Abstract:
The substrate  100  for use in the analysis of a nucleic acid according to the present invention has multiple analysis areas  12  which are partitioned on a substrate  10,  and enables the measurement of the analysis areas  12  while interchanging the analysis areas  12  in turn, said substrate  100  being characterized in that each of the analysis areas  12  consists of an adsorption part  13  onto which a DNA fragment or a carrier having the DNA fragment carried thereon can be adsorbed and a non-adsorption part  14  which is a part outside of the adsorption part  13,  and the non-adsorption part  14  has, formed on at least a part thereof, a marker part  15  that has a specified shape and helps to identify the positions of the analysis areas  12.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a substrate for use in analysis of nucleic acid, a flow cell for use in analysis of nucleic acid, and a nucleic acid analysis device. 
       BACKGROUND ART 
       [0002]    In recent years, as a method of analyzing base sequences of nucleic acid, there is known a method of concurrently analyzing base sequences of multiple DNA fragments. In this method, an absorbent portion capable of absorbing DNA fragments or the like and a non-absorbent portion not capable of the DNA fragment are formed on a substrate, for example, by photolithography or an etching technique. Then, DNA fragments or the like serving as an analysis target are absorbed in the absorbent portion to perform the analysis (for example, see PTL 1). 
         [0003]    In the analysis method described above, excitation light is irradiated onto an analysis area including multiple DNA fragments where fluorochrome-labelled matrices corresponding to bases are introduced, and fluorescence emitted from each DNA fragment is detected to determine the base (for example, see NPL 1). 
         [0004]    In this analysis method, typically, a plurality of analysis areas are provided on a single substrate, and the analysis is performed for overall analysis areas by changing the analysis area whenever the irradiation is performed. Then, a new fluorochrome-labelled matrix is introduced on the basis of a polymerase extension reaction, and each analysis area is analyzed through the aforementioned operation. By repeating this procedure, it is possible to effectively determine the base sequence. 
       CITATION LIST 
     Patent Literature 
       [0000]    
       
         PTL 1: US 2009/0270273 A1 
         NPL 1: Science, 2005, Vol. 309, Pages 1728 to 1732 
       
     
       SUMMARY OF INVENTION 
     Technical Problem 
       [0007]    However, in the background art described above, when the same analysis area is repeatedly analyzed, a positional deviation may occur in the analysis area at every cycle. This positional deviation makes it difficult to map the DNA fragments in every cycle, so that it may be difficult to obtain a suitable base sequence. 
         [0008]    In view of the aforementioned problems, the present invention provides a substrate for use in analysis of nucleic acid, a flow cell for use in analysis of nucleic acid provided with this substrate for use in analysis of nucleic acid, and a nucleic acid analysis device, capable of reproducibly obtaining a position of the analysis area even when the positioning is performed repeatedly for the same analysis area. 
       Solution to Problem 
       [0009]    The invention to solve the above issue is a substrate for use in analysis of nucleic acid having a plurality of analysis areas partitioned on a substrate to perform measurement by sequentially changing each analysis area, 
         [0010]    wherein the analysis area has an absorbent portion capable of absorbing a DNA fragment or a vector where the DNA fragment is borne and a non-absorbent portion other than the absorbent portion, and 
         [0011]    a marker portion having a predetermined shape to calculate a position of the analysis area is provided in at least a part of the non-absorbent portion. 
         [0012]    Furthermore, another invention to solve the above issue is a flow cell for use in analysis of nucleic acid including: 
         [0013]    the substrate for use in analysis of nucleic acid; 
         [0014]    a light-transmitting cover placed to face the substrate for use in analysis of nucleic acid to transmit light; 
         [0015]    a plurality of spacers provided between the substrate for use in analysis of nucleic acid and the light-transmitting cover and separated substantially in parallel with each other; 
         [0016]    a flow passage formed in a portion interposed between the neighboring spacers between the substrate for use in analysis of nucleic acid and the light-transmitting cover to circulate a fluid; 
         [0017]    an inlet port opened in one end of the flow passage to inject the fluid; and 
         [0018]    an outlet port opened in the other end opposite to the inlet port of the flow passage to discharge the fluid. 
         [0019]    In addition, another invention to solve the above issue is a nucleic acid analysis device including: 
         [0020]    the flow cell for use in analysis of nucleic acid; 
         [0021]    a circulation unit that circulates a fluid in a flow passage of the flow cell for use in analysis of nucleic acid; 
         [0022]    a temperature control unit that controls a reactive temperature of the DNA fragment; 
         [0023]    an irradiation unit that irradiates excitation light onto an analysis area serving as an analysis target through the light-transmitting cover; 
         [0024]    a detection unit that detects fluorescence emitted from the DNA fragment by irradiating the excitation light using the light-transmitting cover and detects a position of the marker portion in the analysis area from the detected fluorescence; and 
         [0025]    a carriage unit that carries the flow cell for use in analysis of nucleic acid and shifts the analysis area to a predetermined position with respect to the marker portion. 
         [0026]    Note that, herein, a “predetermined shape” refers to a predefined shape (such as a cross shape) as seen in a plan view, used to determine a position inside the analysis area. In addition, herein, a “predetermined position” refers to a predefined position where the analysis area as an analysis target is shifted. Furthermore, herein, “a plurality of spacers” conceptually include a spacer provided with a single member such as a blanked sheet on which a plurality of spacers are formed by punching as well as a spacer provided with a plurality of members. 
       Advantageous Effects of Invention 
       [0027]    According to the present invention, it is possible to provide a substrate for use in analysis of nucleic acid, a flow cell for use in analysis of nucleic acid having the substrate for use in analysis of nucleic acid, and a nucleic acid analysis device, capable of reproducibly obtaining a position of the analysis area even when the positioning is repeatedly performed for the same analysis area. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0028]      FIG. 1  is a schematic plan view illustrating a substrate for use in analysis of nucleic acid according to a first embodiment of the present invention. 
           [0029]      FIG. 2  is a schematic plan view illustrating a substrate for use in analysis of nucleic acid according to a second embodiment of the present invention. 
           [0030]      FIG. 3( a )  is a schematic plan view illustrating a substrate for use in analysis of nucleic acid according to a third embodiment of the present invention, and  FIG. 3( b )  is a schematic diagram illustrating an exemplary fluorescent image. 
           [0031]      FIG. 4  is a schematic plan view illustrating a substrate for use in analysis of nucleic acid according to a fourth embodiment of the present invention, in which a single analysis area is enlargedly illustrated. 
           [0032]      FIGS. 5( a ) to 5( c )  are schematic diagrams illustrating a state that the fluorescent image obtained from the substrate for use in analysis of nucleic acid of  FIG. 4  is distorted, in which  FIG. 5( a )  shows a deformed state of fluorescent image,  FIG. 5( b )  shows a rotated state of the fluorescent image, and  FIG. 5( c )  shows another rotated state of the fluorescent image. 
           [0033]      FIGS. 6( a ) to 6( f )  are schematic diagrams illustrating a method of manufacturing the substrate for use in analysis of nucleic acid of  FIG. 1 , in which  FIG. 6( a )  shows a state before a hydrophilic membrane is formed,  FIG. 6( b )  shows a state after a hydrophilic membrane is formed,  FIG. 6( c )  shows a state after a resist film is formed,  FIG. 6( d )  shows a state after development,  FIG. 6( e )  shows a state after etching, and  FIG. 6( f )  shows a state after removal of the resist film. 
           [0034]      FIG. 7  is a schematic perspective view illustrating an exemplary flow cell for use in analysis of nucleic acid according to the present invention, in which the light-transmitting cover is partially cut away. 
           [0035]      FIG. 8  is a schematic diagram illustrating an exemplary nucleic acid analysis device according to the present invention. 
           [0036]      FIG. 9  is a flowchart illustrating a control process of the nucleic acid analysis device of  FIG. 8 . 
           [0037]      FIGS. 10( a ) to 10( c )  are schematic diagrams illustrating an exemplary analysis method using the nucleic acid analysis device according to the present invention, in which  FIG. 10( a )  shows a positional relationship of the marker portion in the fluorescent image before movement,  FIG. 10( b )  shows a positional relationship between the target location range and the marker portion before movement, and  FIG. 10( c )  shows a positional relationship between the target location range and the marker portion after the movement. 
           [0038]      FIG. 11  is a schematic diagram illustrating an exemplary fluorescent image using the substrate for use in analysis of nucleic acid of  FIG. 1 . 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0039]    &lt;Substrate for Use in Analysis of Nucleic Acid&gt; 
         [0040]    A substrate for use in analysis of nucleic acid according to the present invention has a plurality of analysis areas partitioned on a substrate, so that measurement is performed by sequentially changing each analysis area. The analysis area includes an absorbent portion capable of absorbing a DNA fragment or a vector where the DNA fragment (hereinafter, the DNA fragment and the vector will be collectively referred to as an “analysis sample”) is borne and a non-absorbent portion other than the absorbent portion. The non-absorbent portion has a marker portion having a predetermined shape for obtaining a position of the analysis area in at least a part thereof. 
         [0041]    Substrates for use in analysis of nucleic acid according to first to fourth embodiments of the present invention will now be described with reference to the accompanying drawings. The present invention is not limited to the first to fourth embodiments and relating drawings. 
       First Embodiment 
       [0042]      FIG. 1  is a schematic plan view illustrating the substrate for use in analysis of nucleic acid according to the first embodiment of the present invention. As illustrated in  FIG. 1 , the substrate for use in analysis of nucleic acid  100  according to this embodiment substantially includes a substrate  10 , a reaction area  11 , an analysis area  12 , an absorbent portion  13 , and a non-absorbent portion  14 . Note that, in  FIG. 1 , the reaction area  11 , the analysis area  12 , and a marker portion  15  (which will be described below) on the substrate  10  are enlargedly illustrated in a hierarchical manner. 
         [0043]    The substrate  10  is a plate-like base material having the reaction area  11  formed on its one surface. This substrate  10  may include a plate having a hydrophobic thin film on its surface, such as a quartz plate, a silicon plate, and a synthetic resin plate. The reaction area  11  is partitioned into a plurality of analysis areas  12  as described below. Note that, in this embodiment, the reaction area  11  is partitioned into one hundred forty analysis areas  12 . 
         [0044]    The analysis area  12  is an area for absorbing an analysis sample s. The analysis area  12  includes a plurality of absorbent portions  13  capable of absorbing the analysis sample s and a non-absorbent portion  14  other than the absorbent portions  13 . 
         [0045]    The absorbent portion  13  is formed of a hydrophilic membrane or the like laminated on the substrate  10  and exposed to the surface in order to allow absorption of the analysis samples s. This hydrophilic membrane may include, for example, a film of inorganic oxide or the like into which a functional group capable of fixing the analysis sample s (such as an amino group) is introduced. This specific inorganic oxide may include, for example, aminosilane. 
         [0046]    As illustrated in  FIG. 1 , each analysis area  12  has a plurality of absorbent portions  13  having a circular shape as seen in a plan view, and each absorbent portion  13  is arranged in a grid-like manner. In this manner, since each analysis area  12  has a plurality of absorbent portions  13 , and each absorbent portion  13  is arranged in a grid-like manner, it is possible to easily and reliably recognize positions of the absorbent portions  13  within the analysis area  12 . 
         [0047]    Note that the absorbent portion  13  typically has a diameter of 0.01 to 10 μm as seen in a plan view. A lower limitation of this diameter is preferably set to 0.05 μm, more preferably 0.1 μm, and most preferably 0.2 μm in terms of easiness in formation of the absorbent portion  13  and improvement of absorbance of the analysis sample. Meanwhile, an upper limitation of the diameter is preferably set to 5 μm, more preferably 1 μm, and most preferably 0.5 μm in terms of improvement of an arrangement density of the absorbent portion  13 . 
         [0048]    The diameter of the absorbent portion  13  as seen in a plan view is preferably set depending on a spatial resolution (pixel dimension) of a detection unit of the nucleic acid analysis device provided with this substrate for use in analysis of nucleic acid  100 . In this case, the diameter is preferably set to one pixel in terms of improvement of the arrangement density of the absorbent portion  13 . 
         [0049]    A pitch of the absorbent portion  13  is typically set to 0.05 to 50 μm. A lower limitation of the pitch is preferably set to 0.1 μm, more preferably 0.5 μm, and most preferably 1 μm in terms of improvement of an anti-interference property between the absorbent portions  13  neighboring in the analysis. Meanwhile, an upper limitation of the pitch is preferably set to 10 μm, more preferably 5 μm, and most preferably 2 μm in terms of improvement of the arrangement density of the absorbent portion  13 . 
         [0050]    Similar to the diameter, the pitch of the absorbent portion  13  is preferably set depending on a spatial resolution (pixel dimension) of a detection unit of the nucleic acid analysis device. In this case, the pitch is preferably set to four to five pixels, and more preferably five pixels in terms of the fluorescence resolution and improvement of the arrangement density of the absorbent portion  13 . 
         [0051]    The non-absorbent portion  14  includes a hydrophobic membrane laminated on the substrate  10  to prevent absorption of the analysis sample s. A compound for forming this hydrophobic membrane may include, for example, a multivalent organic compound, a carboxylic acid compound, a phosphate compound, a sulfate compound, a nitryl compound, and salts thereof. Note that the compound may be employed solely or as a combination of two or more compounds. 
         [0052]    The non-absorbent portion  14  has a marker portion  15  having a cross shape for computing a position of the analysis area  12  in its part. Since a DNA fragment capable of emitting fluorescence is not easily absorbed in this marker portion  15 , the marker portion  15  can be easily distinguished from the absorbent portion  13  using a fluorescent image of the analysis area  12 . 
         [0053]    Next, positioning of the analysis area  12  using the substrate for use in analysis of nucleic acid  100  according to the first embodiment will be described. 
         [0054]    If the substrate for use in analysis of nucleic acid  100  is employed, and excitation light emitting a particular wavelength is irradiated onto the analysis area  12  including the absorbent portion  13  absorbed with the DNA fragment (analysis sample) where a fluorochrome-labelled matrix corresponding to the base is introduced, fluorescence is emitted from an absorbent portion  13  where a particular dye excited by this excitation light exists (hereinafter, also referred to as a “particular absorbent portion”). Note that, for example, in the case of four color fluorescence detection (detection for four different types of fluorescence corresponding to four types of bases), a fluorescence probability caused by irradiation of excitation light having a particular wavelength in an arbitrary absorbent portion  13  becomes about 25%. 
         [0055]    Here, since a marker portion  15  having a predetermined shape for obtaining a position of the analysis area  12  (no fluorescent portion) is provided in at least a part of the non-absorbent portion  14 , the position of the analysis area  12  is determined by finding the marker portion  15  by searching the obtained fluorescent image. Then, the substrate for use in analysis of nucleic acid  100  is shifted on the basis of the determined position of the analysis area  12  to match this analysis area  12  with the fluorescent image range. As a result, it is possible to obtain a fluorescent image of the analysis area  12  serving as an analysis target. 
         [0056]    In this manner, on the substrate for use in analysis of nucleic acid  100 , at least a part of the non-absorbent portion  14  of the analysis area  12  has the marker portion  15  having a predetermined shape for obtaining a position of the analysis area  12 . Therefore, even when the positioning is repeated, it is possible to reproducibly obtain the position of the analysis area  12 . As a result, it is possible to reliably and rapidly analyze base sequences of DNA fragments. 
       Second Embodiment 
       [0057]      FIG. 2  is a schematic plan view illustrating a substrate for use in analysis of nucleic acid according to a second embodiment of the present invention. As illustrated in  FIG. 2 , the substrate for use in analysis of nucleic acid  200  according to the second embodiment substantially includes a substrate  10 , a reaction area  11 , an analysis area  12 , an absorbent portion  13 , and a non-absorbent portion  14 . The substrate for use in analysis of nucleic acid  200  according to the second embodiment is different from that of the first embodiment in that the shape of the marker portion  15  of the non-absorbent portion  14  as seen in a plan view is different depending on the analysis area  12 . Note that the substrate  10 , the reaction area  11 , the analysis area  12 , and the absorbent portion  13  are similar to those of the first embodiment. Therefore, like reference numerals denote like elements as in the first embodiment, and they will not be described repeatedly. 
         [0058]    According to this embodiment, the shape of the marker portion  15  as seen in a plan view is different between at least the neighboring analysis areas  12 . Specifically, as illustrated in  FIG. 2 , the substrate for use in analysis of nucleic acid  200  has two types of analysis areas  12   a  and  12   b  having different shapes of the marker portions  15  as seen in a plan view. The different types of analysis areas  12   a  and  12   b  are alternately arranged (the shape of the marker portion  15  is different between the odd-numbered analysis area  12   a  and the even-numbered analysis area  12   b ). In this embodiment, while the analysis area  12   a  has a marker portion  15   a,  the analysis area  12   b  has a marker portion  15   b  (refer to the shapes of the marker portions  15  in  FIG. 2 ). 
         [0059]    Next, positioning of the analysis area  12  using the substrate for use in analysis of nucleic acid  200  according to the second embodiment will be described. 
         [0060]    If the substrate for use in analysis of nucleic acid  200  is employed, the shapes of the marker portions  15   a  and  15   b  of the odd-numbered analysis area  12   a  and the even-numbered analysis area  12   b  are stored in an external computer (not illustrated) in advance. Then, similar to the positioning of the first embodiment, the positioning is performed for the initial analysis area  12 . 
         [0061]    Then, detection of fluorescence is performed by irradiating excitation light. In this detection of fluorescence, the shape of the marker portion  15  is recognized when the fluorescent image of the target analysis area  12  is obtained. On the basis of the recognized shape, whether this analysis area  12  is the odd-numbered analysis area  12   a  or the even-numbered analysis area  12   b  is determined. For example, if the initial analysis area  12  is set as an odd number, the next analysis area  12  shifted after the analysis of the analysis area  12   a  recognizes the even-numbered marker portions  15   b  unless an erroneous operation such as misalignment occurs in a carriage unit (described below) of the substrate for use in analysis of nucleic acid  200 . If the odd-numbered marker portion  15   a  is recognized instead of the even-numbered marker portion  15   b,  or if no fluorescence is detected from the obtained fluorescent image at all, this means that the corresponding area is not the target analysis area  12 . 
         [0062]    In this manner, the shape of the marker portion  15  on the substrate for use in analysis of nucleic acid  200  as seen in a plan view is different at least between the neighboring analysis areas  12 . Therefore, it is possible to distinguish each analysis area  12  on the basis of the shape of the marker portion  15  and reliably analyze the target analysis area  12 . 
         [0063]    Note that the planar shape of the marker portion  15  is preferably different between overall analysis areas  12 . Specifically, the shape of the marker portion  15  may include, for example, a numeric shape, a shape obtained by modeling a distinguishable symbol, or the like (not shown). 
         [0064]    In this manner, since the planar shape of the marker portion  15  is different between overall analysis areas  12 , it is possible to clearly distinguish each analysis area  12  on the basis of the shape of the marker portion  15  and more reliably analyze the target analysis area  12 . 
       Third Embodiment 
       [0065]      FIGS. 3( a ) and 3( b )  are schematic diagrams illustrating a substrate for use in analysis of nucleic acid according to a third embodiment of the present invention. As illustrated in  FIG. 3( a ) , the substrate for use in analysis of nucleic acid  300  according to the third embodiment substantially includes a substrate  10 , a reaction area  11 , an analysis area  12 , an absorbent portion  13 , and a non-absorbent portion  14 . The substrate for use in analysis of nucleic acid  300  according to the third embodiment is different from that of the first embodiment in the absorbent portion  13  and the non-absorbent portion  14 . Note that the substrate  10 , the reaction area  11 , and the analysis area  12  are similar to those of the first embodiment. Therefore, like reference numerals denote like elements as in the first embodiment, and they will not be described repeatedly. In addition, the positioning of the analysis area  12  using the substrate for use in analysis of nucleic acid  300  according to the third embodiment is similar to that of the first embodiment, and it will not also be described repeatedly. 
         [0066]    According to this embodiment, the entire area of the non-absorbent portion  14  is the marker portion  15 , and the remaining area of the analysis area  12  other than the marker portion  15  is the absorbent portion  13 . Specifically, as illustrated in  FIG. 3( a ) , the non-absorbent portion  14  having a cross shape and serving as the marker portion  15  is formed in an approximate center of the analysis area  12 , and the entire remaining area of this analysis area  12  other than the non-absorbent portion  14  (marker portion  15 ) is the absorbent portion  13 . Note that  FIG. 3( b )  illustrates an exemplary fluorescent image k obtained from the substrate for use in analysis of nucleic acid  300  according to this embodiment. In this drawing, white dots indicate portions corresponding to the analysis samples s emitting fluorescence. 
         [0067]    In this manner, in the substrate for use in analysis of nucleic acid  300 , the entire area of the non-absorbent portion  14  is the marker portion  15 , and the remaining area of the analysis area  12  other than marker portion  15  is the absorbent portion  13 . Therefore, it is possible to densely arrange the analysis sample s and analyze an amount of images at once. 
       Fourth Embodiment 
       [0068]      FIG. 4  is a schematic plan view illustrating a substrate for use in analysis of nucleic acid  400  according to a fourth embodiment of the present invention, in which a single analysis area  12  is enlargedly illustrated. The substrate for use in analysis of nucleic acid  400  according to the fourth embodiment substantially includes a substrate  10 , a reaction area  11 , an analysis area  12 , an absorbent portion  13 , and a non-absorbent portion  14 . As illustrated in  FIG. 4 , the substrate for use in analysis of nucleic acid  400  according to the fourth embodiment is different from that of the first embodiment in that the marker portion  15  is arranged in each of four corners in addition to the center of the analysis area  12 . Note that, since the substrate  10 , the reaction area  11 , and the analysis area  12  are similar to those of the first embodiment, they will not be described repeatedly. Furthermore, the absorbent portion  13  is similar to that of the first embodiment, and a plurality of absorbent portions  13  are not illustrated intentionally in  FIG. 4  for simplicity purposes. 
         [0069]    According to this embodiment, the marker portion  15  is used in positional correction of each absorbent portion  13  in the analysis area  12 . Specifically, the substrate for use in analysis of nucleic acid  400  includes a marker portion  15   c  placed in the center of the analysis area  12  and L-shaped marker portions  15   d  provided in each of four corners of the analysis area  12 . In addition, the marker portions  15   d  placed in the four corners are arranged such that straight lines obtained by linking the neighboring marker portions  15   d  of each corner are separated from the marker portion  15   c  placed in the center at the same distance “a.” Furthermore, although not shown in the drawing, each absorbent portion  13  is arranged in a grid shape at an interval of five pixels on the analysis area other than the marker portion  15 . 
         [0070]    Next, positional correction of each absorbent portion  13  in the analysis area  12  using the substrate for use in analysis of nucleic acid  400  according to the fourth embodiment will be described with reference to  FIGS. 5( a ) to 5( c ) . 
         [0071]    For example, positional correction of the absorbent portion  13  may be performed through the following method if a fluorescent image obtained from the substrate for use in analysis of nucleic acid  400  has a distortion. Specifically, as illustrated in  FIG. 5( a ) , due to a distortion of the fluorescent image k 1 , a portion  15   c ′ corresponding to the marker portion  15   c  in the center of the fluorescent image k 1  (hereinafter, referred to as a “marker mapping portion  15   c ′”) is separated from straight lines obtained by linking portions corresponding to the marker portions  15   d  in the neighboring corners (marker mapping portions  15   d ′) at distances b, c, d, and e. In this case, as a method of estimating the position of the absorbent portion  13  in practice, for example, an interpolation technique such as a linear interpolation technique may be employed, in which interpolation is performed for each of the upper left, upper right, lower left, and lower right regions with respect to the marker mapping portion  15   c′.    
         [0072]    For example, as an example of such a linear interpolation method, an actual pitch is calculated by obtaining a proportion of the distance “a” (refer to  FIG. 4 ) against each of the distance “b, c, d, and e” and multiplying the proportions by five pixels. Specifically, an actual pitch of the BD direction in the B-side region from the marker mapping portion  15   c ′ in  FIG. 5( a )  becomes “b/a×five pixels.” An actual pitch of the CE direction in the E-side region from the marker mapping portion  15   c ′ becomes “e/a×five pixels.” An actual pitch of the BD direction in the D-side region from the marker mapping portion  15   c ′ becomes “d/a×five pixels.” An actual pitch of the CE direction in the C-side region from the marker mapping portion  15   c ′ becomes “c/a×five pixels.” Using these values, positional correction of each absorbent portion  13  is performed. 
         [0073]    Since a distortion of the fluorescent image depends on a detector of the nucleic acid analysis device, the aforementioned calculation may be performed only for the initial analysis area  12  of the first cycle. The correction is performed for the analysis areas  12  to be analyzed after using this calculation result. In addition, if two or more detectors are used in analysis, the calculation and the correction are performed in each detector. 
         [0074]    Meanwhile, for a distortion (deviation) of the fluorescent image in the rotational direction with respect to the optical axis of the excitation light irradiated onto the substrate for use in analysis of nucleic acid  400 , the positional correction of the absorbent portion  13  can be performed in the following way. Specifically, as illustrated in  FIG. 5( b ) , an inclination θ 1  is calculated from the marker mapping portion  15   d ′ of the fluorescent image k 2  obtained by the detector. Then, correction in the rotational direction is performed on the basis of the calculated inclination θ 1 . Note that, since the distortion of the rotational direction in the fluorescent image k depends on the detector of the nucleic acid analysis device, the aforementioned calculation may be performed only for the initial analysis area  12  of the first cycle, and the correction is performed for the analysis areas  12  to be analyzed after using this calculation result. In addition, if two or more detectors are used in the analysis, the calculation and the correction are performed in each detector. For example,  FIG. 5( c )  illustrates a fluorescent image k 3  (inclination θ 2 ) detected from another detector different from the aforementioned detector. 
         [0075]    In this manner, since the marker portion  15  is used in positional correction of each absorbent portion  13  in the analysis area  12 , it is possible to accurately recognize a position of each absorbent portion  13  in the analysis area  12 . 
         [0076]    &lt;Method of Manufacturing Substrate for Use in Analysis of Nucleic Acid&gt; 
         [0077]    Next, a method of manufacturing the aforementioned substrate for use in analysis of nucleic acid will be described.  FIGS. 6( a ) to 6( f )  are schematic diagrams illustrating a method of manufacturing the substrate for use in analysis of nucleic acid  100  of  FIG. 1 . This substrate for use in analysis of nucleic acid  100  may be manufactured, for example, using a method described in JP 2011-99720 A. That is, a substrate  10  having one surface where a hydrophobic membrane is laminated in advance (refer to  FIG. 6( a ) ) is prepared. On this hydrophobic membrane, a hydrophilic membrane  16  formed of specific inorganic oxide or the like is deposited, for example, through vacuum deposition, sputtering, chemic vapor deposition (CVD), physical vapor deposition (PVD), or the like (refer to  FIG. 6( b ) ). 
         [0078]    Then, a resist  17  is coated on the obtained hydrophilic membrane  16  (refer to  FIG. 6( c ) ). Then, predetermined patterning is performed using a photolithographic technique (refer to  FIG. 6( d ) ). An unnecessary part of the hydrophilic membrane is removed through etching by using the patterned resist  17  as a mask (refer to  FIG. 6( e ) ), and the remaining resist  17  is removed by dissolving (refer to  FIG. 6( f ) ). As a result, it is possible to manufacture a substrate for use in analysis of nucleic acid  100  having a desired absorbent portion  13  provided with the hydrophilic membrane  16 . Note that, since the hydrophobic membrane is exposed on a part of the analysis area  12  other than the absorbent portion  13 , this part serves as the non-absorbent portion  14 . 
         [0079]    &lt;Flow Cell for Use in Analysis of Nucleic Acid&gt; 
         [0080]    The flow cell for use in analysis of nucleic acid according to the present invention includes the substrate for use in analysis of nucleic acid, a light-transmitting cover disposed to face the substrate for use in analysis of nucleic acid to transmit light, a plurality of spacers arranged between the substrate for use in analysis of nucleic acid and the light-transmitting cover and separated substantially in parallel with each other, a flow passage formed in a portion interposed by the neighboring spacers between the substrate for use in analysis of nucleic acid and the light-transmitting cover to circulate a fluid, an inlet port opened in one end of the flow passage to inject the fluid, and an outlet port opened in the other end opposite to the inlet port of the flow passage to discharge the fluid. 
         [0081]    The flow cell for use in analysis of nucleic acid according to the present invention will now be described with the accompanying drawings. However, the present invention is not limited to the embodiments illustrated in the drawings. 
         [0082]      FIG. 7  is a schematic perspective view illustrating an exemplary flow cell for use in analysis of nucleic acid according to the present invention, in which the light-transmitting cover is partially cut away. As illustrated in  FIG. 7 , the flow cell for use in analysis of nucleic acid  500  substantially includes a substrate for use in analysis of nucleic acid  100 , a light-transmitting cover  21 , spacers  22 , and a flow passage  23 . Note that, since the substrate for use in analysis of nucleic acid  100  described above is employed as the substrate for use in analysis of nucleic acid in this embodiment, like reference numerals denote like element, and they will not be described repeatedly. 
         [0083]    The light-transmitting cover  21  is a flat cover placed to face the substrate for use in analysis of nucleic acid  100  to transmit light. The light-transmitting cover may be formed of, for example, glass such as soda glass, quartz glass, or sapphire glass, light-transmitting resin such as transparent polyimide resin, or polycarbonate resin, and the like. 
         [0084]    The spacers  22  are placed between the substrate for use in analysis of nucleic acid  100  and the light-transmitting cover  21  and are separated substantially in parallel with each other. The flow cell for use in analysis of nucleic acid  500  has a plurality of spacers  22 . The spacers  22  may be formed of, for example, thermosetting or photosetting epoxy resin, acrylic resin, silicon resin, and the like as disclosed in JP 2006-87974 A. Out of these materials, in terms of improvement of a bonding strength with the glass or the light-transmitting resin, silicon resin is preferable, polysiloxane is more preferable, and polydimethylsiloxane (PDMS) is most preferable. In addition, the spacers  22  preferably have a thickness of 0.05 to 2 mm, and more preferably 0.2 to 1 mm, but not particularly limited thereto. 
         [0085]    The flow passage  23  is a flow passage formed in a portion interposed between the neighboring spacers  22  between the substrate for use in analysis of nucleic acid  100  and the light-transmitting cover  21  to flow a fluid. For example, a fluid such as a reagent that can react with the DNA fragment flows through this flow passage  23 . Specifically, the flow passage  23  is surrounded by the substrate for use in analysis of nucleic acid  100 , the light-transmitting cover  21 , and the spacers  22  to form a rectangular cross section as seen in the flow direction and a substantially rectangular parallel-piped space extending in the flow direction. The flow passage  23  has an inlet port  23   a  opened in one end to inject the fluid and an output port  23   b  opened in the other end opposite to the inlet port  23   a  to discharge the fluid. 
         [0086]    In this manner, since the flow cell for use in analysis of nucleic acid  500  has the substrate for use in analysis of nucleic acid  100 , it is possible to reproducibly obtain the position of the analysis area  12  even when positioning is repeatedly performed during the nucleic acid analysis. As a result, it is possible to reliably and rapidly analyze base sequences of DNA fragments. 
         [0087]    &lt;Method of Manufacturing Flow Cell for Use in Analysis of Nucleic Acid&gt; 
         [0088]    Next, a method of manufacturing the flow cell for use in analysis of nucleic acid  500  will be described. For the flow cell for use in analysis of nucleic acid  500 , for example, a pair of spacers  22  are bonded to the substrate for use in analysis of nucleic acid  100  described above in parallel with each other using an adhesive. Then, the light-transmitting cover  21  is bonded to the bonded spacers  22  using an adhesive. Note that any type of adhesive may be employed without a particular limitation as long as it does not affect the nucleic acid analysis. Through the aforementioned process it is possible to manufacture the flow cell for use in analysis of nucleic acid  500  according to the present invention. 
         [0089]    &lt;Nucleic Acid Analysis Device&gt; 
         [0090]    The nucleic acid analysis device according to the present invention includes the flow cell for use in analysis of nucleic acid, a circulation unit that circulates the fluid in the flow passage of the flow cell for use in analysis of nucleic acid, a temperature control unit that controls an reactive temperature of the DNA fragment, an irradiation unit that irradiates excitation light onto the analysis area as an analysis target through the light-transmitting cover, a detection unit that detects the fluorescence emitted from the DNA fragment by irradiating the excitation light through the light-transmitting cover and detects a position of the marker portion in the analysis area from the detected fluorescence, and a carriage unit that carries the flow cell for use in analysis of nucleic acid and shifts the analysis area to a predetermined position with respect to the marker portion. 
         [0091]    A nucleic acid analysis device according to the present invention will now be described with reference to the accompanying drawings. However, the present invention is not limited to the embodiments illustrated in the drawings. 
         [0092]      FIG. 8  is a schematic diagram illustrating an exemplary nucleic acid analysis device according to the present invention. As illustrated in  FIG. 8 , the nucleic acid analysis device  600  substantially includes a flow cell for use in analysis of nucleic acid  500 , a circulation unit  31 , a temperature control unit  32 , an irradiation unit  33 , a detection unit  34 , and a carriage unit  35 . Note that the flow cell for use in analysis of nucleic acid  500  is similar to the flow cell for use in analysis of nucleic acid  500  described in the paragraph &lt;Flow Cell for Use in Analysis of Nucleic Acid&gt; described above. Therefore, like reference numerals denote like elements, and they will not be described repeatedly. 
         [0093]    The circulation unit  31  circulates the fluid in the flow passage  23  of the flow cell for use in analysis of nucleic acid  500 . The circulation unit  31  has a reagent cooling storage chamber  312  that houses a plurality of reagent containers  311  containing the reagent, a nozzle  313  that accesses the reagent container  311 , a pipe  314  that introduces the reagent into the flow cell for use in analysis of nucleic acid  500 , and a waste liquid reservoir  315  that disposes the reagent reacting with the DNA fragments. 
         [0094]    The temperature control unit  32  controls a reaction temperature of the DNA fragment. The temperature control unit  32  has a temperature control substrate  321  provided on an XY-stage  351  described below to promote a reaction between the DNA fragment (analysis sample s) to be analyzed and the reagent. The temperature control substrate  321  is embedded with, for example, a peltier device. 
         [0095]    The irradiation unit  33  has a light source  331  such as a light emitting diode (LED) serving as the excitation light, a filter switching mechanism  332  capable of selecting an arbitrary wavelength from the excitation light emitted from the light source  331 , a dichroic mirror  333  that reflects the excitation light and transmits the fluorescence described below, an objective lens  334  that irradiates the excitation light onto the analysis sample s to be analyzed, and a Z-stage  335  that drives the objective lens  334  in the Z-axis direction perpendicular to both the X-axis and the Y-axis to adjust a focus of the excitation light. 
         [0096]    The detection unit  34  detects the fluorescence emitted from the DNA fragment by irradiating the excitation light through the light-transmitting cover  21  and a position of the marker portion  15  in the analysis area  12  from the detected fluorescence. The detection unit  34  has an objective lens  334  that recovers the fluorescence emitted from the analysis sample s, a fluorescence separation dichroic mirror  341  that divides parallel light from the objective lens  334  on a wavelength basis, a tube lens  342  that focuses the parallel light, and a detector  343  provided with a sensor such as a complementary metal oxide semiconductor (CMOS) sensor for detecting the focused image. Note that, since the objective lens  334  of the detection unit  34  is shared with the irradiation unit  33 , the same reference numerals are used. 
         [0097]    The carriage unit  35  carries the flow cell for use in analysis of nucleic acid  500  and shifts the analysis area  12  to a predetermined position with respect to the marker portion  15 . The carriage unit  35  has an XY-stage  351  capable of delivering the flow cell for use in analysis of nucleic acid in each of the X-axis and Y-axis directions coplanarly perpendicular to each other and a driving motor (not shown) that drives the XY-stage  351 . Note that the XY-stage  351  is controlled in an open loop manner. 
         [0098]    &lt;Analysis Method&gt; 
         [0099]    Next, a method of analyzing base sequences of the DNA fragments using the nucleic acid analysis device  600  according to the present invention will be described with reference to  FIGS. 8 to 11 . Note that, here, a case where the analysis sample s is a vector that contains the DNA fragment, and the flow cell for use in analysis of nucleic acid  500  provided with the substrate for use in analysis of nucleic acid  100  according to the first embodiment described above is employed will be described by way of example. 
         [0100]    The analysis using the nucleic acid analysis device  600  may be performed, for example, by combining processes described below, including “Preparation of Flow Cell,” “Installation of Flow Cell,” “Introduction of Reagent,” “Temperature Control,” “Shift of Stage,” and “Positioning of Stage.” While each process will be described in details hereinafter, the analysis using the nucleic acid analysis device  600  is not limited to the following aspects. 
         [0101]    &lt;Preparation of Flow Cell&gt; 
         [0102]    In this process, a flow cell for use in analysis of nucleic acid  500  (refer to  FIG. 7 ) where the analysis sample s is borne in advance is prepared. The substrate for use in analysis of nucleic acid  100  of the flow cell for use in analysis of nucleic acid  500  has an absorbent portion  13  and a non-absorbent portion  14  in each analysis area  12  on the substrate  10 , and a marker portion  15  having a cross shape is formed in the center of the non-absorbent portion  14  as illustrated in  FIG. 1 . Note that the analysis sample s is borne only in the absorbent portion  13 . 
         [0103]    &lt;Installation of Flow Cell&gt; 
         [0104]    In this process, the flow cell for use in analysis of nucleic acid  500  prepared in the process &lt;Preparation of Flow Cell&gt; is fixed to the temperature control substrate  321  provided on the XY-stage  351  of the nucleic acid analysis device  600 . 
         [0105]    &lt;Introduction of Reagent&gt; 
         [0106]    In this process, the nozzle  313  of the circulation unit  31  accesses the reagent container  311  of the reagent cooling storage chamber  312  to suction the reagent. Then, the suctioned reagent is injected into the flow passage of the flow cell for use in analysis of nucleic acid  500  through the pipe  314  and the inlet port  23   a,  and the injected reagent comes into contact with the analysis sample s borne in the absorbent portion to generate reaction. Note that the reagent subjected to the aforementioned reaction is disposed to the waste liquid reservoir  315  through the pipe. 
         [0107]    &lt;Temperature Control&gt; 
         [0108]    In this process, a temperature control is performed for the flow cell for use in analysis of nucleic acid  500  using the temperature control substrate  321  to allow the analysis sample s to have a predetermined temperature. Through this temperature control, the reagent reacts with the analysis sample s of the flow cell for use in analysis of nucleic acid  500 . In this case, DNA elongation is performed by suitably repeating the aforementioned processes “Introduction of Reagent” and “Temperature Control.” This elongation is performed by reacting polymerase with four types of nucleotide labeled with different fluorochromes. The nucleotide includes FAM-dCTP, Cy3-dATP, Texas Red-dGTP, or Cy5-dTsTP. The reagent contains polymerase, and only one base of complementary fluorescence nucleotide is incorporated into the DNA fragment. 
         [0109]    &lt;Shift of Stage&gt; 
         [0110]    In this process, in order to observe the reacted analysis sample s, the XY-stage  351  is driven by a driving motor (not shown) to shift the flow cell for use in analysis of nucleic acid  500  to a preset position. Here, the “preset position” refers to an initial target position of the analysis area  12  where the marker portion  15  is to be placed within a fluorescence detection range of the detection unit  34 . Note that the accurate positioning of the stage will be described below in more details in the following paragraph “Positioning of Stage.” 
         [0111]    &lt;Positioning of Stage&gt; 
         [0112]    In this process, first, a focus position of the analysis sample s in the objective lens  334  is adjusted by driving the Z-stage  335  of the detection unit  34 . Then, after shifting the objective lens  334  to the focus position, excitation light having a particular wavelength is irradiated onto the analysis sample s using the filter switching mechanism  332 . In this case, through the irradiation of excitation light, only an analysis sample s corresponding to an excitation wavelength out of the analysis samples s borne in the absorbent portion  13  emits fluorescence. Meanwhile, the marker portion  15  does not emit fluorescence. 
         [0113]    Then, a fluorescent image k is obtained using the detection unit  34 . In this case, for example, when the four colors of fluorescence described above are detected, the analysis sample s emits fluorescence with a probability of “¼.” Therefore, it is possible to perceive a shape of the marker portion  15  that does not emit fluorescence out of the obtained fluorescent image k. Then, as illustrated in FIGS.  10 ( a ) to  10 ( c ), for the detection range of the fluorescent image k, a marker portion  15  having a shape stored in a computer (not shown) in advance is searched. In this case, if the marker portion  15  is searched, pixel values of the X-axis and the Y-axis in the center of the marker portion  15  (coordinates converted into pixels of the marker portion  15  in the fluorescent image k) are calculated (refer to  FIG. 10( a ) ). If the marker portion  15  is not searched, the search area moves to the next analysis area by driving the XY-stage  351 . Note that, if the calculated pixel value is within a target location range  344 , the positioning of the XY-stage  351  is not performed. Meanwhile, if the calculated pixel value is out of the target location range  344 , relative pixel numbers Xa and Ya for this pixel values for the center position within the target location range  344  are calculated (refer to  FIG. 10( b ) ). Then, the pixel numbers Xa and Ya are converted into the number of pulses necessary in the positioning, and the number of pulses is transmitted to the carriage unit  35 . After this transmission, the XY-stage  351  is moved by driving the driving motor of the carriage unit  35  (refer to  FIG. 10( c ) ). 
         [0114]    Then, irradiation of the excitation light and position detection of the marker portion  15  using the detection unit  34  are performed again to check whether or not the marker portion  15  is shifted to the target location range  344 . In this case, if the marker portion  15  is within the target location range  344 , the positioning of the XY-stage  351  is completed. Meanwhile, if the marker portion  15  is out of the target location range  344 , the positioning is performed again. Then, after this positioning is completed, this position (the aforementioned number of pulses) is stored. Note that, if the position of the marker portion  15  does not change after the positioning of the XY-stage  351 , it is considered as misalignment of the driving motor, and the analysis is interrupted by outputting an alarm. 
         [0115]    &lt;Detection of Fluorescence&gt; 
         [0116]    In this process, a focus position is adjusted by driving the objective lens  334  of the detection unit  34  again. Note that this re-adjustment of the focus position is performed to correct a deviation in the vertical direction caused by the movement of the XY-stage. However, if the detection unit  34  and the analysis samples have a sufficient depth of field, this adjustment is not necessary. 
         [0117]    Then, using the filter switching mechanism  332 , the excitation light is irradiated onto the analysis area  12  by switching the excitation light between two wavelength bands of 490 nm and 595 nm as a median, and the fluorescence is detected on each occasion. Here, the excitation light having a wavelength of 490 nm as a median is used to detect fluorescence of FAM-dCTP and Cy3-dATP, and the excitation light having a wavelength of 595 nm as a median is used to detect fluorescence of Texas Red-dGTP and Cy5-dTsTP. 
         [0118]    The fluorescence emitted from the analysis sample  12  is input to a pair of detectors  343  through the fluorescence separation dichroic mirror  341 . Here, since the fluorescence separation dichroic mirror  341  has a smooth reflection characteristic for the four color fluorescence wavelength regions, it is possible to calculate a ratio of the fluorescence intensity at a bright spot emitted from the analysis sample s using a pair of detectors  343 . For this reason, by calculating an intensity ratio on the image planes of a pair of detectors  343 , it is possible to determine which one of the four colors of fluorescence the fluorescence of the analysis sample s belongs to. Note that several ten thousands to several hundred thousands of analysis samples s are borne on the analysis sample  12 , and which position of the analysis samples s emits which fluorescence is detected in a batch through the fluorescence detection. 
         [0119]    Next, an exemplary fluorescent image k detected through the fluorescence detection is illustrated in  FIG. 11 . In  FIG. 11 , reference numeral “ 345 ” denotes pixels of the fluorescent image k, reference numeral “ 13 ′” denotes a portion on the fluorescent image k corresponding to the absorbent portion  13 , and reference numeral “ 15 ′” denotes a portion on the fluorescent image k corresponding to the marker portion  15 .  FIG. 11  illustrates an exemplary fluorescent image when each absorbent portion  13  is arranged at an interval of 1.4 μm, a spatial resolution of the detection unit  34  is set to 0.28 μm/pixel, and the absorbent portion  13  is arranged at the same interval as five pixels of the fluorescent image k. In this example, the analysis sample s has a size of 0.28 μm or larger. For this reason, when the fluorescence of the analysis sample s is detected, a sample position is specified at an interval of five pixels from the edge of the marker portion  15 , and fluorescence of nine pixels (three pixels in each direction) is detected for each position of each analysis sample s. 
         [0120]    After completing the detection of fluorescence for a single analysis area  12 , the detection range is shifted to the next analysis area  12 . Then, the positioning on the XY-stage  351  is performed again, and fluorescence is detected from this analysis area  12 . The movement of the XY-stage  351 , the positioning of the analysis sample s, the storing of the position of the analysis sample s, and the fluorescence detection are repeated until detection is completed for all of the analysis areas  12 . Note that, in order to reduce the analysis time, the positioning of the analysis sample s is performed only for an arbitrary analysis area  12 , and the position information stored as described above may be used for the other analysis areas  12 . Hereinbefore, an operation for a single cycle in this analysis has been described in brief. 
         [0121]    Next, the analysis is performed for the second and subsequent cycles. In the stage movement of the second and subsequent cycles, the positioning is performed by shifting to the position of the initial analysis area  12  obtained in the positioning of the first cycle. This positioning is performed to correct thermal expansion caused by an internal temperature change of the nucleic acid analysis device  600 . After this positioning is completed, the corrected position is stored. 
         [0122]    For the subsequent analysis area  12 , the analysis is performed by shifting to a relative position from the initial analysis area  12  obtained in the first cycle. For example, it is assumed that the initial analysis areas  12  of the first cycle and the subsequent two analysis areas  12  are positioned in 1,000 μm, 2,000 μm, and 3,000 μm, respectively. If the initial analysis area  12  of the second cycle is positioned in 950 μm, the next two analysis areas  12  are positioned in 1,950 μm and 2,950 μm. 
         [0123]    Note that, in this operation, an error or a positional deviation may occur in accuracy of the repeated positioning of the XY-stage  351 . If the accuracy of the repeated positioning of the XY-stage  351  is sufficiently low, a detection loss is not easily generated. However, if the accuracy of the repeated positioning of the XY-stage  351  is high, it is not negligible. If the accuracy of repeated positioning is high, it is necessary to increase the analysis areas  12  for the positioning considering the detection loss and the detection time. 
         [0124]    By repeating the aforementioned cycles, the DNA base sequences of the analysis sample s are analyzed. For example, assuming that a certain analysis sample s emits fluorescence for each cycle in order of Cy3→Texas Red→FAM→Cy5→ . . . , the base sequence of the sample can be determined as “A→G→C→T→ . . . ” from the dNTP corresponding to the fluorochrome. In this manner, fluorescence is detected from several ten thousands to hundred thousands of analysis samples s in a batch, and all of the base sequences of the analysis samples s are determined in parallel. 
         [0125]    Note that, although the analysis of the DNA base sequences of the analysis samples s starts from the first cycle in this example, the first cycle may be a mapping operation of the analysis area  12 . In this mapping operation, if, for example, Texas Red-dGTP is incorporated into the DNA fragments of all of the analysis samples, all of the analysis samples emit fluorescence using excitation light having a wavelength of 595 nm. As a result, it is possible more reliably detect the marker portion. Therefore, it is possible to reduce the analysis area  12  where the positioning is not performed. 
         [0126]    In this manner, the nucleic acid analysis device  600  has the flow cell for use in analysis of nucleic acid  500  provided with this substrate for use in analysis of nucleic acid  100 . Therefore, even when the repeated positioning is performed, it is possible to reproducibly obtain the position of the absorbent portion  13 . As a result, it is possible to reliably and rapidly analyze the base sequences of the DNA fragments. 
         [0127]    Note that the substrate for use in analysis of nucleic acid, the flow cell for use in analysis of nucleic acid, and the nucleic acid analysis device according to the present invention are not limited to the embodiments described above, but the scope of the invention may encompass those described in the claims, their equivalents, and all possible changes. 
         [0128]    For example, although the marker portion  15  has a cross shape or a hook shape by way of example in the aforementioned embodiments, any shape of the marker portion, such as a star shape or a circular shape, may be employed as long as the position of the marker portion  15  can be specified in the analysis area  12 . 
         [0129]    Although the substrate for use in analysis of nucleic acid  400  having the marker portions  15  formed in the center and the four corners of the analysis area  12  has been described in  FIGS. 4 and 5 ( a ) to  5 ( c ), the scope of the invention may also include a substrate having no marker portion  15  in the center or having the marker portions  15  formed in only in opposite corners. 
         [0130]    Although the flow cell for use in analysis of nucleic acid  500  provided with the substrate for use in analysis of nucleic acid  100  has been described in the aforementioned embodiments, any substrate for use in analysis of nucleic acid may also be employed as long as it satisfies the configuration of the substrate for use in analysis of nucleic acid according to the present invention. 
         [0131]    Although the flow cell for use in analysis of nucleic acid  500  provided with a pair of spacers  22  separated substantially in parallel with each other has been described in  FIG. 7 , the flow cell for use in analysis of nucleic acid may have three or more spacers  22  to provide a plurality of lines of flow passages  23 . For example, a flow cell for use in analysis of nucleic acid formed of polydimethylsiloxane (PDMS) or the like and provided with a blanked sheet obtained by blanking the flow passage portion may also be employed. 
         [0132]    While the nucleic acid analysis device  600  provided with the flow cell for use in analysis of nucleic acid  500  has been described in the aforementioned embodiments, any flow cell for use in analysis of nucleic acid may also be employed as long as it satisfies a configuration of the flow cell for use in analysis of nucleic acid according to the present invention. 
         [0133]    While the nucleic acid analysis device  600  having the detection unit  34  provided with a pair of detectors  343  has been described in  FIG. 8 , the nucleic acid analysis device may be additionally provided with a fluorescence separation dichroic mirror, and the nucleic acid analysis device may have the detection unit  34  having three or four detectors. 
       REFERENCE SIGNS LIST 
       [0000]    
       
           10  substrate 
           11  reaction area 
           12  analysis area 
           13  absorbent portion 
           14  non-absorbent portion 
           15  marker portion 
           21  light-transmitting cover 
           22  spacer 
           23  flow passage 
           23   a  inlet port 
           23   b  outlet port 
           31  circulation unit 
           32  temperature control unit 
           33  irradiation unit 
           34  detection unit 
           35  carriage unit 
           100 ,  200 ,  300 ,  400  substrate for use in analysis of nucleic acid 
           500  flow cell for use in analysis of nucleic acid 
           600  nucleic acid analysis device 
         s analysis sample 
         k fluorescent image