JP6143282B2 - Nucleic acid extension / fixation device, nucleic acid extension / fixation method using the device, nucleic acid fixation member, and nucleic acid sample prepared using nucleic acid extension / fixation device - Google Patents

Description

  The present invention relates to a nucleic acid extension / fixation device, a nucleic acid extension / fixation method using the device, a nucleic acid fixing member, and a nucleic acid sample prepared using the nucleic acid extension / fixation device, and in particular, DNA and RNA A nucleic acid capable of efficiently immobilizing an elongated nucleic acid by providing a flow path capable of flowing a solution containing a solution at a predetermined speed and providing a fixing portion for fixing the nucleic acid at a portion facing the flow path And a nucleic acid fixing member that can be used for the device, and a nucleic acid specimen prepared using the nucleic acid extension / fixation device.

  In recent years, analysis of SNPs and DNA methylation has attracted attention in the research fields of lifestyle-related diseases, cancer, aging, or drug effects and side effects. For the analysis of SNPs and DNA methylation, single-molecule imaging of DNA and single-molecule analysis of the interaction between DNA and protein are effective. For this reason, research to extend and immobilize individual DNA molecules has been conducted in Japan and overseas since the end of the 20th century. It is advanced vigorously.

  Techniques for extending and immobilizing DNA include (1) a method of elongating a DNA molecule in molten agarose and immobilizing it during gelation (see Non-Patent Document 1), and (2) applying an alternating electric field. A method of extending DNA molecules in a concentrated acrylamide solution by the above (see Non-Patent Document 2) is known. However, since the methods described in the above (1) and (2) extend DNA in a gel, the linearity of DNA is poor and handling is difficult, and the accuracy is accompanied by unintended DNA cleavage. There are problems such as lowering. Further, the method of extending DNA by applying an electric field also has a problem that when the application of the electric field is stopped, the extended DNA returns to its original form.

  On the other hand, there is also known a technique that does not use a gel when extending and immobilizing DNA. For example, (3) As an example using the principle of a solution and a gas meniscus, a glass substrate is placed in an aqueous solution in which DNA is dissolved. There is known a method (see Non-Patent Document 3) in which DNA is stretched and fixed by utilizing the interfacial movement between an aqueous phase and a gas phase in the process of dipping and then pulling up the glass substrate. Compared with the method using the gel described in the above (1) and (2), the method described in the above (3) can stably extend the DNA and obtain a high throughput. There is a problem that the place to be performed cannot be controlled and is randomly arranged. Further, in order to immobilize DNA, chemical modification of the DNA end and the substrate surface is necessary, and therefore, there is a problem that the DNA once immobilized cannot be transferred to another medium.

  In addition, as a method not using a gel, (4) a method in which one end of a nucleic acid molecule is fixed to a substrate via an amino group and DNA is fixed on the substrate using a centrifugal force by rotation ( Patent Document 1). However, the method described in the above (4) has a problem that it requires modification with an amino group and further requires a centrifuge.

  Unlike the methods described in (3) and (4) above, (5) a method of extending and fixing a DNA molecule on a substrate without modification is also known. As shown in FIG. 1, the method described in the above (5) is performed by sandwiching a DNA solution between a resin substrate having a concave microhole structure and a cover glass and evaporating the solution while sliding the substrate. This is a method of extending and fixing DNA on a substrate (see Non-Patent Document 4). However, the method described in (5) above can efficiently extend and fix DNA at a predetermined position. However, in order to extend and fix DNA, the distance between the substrate and the cover glass must be set small. There is a problem that a special device and technology for adjusting the moving speed of the cover glass and the like are necessary and lack of versatility. In addition, the method described in (5) described above, as indicated by the arrows 1 to 3 in FIG. 1, the DNA at the gas-liquid interface of the DNA solution is vaporized as the cover glass moves, so that the DNA is elongated. Since the DNA solution is fixed, there is a problem that it takes time for the DNA solution to dry and the DNA cannot be fixed quickly. Furthermore, after moving the cover glass, a certain amount of DNA solution remains on the substrate, and as the remaining DNA solution is vaporized, in actuality 4 of FIG. There is a problem that DNA remains on the substrate in addition to the DNA fixed to the supplemental site shown in FIG.

  Furthermore, in order to increase the accuracy in preparing a specimen from a DNA fragment and performing pathological observation with a microscope, it is necessary to obtain a plurality of DNAs having the same fragment length. Further, from the viewpoint of obtaining pathologically advantageous statistical data, it is desirable to analyze at least 100 or more, preferably 200 or more DNA fragments of interest, as described in (3) to (5) above. In this method, DNAs of various fragment lengths contained in the solution are merely immobilized, and DNAs of various fragment lengths contained in the solution can be selectively immobilized. There wasn't.

JP 2006-67921 A

Schwartz DC. et al. , “Ordered restriction maps of Saccharomyces cerevisiae chromosomes structured by optical mapping”, Science, 1993, 262, 110-4. Washizu et al. , “Visualization of a specific sequence on a single large DNA molecule using fluorescence based on a new DNA-stretching method”, Biochem. Biophys. Res. Comm. , 1999, 265, 140-3. Bensimon et al. , “Dynamic Molecular Combing: Stretching the Whole Human for High-Resolution Studies”, Science, 1997, 277, 1518-1523. Craighead HG et al. "Transfer-printing of single DNA molecule arrays on high-resolution for resolution imaging and analysis", Nano Lett. , 2011 Oct 12, 11 (10), 4232-4238 Bensimon, A.D. et al. , "Alignment and sensitive detection of DNA by a moving interface", Science 265, 2096-298 (1994).

  The present invention has been made in order to solve the above-described conventional problems, and as a result of extensive research, a flow channel capable of flowing a solution containing a nucleic acid is provided, and a nucleic acid is applied to a portion facing the flow channel. A modification process is provided by providing a fixing part for fixing, an injection port for injecting a solution containing nucleic acid into the flow path, and a suction port for sucking the solution, and sucking the solution from the suction port at a predetermined speed. Furthermore, the present inventors have newly found that a nucleic acid can be extended and fixed efficiently and with high reproducibility without requiring a drying step. In addition, it has been newly found that DNA of various fragment lengths contained in a solution can be selectively fixed by adjusting the distance between the groove portion to be paired provided in the fixing portion for extending and fixing the nucleic acid. . Furthermore, the present inventors completed the present invention by discovering that a nucleic acid molecule alone can be sampled by subjecting the elongated / fixed nucleic acid to fluorescent staining or the like, and that abnormal DNA contained in cells or blood can be directly observed.

  That is, an object of the present invention is to provide a nucleic acid extension / fixation device, a nucleic acid extension / fixation method using the device, a nucleic acid fixation member, and a nucleic acid specimen prepared using the nucleic acid extension / fixation device. is there.

  The present invention relates to a nucleic acid extension / fixation device, a nucleic acid extension / fixation method using the device, a nucleic acid fixation member, and a nucleic acid specimen prepared using the nucleic acid extension / fixation device shown below.

(1) a nucleic acid fixing member including a fixing part for fixing a nucleic acid,
A channel member including an inlet for injecting a solution containing nucleic acid, a channel for the solution, and a suction port for sucking the solution;
A nucleic acid extension / fixation device comprising:
(2) a nucleic acid fixing member including a flow path of a solution containing a nucleic acid and a fixing portion for fixing the nucleic acid formed in the flow path;
An injection port for injecting a solution containing nucleic acid, and a lid member including a suction port for sucking the solution;
A nucleic acid extension / fixation device comprising:
(3) The nucleic acid extension / fixation device according to (1) or (2) above, wherein the fixing part includes a pair of grooves.
(4) The nucleic acid extension / fixation device according to (3) above, wherein the pair of groove portions has a shape selected from a hole type, a line type, or a zigzag type.
(5) The nucleic acid extension / fixation device according to any one of (1) to (4), further comprising a suction tool capable of sucking the solution at a predetermined speed.
(6) By sucking a solution containing the nucleic acid injected from the injection port of the nucleic acid elongation / fixation device described in (1) to (5) above at a predetermined speed from the suction port, A method for extending / fixing a nucleic acid, comprising extending / fixing the nucleic acid to a fixing part.
(7) A nucleic acid fixing member comprising a fixing part for fixing a nucleic acid, wherein the fixing part includes a pair of grooves.
(8) The nucleic acid fixing member according to (7), wherein the pair of groove portions has a shape selected from a hole type, a line type, or a zigzag type.
(9) The nucleic acid fixing member according to (7) or (8) above, wherein the nucleic acid fixing member has a flow path, and a fixing portion for fixing the nucleic acid is formed in the flow path.
(10) A nucleic acid sample comprising a nucleic acid stretched, fixed and stained on a fixing part of a nucleic acid fixing member by the method described in (6) above, wherein the nucleic acid fixing member is sealed with a cover glass.

  Since the nucleic acid extension / fixation device of the present invention can make the speed of the solution containing the nucleic acid passing over the fixed part constant using, for example, a simple device such as a syringe pump, a conventional cover glass is used. Compared with the moving method, the operation is simple and the reproducibility can be improved. Further, unlike the method of moving the cover glass, the device of the present invention does not require a step of drying the solution containing the nucleic acid, so that the treatment time can be shortened and the nucleic acid can be immobilized efficiently.

  In the present invention, since a solution containing nucleic acid is sucked from the suction port, nucleic acids other than the fixed nucleic acid can be collected together with the solution, and the solution is not left on the nucleic acid fixing member, so that it can be used for other than the fixing portion. Since nucleic acid (residue) does not adsorb nonspecifically, a valuable biological sample can be used efficiently. Furthermore, since no modification process is used for immobilizing nucleic acids, the immobilized nucleic acids can be transferred to another substrate, and can be used for microscopic examinations that require a dedicated substrate for observation, so that it has very high applicability. is there. In particular, as will be described later, in the present invention, a plurality of intended nucleic acids can be fixed together by forming the fixing portion in a specific shape, and thus the plurality of fixed nucleic acids can be collectively transferred to the substrate.・ Observation is effective in obtaining examination and statistical data.

  The fixing portion of the nucleic acid fixing member of the present invention includes a pair of groove portions, and nucleic acid fragments longer than the interval between the groove portions and the groove portions are not fixed. Therefore, based on the length when the nucleic acid fragment to be fixed is extended, the groove portions and groove portion By adjusting the interval, the target nucleic acid fragment can be efficiently immobilized from the nucleic acid solution containing various fragments.

  Since the nucleic acid molecule alone is fixed to the fixing part of the present invention in an extended state, it can be used as it is as a nucleic acid sample, and can be subjected to fluorescence staining and directly observed for pathological examination. . In addition, by providing multiple pairs of grooves and / or lines and zigzags, multiple nucleic acid fragments of the same length can be stretched and fixed in a single operation, and the accuracy of pathological examination is improved. As it goes up, statistical data can be acquired efficiently.

  The fixing part and the flow path of the present invention can be manufactured in large quantities at a low cost by a casting manufacturing method that uses a micropattern designed by CAD or the like and pours a resin into a mold manufactured by photolithography technology. Therefore, various micropattern nucleic acid fixing members can be produced in accordance with the size and application of the nucleic acid to be fixed.

FIG. 1 is a conceptual diagram showing a conventional DNA extension / fixation method. FIG. 2 is a schematic view showing an example of a method of using the nucleic acid elongation / fixation device of the present invention. FIG. 3 is an enlarged schematic view of the cross section of the fixing part 30 of the nucleic acid fixing member 10 of the present invention. FIG. 4A is a diagram showing a schematic configuration of the nucleic acid elongation / fixation device of the present invention. FIG. 4B is a cross-sectional view illustrating an example of an embodiment of the nucleic acid extension / fixation device of the present invention, and FIG. 4C illustrates an example of another embodiment of the nucleic acid extension / fixation device of the present invention. It is sectional drawing. FIG. 5 is a flowchart showing an example of a procedure for creating the nucleic acid fixing member 10 including the fixing unit 30 for fixing the nucleic acid of the present invention. FIG. 6 shows an example of CAD data of a photomask for producing the fixing portion 30, wherein (a) shows a hole type, (b) shows a line type, and (c) shows a zigzag type. FIG. 7 is a drawing-substituting photograph, (a-1) and (a-2) are Example 1, (b) is Example 2, and (c) is the fixing part of the nucleic acid fixing member 10 prepared in Example 3. FIG. FIG. 8 is a drawing-substituting photograph and a photograph of the nucleic acid elongation / fixation device prepared in Example 4. FIG. 9 is a distribution diagram of DNA fragments extended and fixed to the fixing part of the DNA specimen prepared in Example 4. FIG. 10 is a fluorescent photomicrograph of the DNA specimen obtained in Example 5, which is a drawing-substituting photograph. FIG. 11 is a photomicrograph of the DNA specimen obtained in Example 6, which is a drawing-substituting photograph. FIG. 12 is a drawing-substituting photograph and a fluorescence micrograph of the DNA specimen obtained in Example 7. FIG. 13 is a photomicrograph of the DNA specimen obtained in Example 8, which is a drawing-substituting photograph. FIG. 14 is a photomicrograph of the DNA specimen obtained in Example 9, which is a drawing substitute photo.

  The nucleic acid extension / fixation device of the present invention, the nucleic acid extension / fixation method using the device, the nucleic acid fixing member, and the nucleic acid specimen prepared using the nucleic acid extension / fixation device will be described in detail below.

  FIG. 2 is a schematic diagram showing an example of a method for using the nucleic acid extension / fixation device 1 (hereinafter, simply referred to as “device”) of the present invention. A solution 50 containing nucleic acid (hereinafter sometimes simply referred to as “solution”) 50 is injected from the injection port 20 into the flow path 40, and the solution 50 is supplied from the suction port 23 by the suction tool 60 so as to reach a predetermined speed. Then, the nucleic acid contained in the solution 50 is extended and fixed to the fixing unit 30 when the solution 50 passes over the fixing unit 30 (hereinafter, simply referred to as “fixing unit”) 30 that fixes the nucleic acid. be able to. A dotted line is an enlarged view of the fixed portion 30.

  FIG. 3 is an enlarged schematic view of the cross section of the fixed portion 30. In the present invention, the “fixing part” means a region having at least one pair of groove parts 35 and fixing the nucleic acid 36. Further, in the fixing part 30, the “depth” is the depth 37 of the groove part 35, the “width” is the width 38 of the groove part 35, and the “interval” is the distance between the groove part 35 and the groove part 35. Means the length 39 of the part to be.

  FIG. 4A is a diagram illustrating a schematic configuration of the device 1. The device 1 includes a nucleic acid fixing member 10 and a flow path member 20 or a lid member 21 that is used so as to cover the nucleic acid fixing member 10. An inlet 22 for injecting the solution 50 and a suction port 23 for sucking the solution 50 are formed in the upper part of the flow path member 20 or the lid member 21. It communicates with the road 40.

  FIG. 4B is a cross-sectional view showing an example of the embodiment of the device 1, and has a fixing portion 30 on the nucleic acid fixing member 10. A flow path 40 for flowing a solution is formed in the flow path member 20, and an injection port 22 and a suction port 23 (not shown) are formed so as to penetrate the flow path 40. Therefore, the solution 50 can be flowed on the fixing part 30 by combining the nucleic acid fixing member 10 and the flow path member 20 in a liquid-tight manner.

  FIG. 4C is a cross-sectional view showing another embodiment of the device 1 of the present invention. The nucleic acid fixing member 10 is formed with a channel 40 for flowing the solution 50, and the fixing unit 30 is formed on the channel 40. Is formed. On the other hand, an injection port 22 and a suction port (not shown) are formed in the lid member 21 so as to penetrate the lid member 21. Therefore, the solution 50 can be flowed on the fixing part 30 by combining the nucleic acid fixing member 10 and the lid member 21 in a liquid-tight manner.

  The material of the nucleic acid fixing member 10 is not particularly limited as long as it can form the fixing part 30 or can form the flow path 40 and the fixing part 30 can be formed on the flow path 40. PDMS (poly ( dimethylsiloxane)), PMMA (Poly (methyl methacrylate)), PC (polycarbonate), plastic made of hard polyethylene, silicon, glass and the like. Among the materials, the fixed portion 30, the flow path 40, and the fixed portion 30 formed on the flow path 40 are manufactured by first producing a mold using a photolithography technique to be described later and pressing the material against the mold. Then, since it is possible to produce a high-performance nucleic acid fixing member 10 in a large amount at a low cost, plastics and silicon-based materials are preferable. In addition, since the nucleic acid is observed under a microscope after being stretched and fixed, the light transmittance (transparency) is high, and it is preferable that the solution 50 does not remain in the flow path 40 after suction because there is no residue of nucleic acid. PDMS is particularly preferred.

  As the material of the flow path member 20 and the lid member 21, the same material as that of the nucleic acid fixing member 10 can be used, and PDMS is particularly preferable for the same reason as described above. The flow path formed in the flow path member 20 can be produced using the photolithographic technique described later, like the nucleic acid fixing member 10. The inlet 22 and the suction port 23 formed in the channel member 20 and the lid member 21 are not particularly limited in size and shape as long as they communicate with the channel 40, such as a punch, ultrasonic drill, sand blaster, etc. What is necessary is just to produce using.

FIG. 5 is a flowchart showing an example of a procedure for creating the nucleic acid fixing member 10 including the fixing unit 30 shown in FIG.
(1) First, the substrate 31 is prepared.
(2) A photoresist 32 is spin coated on the substrate 31 and prebaked on a hot plate.
(3) It exposes using the photomask which is not shown corresponding to the shape of the fixing | fixed part 30, and performs post-exposure baking on a hotplate.
(4) After developing with a developing solution, rinsing with ultrapure water, removing moisture with a spin dryer or the like, and drying to produce the mold 33.
(5) The nucleic acid immobilization in which the immobilization part 30 including the groove corresponding to the shape of the photomask is formed by transferring the template 33 to the material 34 for the nucleic acid immobilization member 10 and then separating the mold 33. The member 10 is produced.

  The substrate 31 used in the above procedure is particularly a material generally used in the field of photolithography, such as silicon, silicon carbide, sapphire, gallium phosphide, gallium arsenide, gallium phosphide, gallium nitride, etc. There is no limit.

  The photoresist 32 may be appropriately selected from a positive type and a negative type according to the shape of the photomask for producing the fixing portion 30. The positive photoresist is not particularly limited as long as it is a material generally used in the field of photolithography, such as PMER or AZ, and the negative photoresist is SU-8 or KMPR. The developer is not particularly limited as long as it can etch the selected photoresist.

  The flow path member 20 shown in FIG. 4B is manufactured in the same procedure as in FIG. 5 except that a photomask having a shape corresponding to the flow path 40 is used as the photomask in the procedure in FIG. Can do.

When producing the nucleic acid fixing member 10 having the fixing part 30 formed on the flow path 40 shown in FIG. 4C, after the procedures of FIGS. 5 (1) and (2),
(A) Using a photomask for producing the flow path 40, exposure and post-exposure baking on a hot plate are performed.
(B) After developing with a developing solution, rinsing with ultrapure water, removing moisture with a spin dryer or the like and drying, a substrate 31 having convex portions corresponding to the flow path 40 is produced.
(C) The photoresist 32 is spin-coated again, and then a photomask (not shown) corresponding to the shape of the fixed portion 30 is disposed on the convex portion corresponding to the flow path 40. Thereafter, FIG. It can produce according to the procedure of (3)-(5).

  As shown in FIG. 3, the fixing unit 30 includes a pair of groove portions 35 provided on the surface through which the solution 50 flows. The shape and arrangement of the groove 35 are not particularly limited as long as the nucleic acid can be elongated and fixed. On the other hand, when the nucleic acid is extended / fixed to the fixing part 30 using the device 1 of the present invention, the length of the nucleic acid to be extended / fixed can be changed by adjusting the gap 39 between the groove part 35 and the groove part 35. Therefore, for example, when the hole-type groove portions 35 are provided at random, the length of the nucleic acid to be elongated / fixed according to an arbitrary pair of hole-type groove portions 35 in the flow direction of the solution 50 and the interval 39 between the groove portions 35. Changes. Further, the groove portions 35 to be paired are a hole type provided so as to have a constant interval 39 with respect to the flow direction of the solution 50, a line type provided in a direction orthogonal to the flow direction of the solution 50, and a zigzag type. By doing so, it is also possible to fix a plurality of nucleic acids having a constant length in the gap 39 between the groove portions 35 by a single operation. The pair of groove portions 35 is not limited to one pair, and a plurality of pairs of groove portions 35 may be provided. In that case, the intervals 39 between the plurality of pairs of groove portions 35 and the groove portions 35 may be equally spaced, or the intervals 39 for each pair. May be changed. Moreover, the groove part 35 may be provided in a step shape, and nucleic acids having different lengths may be extended and fixed.

  6A and 6B show examples of CAD data for a photomask for producing the fixing portion 30. FIG. 6A shows an example of a hole type, FIG. 6B shows a line type, and FIG. 6C shows an example of a zigzag type.

  The shape, depth 37, width 38 and number of the groove portions 35 of the fixed portion 30 and the width, depth and length of the flow path 40 are not particularly limited as long as the present invention can be carried out. It can be adjusted by the shape and size, and the thickness of the laminated resist. Further, the interval 39 between the groove 35 and the groove 35 may be adjusted as appropriate according to the size of the nucleic acid to be extended / fixed.

  The device 1 of the present invention can be produced by bonding the nucleic acid fixing member 10 produced by the above procedure and the flow path member 20 or the lid member 21 together. When the nucleic acid fixing member 10 and the flow path member 20 or the lid member 21 are made using a resin such as PDMS, it is only necessary to simply bond them together by the adhesion function of the resin itself. In addition, when the nucleic acid fixing member 10 and the flow path member 20 or the lid member 21 are made of a material such as glass that has low adhesion between members, silicon oil or the like may be applied to the edge of the member and bonded together. The solution 50 may drop droplets directly on the inlet 21, or a tube may be connected to a container (not shown) that stores the solution 50, and the other end of the tube may be connected to the inlet 21. There is no particular limitation as long as the solution 50 can be introduced into the inlet 21. The suction tool 60 is not particularly limited as long as it can suck the solution 50 at a constant speed, such as a syringe pump.

  After flowing the solution 50, the nucleic acid fixing member 10 and the flow path member 20 or the lid member 21 are separated, and the nucleic acid extended and fixed to the fixing unit 30 is transferred to YOYO-1, PI, Pyronin Y, SYBR GreenI, etc. A nucleic acid specimen can be prepared by staining with a known fluorescent dye, covered with a cover glass, and observed with a microscope. In addition, when flowing the solution 50, the nucleic acid may be stained in advance.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

[Preparation for photomask formation]
As a photomask material, chrome mask blanks (manufactured by Clean Surface Corporation, thickness 2.3 mm) were used. Photo exposure was performed at 100 mJ / cm ² using a maskless exposure machine (Nano System Solutions, Inc.) from the CAD data of (a) Hall type, (b) Line type, and (c) Zigzag type shown in FIG. A mask (chrome mask) was produced.

[Preparation of nucleic acid fixing member of the present invention]
(Preparation of hole-type nucleic acid fixing member)
<Example 1>
The nucleic acid fixing member of the present invention was produced by the following procedure. First, SU-8 3005 (manufactured by MICRO CHEM) is applied on a silicon substrate (made by Ferrotech Silicon Co., diameter 76 mm, thickness 600 μm) by a spin coater so as to have a thickness of 6 μm, and a hot plate (AS ONE) and prebaked at 95 ° C. for 2 minutes. Next, the hole-type photomask prepared in the above [Preparation of Photomask] is placed on the substrate, exposed to 100 mJ / cm ² using an ultraviolet irradiation device (manufactured by MIKASA), then 65 ° C for 1 minute, 95 ° C. Post exposure baking was performed for 1 minute, and development was performed using SU-8 developer (MICRO CHEM). After the development, the substrate was rinsed with ultrapure water, dried by removing moisture with a spin dryer, and a mold was produced. A polydimethylsiloxane (SILPOT184, manufactured by Toray Industries, Inc.) was poured into the prepared template, and after curing PDMS, the cured PDMS was removed from the template to prepare a nucleic acid fixing member. FIG. 7 (a-1) is an optical micrograph in which the fixing part of the nucleic acid fixing member prepared in Example 1 is enlarged, and FIG. 7 (a-2) is an optical micrograph in which (a-1) is further enlarged. . The depth of the hole-type groove was 6 μm, the diameter was 5.3 μm, and the distance between the holes in the flow direction of the solution was 9.5 μm.

(Production of line-type nucleic acid fixing member)
<Example 2>
A nucleic acid fixing member was produced in the same procedure as in Example 1 except that the line type photomask produced in [Preparation of Photomask] was used in place of the photomask of Example 1. FIG. 7 (b) is an optical micrograph showing an enlargement of the fixing part of the nucleic acid fixing member prepared in Example 2. The depth of the line-type groove is 6 μm, the width of the line-type groove is 15 μm, and the distance between the grooves. Was 15 μm.

(Production of zigzag type nucleic acid fixing member)
<Example 3>
A nucleic acid fixing member was produced in the same procedure as in Example 1 except that the zigzag photomask produced in [Preparation of Photomask] was used in place of the photomask of Example 1. FIG. 7 (c) is an optical micrograph in which the fixing part of the nucleic acid fixing member prepared in Example 3 is enlarged. The depth of the zigzag groove is 6 μm, and the width of the groove (short distance connecting the vertices of the zigzag) is The distance between the grooves (the short distance connecting the vertices of the zigzag) was 15 μm, and the distance between the vertices of one zigzag and the adjacent vertex was 7.3 μm.

[Preparation of flow channel member]
The thickness of SU-8 3050 of Example 1 was applied to be 50 μm, and a photomask having a flow path shape (width 650 μm, length 0.8 cm) was used instead of the photomask of Example 1. A flow path member was produced in the same procedure as in Example 1 except that it was used. The flow path of the obtained flow path member had a width of 650 μm, a depth of 50 μm, and a length of 0.8 cm. Next, an injection port for injecting the solution and a suction port for suction were prepared by a punch (manufactured by Harris). The diameter of the inlet was 1.5 mm, and the diameter of the suction port was 1 mm.

[Sample adjustment]
48.5 kbp λDNA (Nippon Gene) was dissolved in TE buffer (Nippon Gene, pH 8.0) to a concentration of 50 ng / μl, and then stained with 1 mM YOYO-1. The theoretical value of the length when the 48.5 kbp λDNA is extended is about 16.5 μm, but the actual value when it is dyed and extended is 21.5 ± 0.5 μm (Non-patent Document 5). reference).

(Fragment length distribution of extended and fixed sample λ DNA fragment)
<Example 4>
First, the distribution of the length of the DNA fragment extended and fixed using the device of the present invention was examined. As described above, since the actual measurement value of λDNA contained in the stained DNA sample is about 21.5 ± 0.5 μm, the interval between the grooves sufficiently longer than the length (short distance connecting the vertices of zigzag) A photomask having a thickness of 30 μm was prepared, and a nucleic acid fixing member having a zigzag type fixing part was prepared according to the procedure of Example 1 above. The depth of the zigzag groove was 6 μm, the width of the groove (short distance connecting the vertices of the zigzag) was 30 μm, and the distance between one vertex of the zigzag and the adjacent vertex was 7.3 μm.

  Next, the prepared nucleic acid fixing member and the flow channel member prepared in [Preparation of flow channel member] were bonded together to prepare the nucleic acid elongation / fixation device of the present invention. FIG. 8 shows the produced nucleic acid extension / fixation device. Next, 10 μl of TE buffer is dropped into the injection port, and a syringe (HAMILTON (500 μL)) connected to the suction port by a silicon tube is sucked using a syringe pump (AS ONE) into the flow path. Washed. Thereafter, 10 μl of the sample prepared in the above [Preparation of sample] was dropped into the injection port and sucked from the suction port so that the flow rate of the syringe pump was 1.0 μl / min. After the solution was aspirated and collected in the syringe, the nucleic acid fixing member and the flow path member were separated and covered with a cover glass to prepare a DNA specimen.

  FIG. 9 is a distribution diagram of DNA fragments extended and fixed to the fixing part of the DNA specimen prepared in Example 4. As is clear from FIG. 9, the extended and fixed DNA fragments were distributed in the range of 7 to 33 μm with peaks at 20 μm and 21 μm. The peak value was almost the theoretical value, but the fragments shorter than the peak value were cut during the preparation of the λDNA solution and / or while being stretched and fixed using the device of the present invention. Conceivable. On the other hand, a DNA fragment longer than the theoretical value is considered to be elongated and fixed like a single DNA because the fragments are entangled with each other. The λDNA sample of the present invention was prepared by the same procedure as that described in Non-Patent Document 5, but when a DNA fragment was immobilized according to the same principle as in FIG. As shown in FIG. 2, although the DNA fragment has a peak at about 21 μm, a slightly larger peak is observed between 3 and 12 μm, indicating that the DNA fragment was cleaved during extension / fixation. However, since the DNA fragments extended / fixed by the method of Example 4 have few cleaved fragments, the DNA extension / fixation method of the present invention has the effect that the DNA fragments in the sample are difficult to cleave. Became clear.

(Production of device with different shape of immobilization part and nucleic acid extension / immobilization method using the device)
<Example 5>
A nucleic acid fixing member was prepared in the same procedure as in Example 1 except that the distance between the holes in the solution flow direction was 15 μm, and then a DNA sample was prepared in the same procedure as in Example 4.

  FIG. 10 is a fluorescence micrograph of the DNA specimen obtained in Example 5. As is clear from the photograph, the DNA in the solution was stretched and fixed between the holes. However, as shown in FIG. 9, DNA fragments in the sample contain many fragments longer than 15 μm, which is the distance between holes, but DNA fragments longer than the distance between holes are confirmed. There wasn't.

<Example 6>
A DNA specimen was prepared in the same procedure as in Example 4 except that the nucleic acid fixing member prepared in Example 3 was used.

  FIG. 11 is a fluorescence micrograph of the DNA specimen obtained in Example 6. As is apparent from the photograph, the DNA in the solution extended from the top of the zigzag groove and was fixed. Further, as in Example 5, no DNA fragment longer than the zigzag interval was confirmed.

<Example 7>
Instead of the zigzag nucleic acid fixing member prepared in Example 3 above, the depth of the zigzag groove is 6 μm, the width of the groove (short distance connecting the tops of the zigzag) is 15 μm, and the distance between the grooves (zigzag) A nucleic acid-fixing member having a short distance of connecting vertices) of 20 μm and a distance between vertices of one zigzag vertex and the adjacent vertex being 4 μm was prepared and used. Furthermore, the DNA sample was prepared by the same procedure as in Example 4 except that the sample was stirred for 10 to 20 seconds with a vortex mixer to obtain a sample containing more DNA fragments shorter than the sample prepared in [Sample preparation] above. Was made.

  FIG. 12 is a fluorescence micrograph of the DNA specimen obtained in Example 7. As is apparent from the photograph, the DNA in the solution extended from the top of the zigzag groove and was fixed. Further, one end of a DNA molecule having an interval shorter than the interval between the groove portions was caught and fixed in the groove portion, but DNA longer than the zigzag interval was not confirmed.

<Example 8>
Instead of the zigzag type nucleic acid fixing member prepared in Example 7, the depth of the zigzag groove is 6 μm, the width of the groove (short distance connecting the tops of the zigzag) is 15 μm, and the distance between the grooves (zigzag) The DNA sample was prepared in the same manner as in Example 7, except that a nucleic acid-fixing member having a short distance between the vertices of 20 μm and a distance between the vertices of one zigzag vertex and the adjacent vertex of 7.3 μm was prepared and used. Was made.

  FIG. 13 is a fluorescence micrograph of the DNA specimen obtained in Example 8. As is clear from the photograph, as in Example 7, the DNA in the solution extended from the apex of the zigzag groove and was fixed. Further, one end of a DNA molecule having an interval shorter than the interval between the groove portions was caught and fixed in the groove portion, but DNA longer than the zigzag interval was not confirmed.

<Example 9>
Instead of the zigzag type nucleic acid fixing member prepared in Example 7, a nucleic acid fixing member having a line-type groove portion depth of 50 μm, a groove portion width of 1.3 μm, and a groove-to-groove interval of 2.6 μm was prepared / A DNA specimen was prepared in the same procedure as in Example 7 except that it was used.

  FIG. 14 is a fluorescence micrograph of the DNA specimen obtained in Example 9. As described in Example 7, although the sample contains a lot of short DNA fragments by stirring the sample with a vortex mixer, as shown in FIG. 12 and FIG. DNA fragments having a length close to 20 μm, which is the distance between the grooves used in 7 and 8, are also included. However, as is clear from the photograph of FIG. 14, DNA longer than 2.6 μm, which is the distance between the groove portions, was not fixed. On the other hand, a small-sized DNA fragment was fixed between the groove portions.

  From the results of Examples 4 to 9 above, DNA fragments longer than the interval between the groove portions are not elongated or fixed. Therefore, by selecting the desired length as the interval, a solution containing various DNA fragments can be selected. In particular, DNA can be elongated and fixed. Furthermore, when a zigzag type is adopted as the fixing part, DNA fragments having relatively uniform lengths can be extended and fixed at a fixed interval, and thus is particularly useful for pathological examinations and collection of statistical data.

  The nucleic acid extension / fixation device of the present invention can simultaneously extend and fix a plurality of nucleic acids of a certain length from a nucleic acid solution containing various fragment lengths by a simple operation, and further stains. Thus, a single DNA molecule can be sampled for pathological examination. Therefore, it is useful for simple and rapid preparation and examination of specimens at medical institutions, universities, companies, research institutions, and the like.

Claims (8)

A nucleic acid fixing member including a fixing part for fixing the nucleic acid;
A channel member including an inlet for injecting a solution containing nucleic acid, a channel for the solution, and a suction port for sucking the solution;
Including
The nucleic acid elongation / fixation device, wherein the fixing part includes a pair of grooves. A nucleic acid fixing member including a flow path of a solution containing the nucleic acid and a fixing portion for fixing the nucleic acid formed in the flow path;
An injection port for injecting a solution containing nucleic acid, and a lid member including a suction port for sucking the solution;
Including
The nucleic acid elongation / fixation device, wherein the fixing part includes a pair of grooves.   The nucleic acid extension / fixation device according to claim 1 or 2, wherein the pair of grooves has a shape selected from a hole type, a line type, and a zigzag type.   The nucleic acid extension / fixation device according to any one of claims 1 to 3, further comprising a suction tool capable of sucking the solution at a predetermined speed. The nucleic acid fixing member is fixed by aspirating a solution containing the nucleic acid injected from the injection port of the nucleic acid extending / fixing device according to any one of claims 1 to 4 at a predetermined speed. A method for extending / fixing a nucleic acid, comprising extending / fixing the nucleic acid to a part.   A nucleic acid fixing member comprising a channel and a fixing part for fixing a nucleic acid formed in the channel, wherein the fixing part includes a pair of grooves.   The nucleic acid fixing member according to claim 6, wherein the pair of grooves has a shape selected from a hole type, a line type, or a zigzag type. The nucleic acid extension / fixation device according to claim 1,
A nucleic acid stretched, fixed and stained in a fixing portion of a nucleic acid fixing member of the nucleic acid extension / fixation device ; and
A cover glass sealed the nucleic fixing member,
A nucleic acid sample characterized by comprising: