JP2005245401A - Nucleic acid isolation member and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid isolation member (chip) producible at a low cost, hardly causing stain of DNA, capable of decreasing the amount of a specimen to be purified, requiring little labor because employing a throughput type, and requiring short time for treatment, and to provide a method for producing the chip. <P>SOLUTION: The chip comprises a main body 3 and a cover 4 covering the upper face of the main body 3. The main body 3 has, as a nucleic acid separator 3b, a holder 5, a nucleic acid isolator 15, a waste liquid holder 9, a holder 11, paths 27 and 29, and a valve 51. In the nucleic acid isolator 15, many columnar projects 21 are formed and silica elements 39 are exposed on the surface of the projects. The chip has, as an amplifying part 3c, a path 25, a mixer 57, a guide path 26, a valve 55, a PCR amplifier 23, a path 31, and a holder 13. A biomaterial is fed from the holder 5 to the nucleic acid isolator 15 and DNA in the biomaterial is adsorbed on the projects 21. The adsorbed DNA is eluted with a reagent and amplified by the PCR amplifier 23. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a nucleic acid isolation member for isolating a nucleic acid from a biological material containing the nucleic acid and a method for producing the same.

When performing DNA analysis or the like, it is necessary to first isolate the nucleic acid. As this method, there is a method described in Japanese Patent No. 2680462 (Patent Document 1). In recent years, filtration processing parts in which a filter formed of silica is carried on a cylindrical plastic member are commercially available as instruments for isolating nucleic acids. In this filtered part, a solution containing a biomaterial is poured through a filter part made of silica to adsorb nucleic acid. Then, the nucleic acid is isolated by eluting the nucleic acid from the silica portion.
Japanese Patent No. 2680462

  However, in conventional processing parts, the silica filter that adsorbs nucleic acids is made porous, and the method for making it porous is, for example, mixing organic substances into silica and then removing the organic substances at high temperatures. In addition, since a method for mounting the filter on a plastic cylindrical part is also required, there is a problem that the manufacturing process is many and expensive.

  In addition, since the conventional processing parts are instruments that only isolate nucleic acids, it has been necessary to transfer the nucleic acids to other instruments in order to analyze the isolated nucleic acids. In this transfer, there is a problem that the nucleic acid may be contaminated.

  Furthermore, since analysis in biochemistry is expensive, it is desirable to reduce the amount of nucleic acid sample, but when transferring nucleic acid as described above, liquid residue and adhesion to the instrument occur. Numerous nucleic acids had to be isolated, and there was a problem that it was difficult to reduce the amount of specimen.

  The present invention has been made in view of the above points, is a technology that has few manufacturing processes, is rich in mass productivity, can be manufactured at low cost, is unlikely to be contaminated with nucleic acids, and makes the amount of a sample small. It is an object of the present invention to provide a nucleic acid isolation member that can be used in a throughput method and requires less time and requires less time for processing, and a method for producing the same.

(1) The invention of claim 1
A portion to be supplied with a biomaterial containing nucleic acid, and a protrusion provided at a position in contact with the biomaterial in the portion to be supplied, the protrusion including a resin and silica, and The gist of the nucleic acid isolation member is characterized in that the silica is exposed on at least a part of the surface of the protrusion.

  In the nucleic acid isolation member of the present invention, silica that easily adsorbs nucleic acid is exposed on at least a part of the surface of the protrusion. Therefore, the nucleic acid can be efficiently isolated by adsorbing the nucleic acid to the exposed silica.

  Moreover, since the protrusion part contains resin, it can be easily formed by, for example, a method using a mold. Therefore, the nucleic acid isolation member of the present invention requires only a small number of manufacturing steps, is rich in mass productivity, is easy to manufacture, and can be manufactured at low cost.

  Furthermore, the nucleic acid isolation member of the present invention can be formed with a portion for performing treatment using the purified nucleic acid (for example, amplification of nucleic acid or analysis of nucleic acid) in addition to the portion for isolating nucleic acid. . This eliminates the need to transfer the nucleic acid to another instrument when processing the purified nucleic acid. As a result, the nucleic acid is not contaminated during transfer, and the analysis accuracy can be improved. In addition, since there is no loss of nucleic acid during transfer, the amount of sample to be purified can be reduced, and the amount of expensive reagent used for nucleic acid purification can be reduced. Furthermore, the purification of the nucleic acid and the treatment using the purified nucleic acid can be automated, and the time required for the treatment can be shortened.

-As said resin, a thermoplastic resin, a thermosetting resin, a thermoplastic elastomer, rubber | gum etc. are mentioned, for example.
(2) The invention of claim 2
The gist of the nucleic acid isolation member according to claim 1, wherein the protruding portion has a substantially cylindrical shape.

In the present invention, since the projecting portion has a substantially cylindrical shape, it can be formed by a mold and can be mass-produced.
(3) The invention of claim 3
The gist of the nucleic acid isolation member according to claim 1, wherein the shape of the protruding portion is a substantially truncated cone shape or a shape having a tapered portion.

In the present invention, since the shape of the protruding portion is a substantially truncated cone shape or a shape having a tapered portion, there is a portion that is inclined with respect to the protruding direction of the protruding portion, and the inclined portion depends on the mold. Since it corresponds to the draft angle in formation and is inclined like that, the area for adsorbing nucleic acid is increased in the protruding portion compared to the case where it is configured parallel to the protruding direction. Separation can be performed quickly.
(4) The invention of claim 4
The protrusion is composed of the resin and a silica member having at least one of a granular shape, a flake shape, and a linear shape, and the size of the silica member is ½ or less of the width of the protrusion portion. The gist of the nucleic acid isolation member according to any one of claims 1 to 3.

  In this invention, since the magnitude | size of a silica member is 1/2 or less of the width | variety of a protrusion part, two or more silica members can be arrange | positioned in the predetermined width direction in a protrusion part, and the magnitude | size of a silica member is Silica is exposed more than when the width exceeds 1/2 of the protrusion. Therefore, in the present invention, the nucleic acid can be efficiently adsorbed to the protruding portion.

The size of the silica member is the diameter in the case of a sphere, the length in the longitudinal direction in the case of a flake, and the case of a line, as indicated by arrows in FIGS. 5 (a) to 5 (c). Is the diameter of the cross section.
(5) The invention of claim 5
The gist of the nucleic acid isolation member according to claim 1, wherein the nucleic acid isolation member has a plurality of the protruding portions.

In the present invention, the contact area between the protrusion and the biomaterial can be increased by having a plurality of protrusions. As a result, the nucleic acid isolation member of the present invention can efficiently isolate a nucleic acid.
(6) The invention of claim 6
The gist of the nucleic acid isolation member according to claim 5, wherein the protrusions are arranged along a direction intersecting a moving direction of the biomaterial in the supplied part.

In the present invention, since the projecting portions are arranged along the direction intersecting the moving direction of the biomaterial in the supplied portion, the projected area of the projecting portion increases and moves when viewed from the moving direction of the biomaterial. The probability that the biomaterial to be in contact with the protruding portion increases. As a result, the nucleic acid isolation member of the present invention can efficiently isolate a nucleic acid.
(7) The invention of claim 7
The gist of the nucleic acid isolation member according to claim 5 or 6, wherein the protrusions are arranged in a staggered pattern.

In the present invention, since the projecting portions are arranged in a staggered pattern, the projecting portions in a certain row of the staggered lattice are positioned between the projecting portions in the adjacent rows as viewed from the moving direction of the biomaterial. It is supposed to be. Therefore, when viewed from the moving direction of the biomaterial, the projected area of the protrusion is increased, and the probability that the moving biomaterial and the protrusion are in contact with each other increases. As a result, the nucleic acid isolation member of the present invention can efficiently isolate a nucleic acid.
(8) The invention of claim 8
The gist of the nucleic acid isolation member according to any one of claims 5 to 7, wherein an interval between adjacent protrusions is in a range of 0.001 to 1 mm.

In the present invention, when the biomaterial flows through the supply part, the distance between the protrusions is as narrow as 1 mm or less, so that the nucleic acid in the biomaterial passing between the protrusions can be adsorbed to the protrusions.
In the present invention, since the interval between the protrusions is 0.001 mm or more, even long-chain DNA can pass through the protrusions.

The distance between the protrusions is preferably in the range of 0.001 to 0.05 mm. By setting it within this range, it is difficult for the biomaterial to flow unnecessarily between the protrusions.
(9) The invention of claim 9
The protruding portion is composed of the resin and a silica member having at least one of a granular shape, a flake shape, and a linear shape dispersed in the resin, and a part of the silica member is a portion of the protruding portion. The gist of the nucleic acid isolation member according to any one of claims 1 to 7, which is exposed on the surface.

In the nucleic acid isolation member of the present invention, nucleic acid is efficiently isolated by adsorbing the nucleic acid to a silica member having at least one of granular, flake, and linear shapes exposed on the surface of the protrusion. be able to.
(10) The invention of claim 10
The nucleic acid isolation member according to claim 9, wherein a weight ratio of the silica member to the entire protrusion is in a range of 5 to 90% by weight.

  In the present invention, since the weight ratio of the silica member to the entire protrusion is 5% by weight or more, the amount of the silica member exposed on the surface of the protrusion is sufficiently large, and the nucleic acid adsorption efficiency is high. Therefore, the nucleic acid isolation member of the present invention can efficiently isolate a nucleic acid.

In the present invention, since the weight ratio of the silica member to the entire protrusion is 90% by weight or less, the resin and silica can be easily kneaded and dispersed.
(11) The invention of claim 11
The nucleic acid isolation member according to any one of claims 1 to 8, wherein the protruding portion includes a central portion made of a resin and a silica layer containing silica covering a surface of the central portion. And

  In the present invention, since the silica layer covers the surface of the protrusion, the contact area between the silica and the biomaterial is large, and the nucleic acid adsorption efficiency is high. Therefore, the nucleic acid isolation member of the present invention can efficiently perform nucleic acid isolation.

In the present invention, since the central portion of the projecting portion is made of resin, the central portion can be easily formed by a method using a mold or the like. Therefore, the nucleic acid isolation member of the present invention is easy to manufacture.
(12) The invention of claim 12
The gist of the nucleic acid isolation member according to any one of claims 1 to 11, further comprising a cover member that covers the protruding portion.

In the present invention, the protrusion is covered by the cover member. For example, when the protruding portion is provided so as to protrude upward, the upper surface of the protruding portion is covered with a cover member. As a result, the solution can flow into the flow path by pressurization, capillary action, tilting the nucleic acid isolation member, and rotating the nucleic acid isolation member. In addition, foreign matter can enter during the experiment, and evaporation of the liquid can be prevented.
(13) The invention of claim 13
The gist of the nucleic acid isolation member according to claim 12, wherein the cover member is made of the same resin as the main body of the nucleic acid isolation member.

In the present invention, since the cover member is made of the same resin as the main body of the nucleic acid isolation member, it can be easily joined to the nucleic acid isolation member.
-The main body of the said nucleic acid isolation member means parts other than a cover member.
(14) The invention of claim 14
The said cover member and the main body of the said nucleic acid isolation member are joined by at least 1 method of the heat welding, the ultrasonic welding, and the adhesion | attachment method, The Claim 12 or 13 characterized by the above-mentioned. The gist of the nucleic acid isolation member.

In the present invention, by using the above-described joining method, the cover member and the main body of the nucleic acid isolation member can be firmly joined.
(15) The invention of claim 15
It is a manufacturing method of the metal mold | die used for manufacture of the nucleic acid isolation member in any one of Claims 1-14, Comprising: The surface of a resist is covered with the mask corresponding to the shape of the said nucleic acid isolation member, and it exposes with a light beam. A first step of developing, a second step of developing the exposed resist, a third step of electroforming the resist with a mold material, and a fourth step of removing the resist. The gist of the present invention is a method for producing a mold for a nucleic acid isolation member.

In the present invention, since the lithography technique and the electroforming technique as described above are used, an accurate mold can be easily manufactured even when the protrusion has a fine shape.
(16) The invention of claim 16
The gist of the method for producing a mold according to claim 15, wherein the light beam is an X-ray.

In the present invention, since exposure is performed using X-rays, the diameter of the protrusions is made fine and the height is high due to the high X-ray transparency and straightness of the portion of the mold corresponding to the protrusions. That is, a mold for a nucleic acid isolation member having a large surface area of the protrusion can be produced.
(17) The invention of claim 17
The method for producing a nucleic acid isolation member according to any one of claims 1 to 14, wherein a step A in which a material comprising the resin and the silica is poured into a mold for the nucleic acid isolation member, and the solidified material. A step of separating the B from the mold, and a method for producing a nucleic acid isolation member.

According to the present invention, a nucleic acid isolation member can be easily produced using a mold.
(18) The invention of claim 18
The method for producing a nucleic acid isolation member according to claim 11, wherein the silica layer is formed using CVD.

In the present invention, the protrusion can be covered with a silica layer by using CVD (Chemical Vapor Deposition). Therefore, the nucleic acid isolation member produced in the present invention has a large contact area between the silica layer and the biomaterial, and can efficiently isolate the nucleic acid.
(19) The invention of claim 19
19. The method for producing a nucleic acid isolation member according to claim 18, wherein the shape of the protruding portion that forms the silica layer using CVD is a truncated cone shape or a shape having a tapered portion. .

  In the present invention, the silica layer is formed by CVD on the frustoconical shape or the tapered protrusion, so that the silica layer is formed on the side surface of the vertical protrusion by using CVD. The silica layer can be easily formed as compared with the case where it is formed.

The best mode for carrying out the invention will be described with reference to examples.

  a) First, a chip comprising a nucleic acid isolation part that adsorbs, purifies, and elutes DNA from a solution containing DNA in Example 1 and an amplification part that amplifies the isolated DNA by PCR. The configuration will be described with reference to FIGS.

  The chip is composed of a main body portion 3 shown in FIG. 1 and a cover portion 4 shown in FIG. First, the main body 3 will be described with reference to FIG. The main body 3 has a plate shape, and includes a nucleic acid isolation unit 3b for isolating nucleic acid and an amplification unit 3c for performing amplification by a PCR method on the upper surface 3a.

The nucleic acid isolation unit 3 b includes a holding unit 5, a nucleic acid isolator 15, a waste liquid holding unit 9, a holding unit 11, flow paths 27 and 29, and a valve 51.
Here, the holding unit 5, the waste liquid holding unit 9, and the holding unit 11 are each a concave portion that opens upward on the upper surface 3 a of the main body unit 3. The nucleic acid isolator 15 is provided with a protrusion 21 in a groove formed in the upper surface 3a. The nucleic acid isolator 15 is connected to the holding unit 5 at one end thereof, and is connected to the waste liquid holding unit 9 through the channel 27 at the opposite end and through the channel 29. And connected to the holding unit 11. The flow paths 27 and 29 are grooves provided in the upper surface 3a. The valve 51 is provided at the connection between the nucleic acid isolator 15 and the flow paths 27 and 29. When the valve 51 is opened between the nucleic acid isolator 15 and the flow path 27, the space between the nucleic acid isolator 15 and the flow path 29 is closed. When the space between the passage 27 is closed, the valve opens between the nucleic acid isolator 15 and the flow passage 29. The valve 53 is provided at a connection portion between the flow path 29 and the holding portion 11.

Next, the amplification unit 3c will be described. The amplification unit 3 c includes a flow channel 25, a mixing unit 57, an introduction flow channel 26, a valve 55, a PCR amplifier 23, a flow channel 31, and a holding unit 13.
The channel 25 is a groove formed in the upper surface 3 a and connects the holding unit 11 and the mixing unit 57. The valve 55 is a valve that is positioned at a connection portion between the holding unit 11 and the flow path 25 and opens and closes between the holding unit 11 and the flow path 25. The mixing portion 57 is a groove formed in the upper surface 3a, and is provided to have a bent shape. The introduction channel 26 is a groove formed in the upper surface 3 a and connects the mixing unit 57 and the PCR amplifier 23.

  The PCR amplifier 23 includes a flow path 23a and heaters 23b and 23c. Here, the channel 23 a is a groove formed in the upper surface 3 a and connects the introduction channel 26 and the channel 31. The flow path 23 a has a shape that is folded 10 times as many times as the number of cycles of the PCR amplifier 23. The heater 23b and the heater 23c are each provided in the bottom part of the flow path 23a. The temperature of the heater 23b is 60 ° C., and the temperature of the heater 23c is 95 ° C. The ratio of the length of the flow path 23a in contact with the heater 23b and the length of the flow path 23a in contact with the heater 23c is 1: 4.

The holding unit 13 is a groove formed in the upper surface 3 a and connects the PCR amplifier 23 and the holding unit 13. The holding part 13 is a concave part whose upper side is open on the upper surface 3a.
As shown in FIG. 2, the cover portion 4 constituting the chip has four holes 4a, 4b, 4c, and 4d in the plate member. This cover part 4 is joined to the upper surface 3a of the main-body part 3 by heat welding. At this time, the four holes 4a, 4b, 4c, and 4d are located in the holding unit 5, the waste liquid holding unit 9, the holding unit 11, and the holding unit 13, respectively. Therefore, among the upper surface 3a of the main body 3, the upper part of the upper surface of the holding unit 5, the waste liquid holding unit 9, the holding unit 11, and the holding unit 13 is open upward, but the other parts are Is closed.

b) Next, the configuration of the nucleic acid isolator 15 and the protrusion 21 will be described in detail with reference to FIGS.
3A is a plan view of the nucleic acid isolator 15 as viewed from above, and FIG. 3B is an enlarged view of a part of the protrusion 21 shown in FIG. 3A. is there. FIG. 4 is a cross-sectional view taken along the line AA in FIG.

As shown in FIG. 3A, the nucleic acid isolator 15 is provided with a plurality of columnar protrusions 21 standing upright. Further, cylinders 22 having different diameters are erected in some places.
The protrusions 21 are arranged in a staggered pattern as shown in FIG. That is, the protrusion 21 belongs to one of a plurality of rows orthogonal to the liquid flow direction of the nucleic acid isolator 15, and when viewed along the liquid flow direction, the protrusion 21 belongs to the (n + 1) th row. 21 are arranged so as to be positioned between the protrusions 21 belonging to the nth row. Note that n is a natural number indicating the row number, and the curved channel portion of the nucleic acid isolator 15 and the vicinity of the standing column 22 with different diameters are arranged so as to be approximately in the middle.

  The shape of the protrusion 21 is cylindrical as shown in FIG. 3B and FIG. Its diameter is 0.03 mm and its height is 0.15 mm. The interval between adjacent protrusions 21 is 0.03 mm. The direction in which the protrusion 21 protrudes is a direction orthogonal to the bottom surface of the nucleic acid isolator 15.

  As shown in FIG. 4, the nucleic acid isolator 15 including the protrusions 21 includes a resin 37 and a silica member 39 dispersed in the resin 37, and the silica member 39 is exposed on the surface of the protrusion 21. ing. The weight ratio occupied by the silica member 39 in the nucleic acid isolator 15 including the protrusions 21 is 60% by weight. The size of the silica member 39 is approximately φ0.003 mm, which is not more than ½ of the diameter d of the protrusion 21.

  The shape of the silica member 39 may be any of the spherical shape shown in FIG. 5 (a), the flake shape shown in FIG. 5 (b), and the linear shape shown in FIG. 5 (c). As shown by the arrows in FIGS. 5A to 5C, the size of the silica member 39 is the diameter in the case of a sphere, the length in the longitudinal direction in the case of a flake, and the length in the case of a line. The diameter of the cross section.

c) Next, a chip manufacturing method will be described.
First, a mold corresponding to the shape of the main body 3 constituting the chip is manufactured using a known lithography technique. Specifically, first, a resist is applied to a conductive substrate, a resist layer is formed, the surface of the resist is covered with a mask corresponding to the shape of the nucleic acid isolation member 1, and exposed to ultraviolet rays (first). 1 step). Next, the exposed resist is developed (second step), and the developed resist is electroformed with a mold material (third step). Finally, the mold is manufactured by removing the resist and the base material (fourth step).

  Then, the molding material in which the resin 37 and the silica member 39 are kneaded is melted and heated in a cylinder of an injection molding machine using an injection molding method, injected into the mold, filled, cooled and solidified, and molded. Thus, the main body 3 was manufactured. The resin 37 was molded using PA66 at a mold temperature of 80 ° C. Thereafter, the valves 51, 53, and 55 are attached to the main body 3 from the back surface of the chip (opposite side of 3a).

  Next, the film is a PA66 made of the same material as the resin 37 to which no silica member is added, and the portions corresponding to the upper surfaces of the holding unit 5, the waste liquid holding unit 9, the holding unit 11, and the holding unit 13 are cut out. The film and the main body part 3 were heated and joined to the upper surface 3a of the main body part 3 to form the cover part 4 to complete the chip. The cover unit 4 has a holding unit 5, a waste liquid holding unit 9, a holding unit 11, and a holding unit 13 with open upper surfaces, a plurality of channels, a nucleic acid isolator 15, a valve 51, a mixing unit 57, and a PCR. The upper part of the amplifier 23 is closed. In addition, the joining method of the cover part 4 may be adhesion by ultrasonic welding or an adhesive other than thermal welding.

d) Next, a method for isolating DNA using a chip will be described.
Regarding the detection of DNA after isolation, if the final concentration cannot be predicted, the initial DNA concentration cannot be properly adjusted, and the luminance of the band in electrophoresis may not be properly produced.

That is, there is a possibility that the DNA concentration at the time of chip insertion is too high than the adsorption capacity and flows out, and the adsorption efficiency is apparently low.
Therefore, an attempt was made to estimate the DNA concentration of the solution after PCR amplification so that the DNA concentration could be estimated.

  As a preliminary experiment, PCR was performed for 25 cycles using primers (In-F, In-R) from pRSET-scFv to produce an approximately 1 kbp PCR product a (ScFV fragment, 0.1 μg / μl). a is diluted so that the final concentration is 0.005 ng / μl to 0.5 ng / μl, PCR is performed for 10 cycles with the same primer, and the DNA concentration after amplification is determined by electrophoresis to amplify the PCR product a The relationship between the DNA concentration before and after amplification was calculated.

  The DNA used in this adsorption experiment is the above PCR product a, and the eluate from which the DNA is isolated is diluted 10-fold from the stock solution, PCR is performed for 10 cycles with the same primer, and electrophoresis is performed to determine the DNA concentration after amplification. The concentration of the eluate before amplification was estimated from the relationship between the DNA concentration before and after amplification of the PCR product a obtained in the preliminary experiment.

 i) Into an Eppendorf tube, add 60 μl of buffer 1, 60 μl of buffer 2, 84 μl of buffer 3, add 10 μl of PCR product a, and mix gently by inversion several times. Here, the buffer 1 is a reagent for removing RNA in the sample, and is a solution containing RNase A. Buffer 2 is a solution for cell lysis and is a solution containing NaOH and SDS. The buffer 3 is a solution containing guanidine hydrochloride and acetic acid, and has a function of precipitating unwanted substances and adsorbing DNA with a chaotropic salt (guanidine hydrochloride).

  ii) The above mixed solution is centrifuged at 15,000 rpm for 10 minutes, 200 μl of the supernatant is removed with a pipette, and poured into the holding part 5 of the chip. The poured solution is a solution containing 1 kbp DNA at a concentration of 0.0375 ng / μl.

Next, the solution poured into the holding unit 5 is pushed out by air using a syringe pump, thereby moving the nucleic acid refiner 15, the valve 51, the channel 27, and the waste liquid holding unit 9 in this order.
The solution moves between the protrusions 21 in the nucleic acid isolator 15. At this time, the DNA in the solution is adsorbed to the silica member 39 exposed on the surface of the protrusion 21.

  iii) After the solution moves to the waste liquid holding unit 9, the buffer 4 is poured into the 0.5ml holding unit 5. Then, the buffer 4 is driven by air by a syringe pump in the same manner as in the above vi), and is moved to the waste liquid holding unit 9 through the nucleic acid refiner 15, the valve 51, and the flow path 27. The buffer 4 is a solution containing guanidine hydrochloride and isopropanol, and has an action of removing nuclease.

  iv) Next, the buffer 5 is poured into the 0.75 ml holding unit 5. Then, the buffer 5 is driven by air by a syringe pump in the same manner as in the above ii), and is moved to the waste liquid holding unit 9 through the nucleic acid refiner 15, the valve 51, and the flow path 27. The buffer 5 is a solution containing ethanol and has the same action as ethanol precipitation.

v) The valve 51 is rotated so that the nucleic acid isolator 15 and the flow path 29 are connected. The valve 53 is opened and the valve 55 is closed.
vi) Next, the buffer 6 is poured into the 50 μl holder 5. This buffer 6 is driven by air by a syringe pump, and the valve 53 is opened via the nucleic acid refiner 15, the valve 51, and the flow path 29 and moved to the holding unit 11. The buffer 6 is a pH 8.5 solution containing 10 mM Tris and HCl, and elutes the DNA adsorbed on the silica of the protrusions 21 of the nucleic acid isolator 15. After all the buffer 6 containing the eluted DNA has moved to the holding unit 11, the valve 53 is closed.

e) Next, a method for amplifying nucleic acid eluted by the PCR method will be described.
The PCR reaction solution is added from the holding unit 11 to the buffer 6 containing the eluted DNA that has been moved to the holding unit 11. The PCR reaction solution is composed of enzymes, primers (In-F and In-R), substrates dNTP (dATP, dGTP, dCTP, dTTP mix), buffer (ExTaq buffer), and sterilized water. ExTaq DNA polymerase was used as the enzyme, and 450 μl of the PCR reaction solution was poured.

  Thereafter, the valve 55 is opened, and air is pushed out from the holding unit 11 by using a syringe pump, so that the buffer 6, the PCR reaction solution and the enzyme are well mixed in the mixing unit 57 through the flow path 25. The mixed liquid moves to the flow path 26.

  From the middle of the flow path 26, the mixed solution is thermally denatured by the heater 23c set at 95 ° C. on the bottom surface of the chip. The PCR amplifier 23 employs a shuttle PCR method in which primer annealing reaction and extension reaction are simultaneously performed at the same temperature.

  When the mixed solution passes through the flow path 23a of the PCR amplifier 23 by a syringe pump controlled at a constant feed rate appropriate for the PCR reaction, a 60 ° C. heater 23b installed on the bottom surface of the PCR amplifier 23. And 95 ° C. heater 23c. At this time, since the ratio of the length in contact with the heater 23b to the length in contact with the heater 23c in the flow path 23a is 1: 4, the time during which the mixed solution is heated by the heater 23b and the time during which the heater 23c is heated The ratio is 1: 4.

The above heating is performed for 10 cycles, which is the number of repetitions of the flow path 23 a of the PCR amplifier 23, and the liquid mixture obtained by amplifying the DNA moves to the holding unit 13 through the flow path 31.
The solution that has moved to the holder 13 is immediately collected with a pipette and stored at -20 ° C.

  The above is the procedure when the eluate from which DNA is isolated is used as a stock solution. In this example, in order to ensure accuracy, an experiment was also conducted with a 10-fold dilution of the eluate from which DNA was isolated. The same procedure is performed until vi), and the buffer 6 is poured into the 500 μl holder 5 from vi). The subsequent procedure is the same as described above. As a result of performing electrophoresis based on this solution and measuring the recovery rate, the ratio of the amount of DNA contained in buffer 6 to the amount of DNA contained in the supernatant poured in ii) (recovery rate) ) Was 86%.

e) Next, effects produced by the chip of the first embodiment will be described.
i) In the chip of Example 1, the silica member 39 that easily adsorbs DNA is exposed on the surface of the protrusion 21. Therefore, DNA can be isolated efficiently by adsorbing DNA to the silica member 39.

  ii) In the chip of the first embodiment, the size of the spherical silica member 39 is not more than ½ of the diameter d (width) of the protrusion 21. Therefore, two or more silica members 39 are arranged in the width direction of the protrusion 21 in the protrusion 21. In this case, since each of the two or more silica members 39 can be exposed on the surface of the protrusion 21, the exposure of the silica member 39 can be increased. Thereby, DNA can be efficiently adsorbed to the protrusions 21.

  iii) Since the chip of the first embodiment has the plurality of protrusions 21, the contact area between the protrusion 21 and the sample can be increased. Thereby, DNA can be isolated efficiently.

  iv) In the first embodiment, the protrusions 21 are arranged along the direction intersecting the moving direction of the biomaterial in the nucleic acid isolator 15. Therefore, when viewed from the moving direction of the biomaterial, the projected area of the protrusion 21 increases, and the probability that the moving biomaterial and the protrusion 21 come into contact with each other increases. As a result, the chip of Example 1 can efficiently isolate DNA.

  v) In the first embodiment, the protrusions 21 are arranged in a staggered pattern, and the protrusions 21 in a certain row of the staggered lattice are the protrusions 21 in the adjacent rows as viewed from the moving direction of the biomaterial. It is designed to be in between. Therefore, when viewed from the moving direction of the biomaterial, the projected area of the protrusion 21 increases, and the probability that the moving biomaterial and the protrusion 21 come into contact with each other increases. As a result, the chip of Example 1 can efficiently isolate DNA.

  vi) In the chip of Example 1, the distance S between the protrusions 21 is as narrow as 0.03 mm, so that DNA in the biomaterial passing between the protrusions 21 is easily adsorbed to the protrusions 21. Further, since the interval S between the protrusions 21 is the above value, even long-chain DNA can pass through between the protrusions 21.

  vii) In the chip of Example 1, the weight ratio of the silica member 39 to the entire protrusion 21 is 60% by weight, so that the amount of the silica member 39 exposed on the surface of the protrusion 21 is sufficiently large, and the DNA adsorption efficiency Therefore, DNA can be isolated efficiently. Further, since the weight ratio of the silica member 39 to the entire protrusion 21 is the above value, the resin 37 and the silica member 39 can be easily kneaded and dispersed.

  viii) Since the chip of Example 1 can be easily manufactured using a mold, it can be manufactured at low cost. And since the lithography technique and the electroforming technique are used in the manufacture of the mold, an accurate mold can be manufactured and the chip can be easily manufactured even when the protrusion 21 has a fine shape. .

  ix) In addition to the nucleic acid isolator 15 which is a part for isolating DNA, the chip of Example 1 is provided with a PCR amplifier 23 which is a part for amplifying purified DNA. Therefore, when amplifying the purified DNA, it is not necessary to transfer the DNA to another amplification instrument. As a result, the DNA is not contaminated during transfer, and the DNA analysis accuracy can be improved. In addition, since there is no loss of DNA during transfer, the amount of specimen to be purified can be reduced, and in addition, the amount of expensive reagents used for DNA purification can be reduced. Furthermore, DNA purification and amplification can be automated to shorten the time required for DNA purification and amplification.

a) The configuration of the chip of the second embodiment is basically the same as that of the first embodiment. However, in the second embodiment, except for the protrusions 21 in the main body 3 of the chip, only the resin 37 is formed, and the protrusions 21 include a central part 21a made of resin and a central part 21a as shown in FIG. It is comprised from the silica layer 21b which covers the surface of this. FIG. 6 is a cross-sectional view taken along the line AA in FIG.

  b) In order to manufacture the chip of the second embodiment, first, the main body 3 is manufactured by pouring only the resin 37 in which the silica member 39 is not kneaded into the same mold as that of the first embodiment. At this stage, only the central portion 21 a made of the resin 37 is formed on the protrusion 21. Next, a silica layer 21b is formed on the surface of the central portion 21a of the protrusion 21 using CVD (Chemical Vapor Deposition), and the nucleic acid isolator 15 is completed. The thickness of the silica layer 21b can be controlled by the CVD conditions. When the silica layer 21b is formed, the 3a surface of the chip other than the nucleic acid isolator 15 may be shielded.

c) The chip of the second embodiment can be used in the same manner as the chip of the first embodiment, and has the same effect.
Furthermore, in the chip of Example 2, since the silica layer 21b uniformly covers the surface of the protrusion 21, the contact area between the silica layer 21b and the biological material is increased, and the DNA adsorption efficiency is high. Therefore, DNA can be isolated more efficiently.

  Further, in the second embodiment, the portion other than the silica layer 21b in the main body 3 is made of the resin 37 in which the silica member 39 is not kneaded. Therefore, the mold is more easily performed in the injection molding than in the first embodiment. Can be filled. Therefore, in the chip of Example 2, it is easy to form the fine protrusions 21 in the injection molding.

  a) The chip configuration and manufacturing method of the third embodiment are basically the same as those of the second embodiment. However, in the third embodiment, the shape of the protrusion 21 is not a cylindrical shape, but is one of the shapes shown in FIG. 7 or FIG.

  The protrusion 21 shown in FIG. 7A has a truncated cone shape. That is, the protrusion 21 includes a circular bottom surface 21c, a top surface 21d that is a circle having a smaller diameter than the bottom surface 21c, and a side surface 21e. An angle formed by the side surface 21e and the protruding direction of the protrusion 21 (a direction orthogonal to the bottom surface 21c and the upper surface 21d) is always constant from the bottom to the top of the protrusion 21. The side surface 21e is a tapered portion that is inclined with respect to the protruding direction.

  The protrusion 21 shown in FIG. 7B has a substantially truncated cone shape. That is, the protrusion 21 includes a circular bottom surface 21c, a top surface 21d that is a circle having a smaller diameter than the bottom surface 21c, and a side surface 21e. An angle formed by the side surface 21e and the protruding direction of the protrusion 21 (a direction orthogonal to the bottom surface 21c and the top surface 21d) is smaller as the surface 21d is closer to the upper surface 21d. The side surface 21e is a tapered portion that is inclined with respect to the protruding direction.

  The protrusion 21 shown in FIG. 7C has a substantially truncated cone shape. That is, the protrusion 21 includes a circular bottom surface 21c, a top surface 21d that is a circle having a smaller diameter than the bottom surface 21c, and a side surface 21e. An angle formed by the side surface 21e and the protruding direction of the protrusion 21 (a direction orthogonal to the bottom surface 21c and the top surface 21d) increases as the surface approaches the top surface 21d. The side surface 21e is a tapered portion that is inclined with respect to the protruding direction.

  The protrusion 21 shown in FIG. 8A includes a truncated cone-shaped base portion 21f and a cylindrical upper portion 21h standing in a protruding direction from the upper surface 21g of the base portion 21f. The angle formed by the side surface 21i and the protruding direction in the base 21f is always constant from the bottom to the top of the base 21f. The side surface 21i is a tapered portion that is inclined with respect to the protruding direction.

  The protrusion 21 shown in FIG. 8B includes a base part 21f having a substantially truncated cone shape and a cylindrical upper part 21h erected in a protruding direction from the upper surface 21g of the base part 21f. The angle formed between the side surface 21i and the protruding direction in the base portion 21f becomes smaller as it approaches the upper surface 21g of the base portion 21f. The side surface 21i is a tapered portion that is inclined with respect to the protruding direction.

  The protrusion 21 shown in FIG. 8C includes a substantially truncated cone-shaped base portion 21f and a cylindrical upper portion 21h erected in the protruding direction from the upper surface 21g of the base portion 21f. The angle formed between the side surface 21i and the protruding direction in the base portion 21f increases as it approaches the upper surface 21g of the base portion 21f. The side surface 21i is a tapered portion that is inclined with respect to the protruding direction.

b) The chip of the third embodiment can be used in the same manner as the chip of the second embodiment, and has the same effect.
In the chip of the third embodiment, the protrusion 21 has a truncated cone shape as shown in FIG. 7 or FIG. 8 or a shape having a tapered portion, and has a portion inclined with respect to the protruding direction. . Such inclined portions are more likely to form the silica layer 21b by CVD compared to vertical portions (portions parallel to the protruding direction), which increases the efficiency of DNA adsorption in this Example 3. To increase.

  In addition, since the shape of the protrusion 21 is a substantially truncated cone shape or a shape having a tapered portion, there is a portion that is inclined with respect to the protruding direction of the protrusion 21, and the inclined portion is formed in a mold. Corresponds to draft. For this reason, it is easy to release the chip body 3 from the mold, which facilitates manufacture.

  In addition, since the protrusion 21 has such an inclined portion, the area on which the DNA is adsorbed is increased in the protrusion 21 as compared with the case where the side surface of the protrusion 21 is composed only of a portion parallel to the protrusion direction. The efficiency of DNA adsorption can be increased.

Needless to say, the present invention is not limited to the above-described embodiments, and can be implemented in various modes without departing from the scope of the present invention.
For example, the shape of the protrusion 21 in the first embodiment may be the one described in FIG. 7 or FIG.

  Moreover, in the said Example 1, the silica member contained in the processus | protrusion 21 may be a flake-shaped thing which has various shapes, as shown in FIG.

It is a perspective view showing the structure of the chip | tip which united the nucleic acid isolation part and the PCR amplification part. It is a perspective view showing the structure of a cover part. It is a top view showing the structure of a nucleic acid isolation part. It is sectional drawing showing the structure of protrusion vicinity. It is explanatory drawing showing the shape of a silica member. It is sectional drawing showing the structure of protrusion vicinity. It is a perspective view showing the shape of a processus | protrusion. It is a perspective view showing the shape of a processus | protrusion. It is sectional drawing showing the structure of protrusion vicinity.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Nucleic acid isolation member 3 ... Main-body part 4 ... Cover part 5 ... Holding part 9 ... Waste liquid holding | maintenance part 15 ... Nucleic acid isolator 25, 26, 27, 29, 31 ... Flow path 21 ... Protrusion 21a ... Center part 21b ... Silica layer 23 ... PCR amplifier 37 ... Resin 39 ... Silica member

Claims (19)

A to-be-supplied part to which a biomaterial containing nucleic acid is supplied;
A protrusion provided at a position in contact with the biomaterial in the supplied portion;
The protruding portion contains a resin and silica, and the silica is exposed at least at a part of the surface of the protruding portion.   The nucleic acid isolation member according to claim 1, wherein the protruding portion has a substantially cylindrical shape.   The nucleic acid isolation member according to claim 1, wherein the shape of the protruding portion is a substantially truncated cone shape or a shape having a tapered portion.   The protrusion is composed of the resin and a silica member having at least one of a granular shape, a flake shape, and a linear shape, and the size of the silica member is ½ or less of the width of the protrusion portion. The nucleic acid isolation member according to any one of claims 1 to 3.   The nucleic acid isolation member according to claim 1, wherein the nucleic acid isolation member has a plurality of the protruding portions.   The nucleic acid isolation member according to claim 5, wherein the protrusions are arranged along a direction intersecting a moving direction of the biomaterial in the supply target part.   The nucleic acid isolation member according to claim 5 or 6, wherein the protrusions are arranged in a staggered pattern.   The nucleic acid isolation member according to any one of claims 5 to 7, wherein an interval between the adjacent protrusions is in a range of 0.001 to 1 mm.   The protruding portion is composed of the resin and a silica member having at least one of a granular shape, a flake shape, and a linear shape dispersed in the resin, and a part of the silica member is a portion of the protruding portion. The nucleic acid isolation member according to any one of claims 1 to 7, which is exposed on the surface.   The nucleic acid isolation member according to claim 9, wherein a weight ratio of the silica member to the entire protrusion is in a range of 5 to 90% by weight.   The nucleic acid isolation member according to any one of claims 1 to 8, wherein the protruding portion includes a central portion made of a resin and a silica layer containing silica covering a surface of the central portion.   The nucleic acid isolation member according to any one of claims 1 to 11, further comprising a cover member that covers the protruding portion.   The nucleic acid isolation member according to claim 12, wherein the cover member is made of the same resin as a main body of the nucleic acid isolation member.   The said cover member and the main body of the said nucleic acid isolation member are joined by at least 1 method of the heat welding, the ultrasonic welding, and the adhesion | attachment method, The Claim 12 or 13 characterized by the above-mentioned. Nucleic acid isolation member. It is a manufacturing method of the metal mold | die used for manufacture of the nucleic acid isolation member in any one of Claims 1-14,
A first step of covering the surface of the resist with a mask corresponding to the shape of the nucleic acid isolation member and exposing with a light beam;
A second step of developing the exposed resist;
A third step of electroforming the resist with a mold material;
And a fourth step of removing the resist, and a method for producing a mold for a nucleic acid isolation member.   The mold manufacturing method according to claim 15, wherein the light beam is an X-ray. In the manufacturing method of the nucleic acid isolation member in any one of Claims 1-14,
A step of pouring a material composed of the resin and the silica into a mold for the nucleic acid isolation member;
B step of separating the solidified material from the mold;
A method for producing a nucleic acid isolation member, comprising: The method for producing a nucleic acid isolation member according to claim 11,
A method for producing a nucleic acid isolation member, wherein the silica layer is formed using CVD.   The method for forming a nucleic acid isolation member according to claim 18, wherein the shape of the protruding portion that forms the silica layer using CVD is a truncated cone shape or a shape having a tapered portion.

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