured from the surface of the substrate and one or more template images cut out from one or more positions of a reference image. A pattern of said spots in four or more said blocks adjacent to each other is included .
A substrate for nucleic acid analysis. The nucleic acid analysis substrate according to claim 1.
The shape or size of the blocks is different between the first block and the second block adjacent to each other.
A substrate for nucleic acid analysis. The nucleic acid analysis substrate according to claim 1.
The particular pattern is a hexagonal lattice pattern,
A substrate for nucleic acid analysis. The substrate for nucleic acid analysis according to claim 1.
In the region on the surface of the substrate where the spot is not formed, there is a blocking layer to which the biopolymer cannot adhere.
A substrate for nucleic acid analysis. The substrate for nucleic acid analysis according to claim 1.
The diameter of the spot and the pitch of the spot are 50 to 1000 nanometers.
A substrate for nucleic acid analysis. A substrate for nucleic acid analysis according to any one of claims 1 to 5, a hollow sheet, and a second substrate are bonded together.
A flow cell for nucleic acid analysis. With the board
A pattern of spots to which a biopolymer adheres, which is formed on the surface of the substrate, is provided.
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 shape or size of the blocks differs between the first block and the second block that are adjacent to each other.
At least one target image cut out from one or more positions of a light emission image captured from the surface of the substrate and one or more template images cut out from one or more positions of a reference image. A pattern of said spots in four or more said blocks adjacent to each other is included .
A substrate for nucleic acid analysis. The nucleic acid analysis substrate according to claim 7.
The patterns are different between the first block and the second block adjacent to each other.
A substrate for nucleic acid analysis. The nucleic acid analysis substrate according to claim 7.
One of the one or more specific patterns is a hexagonal grid pattern,
A substrate for nucleic acid analysis. The nucleic acid analysis substrate according to claim 7.
In the region on the surface of the substrate where the spot is not formed, there is a blocking layer to which the biopolymer cannot adhere.
A substrate for nucleic acid analysis. The substrate for nucleic acid analysis according to claim 7.
The diameter of the spot and the pitch of the spot are 50 to 1000 nanometers.
A substrate for nucleic acid analysis. A substrate for nucleic acid analysis according to any one of claims 7 to 11, a hollow sheet, and a second substrate are bonded together.
A flow cell for nucleic acid analysis. It ’s an analysis method,
A pattern of spots on which biopolymers adhere is formed on the surface of the substrate,
The area where the spot pattern is formed is divided into a plurality of blocks.
A specific pattern of spots is formed on the block,
In the first block and the second block adjacent to each other, the spot patterns are the same and there is a phase shift.
Between one or more target images cut out from one or more positions of the luminescence image captured 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 and
The template image and the target image include patterns of the spots of at least four or more blocks adjacent to each other .
An analysis method characterized by that. The analysis method according to claim 13.
The reference image is created based on the position information of the spot pattern.
An analysis method characterized by that. The analysis method according to claim 13.
The reference image is one of the plurality of emission images.
An analysis method characterized by that. The analysis method according to claim 13.
The shape or size of the blocks is different between the first block and the second block adjacent to each other .
An analysis method characterized by that.

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