JP2007244389A - Nucleic acid-amplifying substrate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a nucleic acid-amplifying substrate for carrying out a nucleic acid-amplifying reaction such as a PCR on the substrate to amplify the nucleic acid, and free from the leakage of a reaction liquid in the nucleic acid-amplification reaction process. <P>SOLUTION: The nucleic acid-amplifying substrate 10 is obtained by laminating a first layer 11 comprising a glass plate and a second layer 12 comprising a silicone rubber. The second layer 12 has fine groove work formed on the face in contact with the first layer 11 to form spaces 6 between the first layer 11 and the second layer 12. Four systems of nucleic acid-amplifying/separating flow channels 15, 16, 17 and 18 are formed by the pattern (groove pattern) of the spaces in the nucleic acid-amplifying substrate 10. The nucleic acid-amplifying/separating flow channel 18 has a reaction liquid-storing part 20 and an electrophoresis part 21, and is obtained by forming a reaction liquid reciprocating flow channel 22, a reaction liquid-sucking flow channel 23 and a nucleic acid-feeding flow channel 25. The reaction liquid-storing part 20 is obtained by forming a maze-shaped zigzag flow channel in a nearly square space in front view. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid amplification substrate that performs nucleic acid amplification reaction such as PCR in one substrate to amplify nucleic acid.

In recent years, diagnosis using DNA has attracted attention in the medical field. DNA testing is also used to determine genetically modified crops and varieties in the agricultural field.
Diagnosis and variety determination as described above are performed based on whether or not the target DNA is present in the blood or crop sample, but the target DNA in the sample is often in trace amounts. Therefore, it is necessary to amplify the target DNA contained in the sample prior to DNA diagnosis or DNA identification. Here, various nucleic acid amplification reactions have been proposed as means for specifically amplifying target DNA in a sample. In particular, a nucleic acid amplification reaction by PCR (Polymerase Chain Reaction) is a basic technology in the biotechnology field.
The PCR method is a method of amplifying a target DNA by adjusting the temperature of a reaction solution in which a template DNA, a primer, a substrate, a heat-resistant polymerase enzyme, etc. are mixed, and sequentially changing the temperature to three predetermined temperatures. It is.

  That is, the temperature of the reaction solution is adjusted to a temperature at which a dynalation reaction that dissociates double-stranded DNA into single-stranded DNA is performed, and then the temperature is adjusted to a temperature at which an annealing reaction that associates a primer with single-stranded DNA is performed. Subsequently, the temperature is adjusted to a temperature at which a double-strand elongation reaction with a thermostable polymerase enzyme is performed. In this way, DNA can be amplified by sequentially adjusting the temperature of the reaction solution to three stages.

  Specifically, a large amount of DNA replication product is obtained by repeating the step of adjusting the temperature of the reaction solution to each set temperature about 30 times. Note that the reaction temperature for annealing, annealing temperature, and double-strand elongation are about 95 ° C., 50 to 55 ° C., and 72 ° C., respectively.

Traditionally, the PCR method has been performed using a microtube. A reaction solution containing a sample and a PCR primer is placed in the microtube, and the microtube is set to a predetermined temperature atmosphere.
However, the method using a microtube requires a corresponding amount of sample, but there are cases where only a very small amount of sample can be obtained, and DNA identification or the like cannot be performed.
Also, the method using a microtube has a problem that it takes time to amplify DNA.
Furthermore, in order to analyze the amplified DNA, it was necessary to extract the reaction solution from the microtube and separately apply it to a separation device, and there was also a complaint that it took time and effort.

On the other hand, a method has been proposed in which DNA is amplified by a PCR method on a substrate, and further DNA is separated on the same substrate.
The following patent documents all disclose techniques for performing amplification and separation of DNA on a single substrate.
JP 2003-83965 A JP 2002-306154 A JP 2003-270204 A

The substrate disclosed in Patent Document 1 has a recess or groove formed on the surface of a glass plate or the like, and is provided with an open PCR chamber. The substrate disclosed in Patent Document 1 includes a PCR tank in a part thereof, but the upper surface of the PCR tank is open.
In the technique disclosed in Patent Document 1, the above-described PCR tank is filled with a reaction solution or the like. Then, a reaction solution or the like in the PCR tank is heated in a non-contact state by a heat source installed at the upper portion to perform a daturation reaction.
Subsequently, the reaction solution or the like in the PCR tank is cooled by a cooling device installed at the lower portion, and annealing is performed.
Furthermore, the reaction solution in the PCR tank is heated in a non-contact state by a heat source installed at the upper part to carry out a double-strand extension reaction.
These steps are repeated to amplify the target DNA in the sample.

The substrate disclosed in Patent Document 2 is also provided with a PCR tank with an open top surface, and PCR is performed by filling the PCR tank with a reaction solution or the like.
In the technique disclosed in Patent Document 2, the temperature of the substrate is controlled only from the bottom surface side.

Also for the substrate disclosed in Patent Document 3, a PCR tank with an open top surface is provided, and PCR is performed by filling the PCR tank with a reaction solution or the like.
In the technique disclosed in Patent Document 3, the substrate is heated from the upper and lower surfaces, and at that time, the upper surface of the PCR tank is closed by a seal portion provided on the temperature control device side.

It is expected that the demand for DNA diagnosis and appraisal will increase in the future. Therefore, there is an urgent need to develop a technique capable of performing DNA amplification by a simpler method. Desirably, a desired diagnosis or the like is performed with a small amount of sample.
Furthermore, since DNA diagnosis is often used for diagnosis of infectious diseases, DNA amplification must be performed quickly.
In view of this, the present inventors examined a method of heating and cooling from both sides of the substrate in order to more quickly adjust the temperature during PCR. More specifically, a method of adjusting the temperature of the substrate by using a pair of heat blocks on the top and bottom and sandwiching the substrate with the heat block was examined.
Furthermore, in order to further speed up the temperature control, three sets of the heat block described above are prepared, and the temperature of each of these heat blocks is adjusted in advance to the reaction temperature of annealing temperature, annealing temperature, and double-strand elongation, respectively. We examined the measures to sequentially adjust the temperature to three stages by sequentially replacing the sandwiched heat blocks.

Here, since the upper surface of the PCR tank is open in all of the prior art substrates, the heat block cannot be brought into contact with the upper portion of the substrate. In addition, if the apparatus disclosed in Patent Document 3 is used, the heat block can be brought into contact with the upper part of the substrate. However, in the apparatus disclosed in Patent Document 3, there is a seal member on the temperature control device side. The seal member is contaminated with the reaction solution. For this reason, when the temperature of different substrates is adjusted, the sealing member must be washed one by one, which is not practical.
Furthermore, if the heat block sandwiching the substrate is sequentially replaced on the basis of the idea of the present inventors and the temperature is adjusted to three stages in sequence, it is inevitable that the substrate is subjected to vibration and impact. On the other hand, in the substrate of the prior art, since the upper surface of the PCR tank is open, there is a risk that the reaction liquid or the like spills from the PCR tank and contaminates the surroundings or mixes with other reaction liquids.
Accordingly, the present invention pays attention to the above-mentioned problems of the prior art, and an object of the present invention is to develop a substrate in which a reaction solution hardly leaks in a nucleic acid amplification reaction step such as PCR.

  In the invention according to claim 1 for solving the above-described problem, a reaction liquid reservoir is formed in a part of a substrate, and the reaction liquid reservoir is filled with a reaction liquid containing components necessary for a nucleic acid amplification reaction. In the nucleic acid amplification substrate that adjusts the temperature of the reaction solution reservoir and performs a nucleic acid amplification reaction in the reaction solution reservoir to amplify the nucleic acid, the reaction solution reservoir is formed by a gap provided in the substrate. A nucleic acid amplification substrate comprising an electrophoresis unit that separates nucleic acids amplified in a reaction solution reservoir by electrophoresis.

In the nucleic acid amplification substrate of the present invention, the reaction liquid reservoir is constituted by a gap provided inside the substrate and is in a semi-sealed state. Therefore, even if an object touches the surface of the substrate, it is not contaminated by the reaction solution. Even if the substrate is vibrated, the reaction solution does not spill from the reaction solution reservoir.
In addition, the nucleic acid amplification substrate of the present invention includes an electrophoresis unit that separates nucleic acids amplified in the reaction liquid reservoir by electrophoresis in the same substrate. Therefore, according to the nucleic acid amplification substrate of the present invention, nucleic acid amplification and separation can be performed in the same substrate.
Examples of the nucleic acid amplification reaction performed using the nucleic acid amplification substrate of the present invention include a PCR method, an LTR (Ligase Chain Reaction) method, an SDA (Standard Displacement Amplification) method, etc. The PCR method is typical. . Examples of components necessary for the nucleic acid amplification reaction include template DNA, primers, deoxynucleotide triphosphates, and heat-resistant DNA polymerase in the case of PCR.

  The invention according to claim 2 includes a reaction liquid introduction part that introduces a reaction liquid, and a reaction liquid flow path that is formed by a gap provided inside the substrate and connects the reaction liquid introduction part and the reaction liquid reservoir, 2. The nucleic acid amplification substrate according to claim 1, wherein the reaction solution is introduced into the reaction solution introduction part, and further filled into the reaction solution reservoir part through the reaction solution channel.

  The nucleic acid amplification substrate of the present invention includes a reaction liquid introduction part for introducing a reaction liquid, and the reaction liquid is introduced into the substrate from the reaction liquid introduction part. In the nucleic acid amplification substrate of the present invention, since the reaction liquid flow path is provided between the reaction liquid introduction part and the reaction liquid reservoir part, there is a considerable distance between the reaction liquid reservoir part and the reaction liquid introduction part. The reaction liquid in the liquid reservoir is difficult to leak.

  According to a third aspect of the present invention, the substrate is formed by laminating two or more layers, and a groove is provided on one of the two laminated layers, and the gap is formed inside the substrate by the groove. The nucleic acid amplification substrate according to claim 1, wherein the nucleic acid amplification substrate is formed.

  The nucleic acid amplification substrate of the present invention can easily form a void in the substrate by a simple method.

In addition, the present inventors made prototypes of various shapes of substrates, but the prototype at the beginning of the development was a nucleic acid amplification substrate 2 having a reaction liquid reservoir portion 1 constituted by a single cavity as shown in FIG. Met.
The nucleic acid amplification substrate 2 shown in FIG. 13 did not spill the reaction solution even when subjected to vibrations, and was able to achieve the original purpose, but had an unexpected problem at the beginning of development.
That is, using the nucleic acid amplification substrate 2 shown in FIG. 13, as described above, the heat blocks sandwiching the nucleic acid amplification substrate 2 are sequentially replaced, and the temperature is sequentially adjusted to three stages, and this is repeated about 30 times. The reaction solution disappeared from the liquid reservoir 1.

When this cause was examined, it became clear that it was based on two main reasons.
One reason was that the reaction solution expanded and contracted as the temperature of the reaction solution was repeatedly raised and cooled, and the reaction solution moved and escaped outside the reaction solution reservoir. .
Another cause is that, when sandwiched between the upper and lower heat block pieces 3 and 4 as shown by a two-dot chain line in FIG. This was caused by being pushed out of the reaction liquid reservoir 1.
Therefore, the present inventors have invented the inventions described in claims 4 to 7 as a countermeasure.

  That is, the invention described in claim 4 is characterized in that a movement suppressing means for suppressing the movement of the reaction liquid is provided in the reaction liquid reservoir or in the vicinity of the inlet / outlet of the reaction liquid reservoir. The nucleic acid amplification substrate according to 1.

The nucleic acid amplification substrate of the present invention is provided with a movement suppressing means for suppressing the movement of the reaction solution. Therefore, the reaction liquid is prevented from escaping to the outside of the reaction liquid reservoir.
As the movement restraining means, for example, a configuration for increasing the flow path resistance is conceivable.

  The invention according to claim 5 is characterized in that a flow path including a plurality of curved paths is formed in the reaction liquid reservoir or in the vicinity of the inlet / outlet of the reaction liquid reservoir. The nucleic acid amplification substrate as described.

In the nucleic acid amplification substrate of the present invention, since the flow path including a plurality of curved paths is formed in the reaction liquid reservoir or in the vicinity of the inlet / outlet of the reaction liquid reservoir, the flow path resistance in or near the reaction liquid reservoir is high. Therefore, the reaction solution is prevented from escaping to the outside of the reaction solution reservoir.
Note that the flow path including the curved path also functions as “movement restraining means”.
In particular, when a flow path is formed in the reaction liquid reservoir, the deflection of the front and back surfaces of the reaction liquid reservoir is reduced by the wall surface forming the flow path. Therefore, the outflow of the reaction solution due to the latter cause is also prevented.

  The invention according to claim 6 is characterized in that a serial flow path including a plurality of curved paths is formed in the reaction liquid reservoir, and the nucleic acid amplification substrate according to any one of claims 1 to 4 It is.

Since the flow path including a plurality of curved paths is formed in the reaction liquid reservoir also for the nucleic acid amplification substrate of the present invention, the flow path resistance is high, and the deflection of the front and back surfaces of the reaction liquid reservoir is reduced. Is prevented from flowing out.
In particular, in the nucleic acid amplification substrate of the present invention, since the flow paths in the reaction liquid reservoir are in series, the reaction liquid reservoir can be filled closely with the reaction liquid.

  The invention according to claim 7 is characterized in that a compression resistant part is provided in the reaction liquid reservoir, and when the reaction liquid reservoir is compressed in the front and back direction, the compression force is borne by the compression resistant part. A nucleic acid amplification substrate according to any one of claims 1 to 6.

In the nucleic acid amplification substrate of the present invention, the compression resistant part is provided in the reaction liquid reservoir, and the compressing part bears part or all of the compressive force. Therefore, the deflection of the front and back surfaces of the reaction liquid reservoir is reduced and the reaction liquid is prevented from flowing out.
Note that the flow path provided in the reaction liquid reservoir also functions as a compression resistant part.

  The reaction liquid reservoir and the electrophoresis part are separated, and the distance between the two is preferably at least three times the thickness of the thickest part of the substrate.

That is, the electrophoresis unit is filled with a carrier for separating nucleic acids such as DNA amplified by a nucleic acid amplification reaction such as PCR. This carrier is often a gel. Here, the nucleic acid amplification substrate of the present invention performs the nucleic acid amplification reaction in the same substrate, and the temperature of the reaction solution reservoir in which the nucleic acid amplification reaction such as PCR is performed is increased. For this reason, if the electrophoresis part is located near the reaction liquid reservoir, the electrophoresis part is heated when the temperature of the reaction liquid reservoir is raised, and the water in the gel inside is evaporated. As a result, the composition of the gel changes, and the resolution during electrophoresis is adversely affected.
On the other hand, in the invention according to claim 8, the reaction liquid reservoir and the electrophoresis part are separated, and the distance between the two is more than three times the thickness of the thickest part of the substrate. Even if the reservoir is heated, the electrophoretic part is not affected by the heat, and the separation performance during electrophoresis is not adversely affected.

  The invention according to claim 9 is the nucleic acid amplification substrate according to any one of claims 1 to 8, wherein the thickness of the reaction liquid reservoir is smaller than the thickness of the electrophoresis part. .

  In the nucleic acid amplification substrate of the present invention, the thickness of the reaction liquid reservoir is thinner than the thickness of the electrophoresis part. Therefore, it is easy to control the temperature of the reaction liquid reservoir from the outside. On the contrary, since the electrophoretic part is thick, the electrophoretic part is not easily affected by heat from the outside.

  The region from the reaction solution introduction part to the reaction solution reservoir is preferably hydrophilic.

  That is, in the invention according to the tenth aspect, since the part is hydrophilic, the sample is easily adapted to the surface, and the sample can be easily introduced into the substrate. Furthermore, since the heat conduction from the substrate to the sample is good in the reaction liquid reservoir, the nucleic acid can be amplified more reliably by PCR or the like.

In the nucleic acid amplification substrate of the present invention, there is little leakage of the reaction solution in the nucleic acid amplification reaction step, and there is an effect that there is no surrounding contamination or mixing of different samples. Furthermore, nucleic acid amplification and separation can be performed with a single substrate, which has the effect of high work efficiency.
In particular, the nucleic acid amplification substrate according to any one of claims 4 to 7 has an effect of preventing the reaction solution from flowing out of the reaction solution reservoir when the substrate is sandwiched between heat blocks or the like. Is possible.

  In particular, the nucleic acid amplification substrate according to claim 9 has an excellent effect that the temperature in the reaction liquid reservoir can be rapidly changed and the thermal influence on the electrophoresis portion is small.

  In particular, the nucleic acid amplification substrate according to claim 10 is advantageous in that the introduction of the sample is easy and the nucleic acid can be amplified from a smaller amount of sample because the loss of the sample is small.

Embodiments of the present invention will be further described below.
FIG. 1 is a front view of a nucleic acid amplification substrate according to an embodiment of the present invention. FIG. 2 is a perspective view of the nucleic acid amplification substrate of FIG. FIG. 3 is a front view of two systems of nucleic acid amplification / separation flow paths of the nucleic acid amplification substrate of FIG. 4 is an exploded perspective view of the first layer and the second layer around the reaction liquid reservoir of the nucleic acid amplification substrate of FIG. FIG. 5 is an enlarged cross-sectional view around the reaction liquid reservoir of the nucleic acid amplification substrate of FIG. FIG. 6 is an enlarged perspective view of the vicinity of the reaction solution inlet of the nucleic acid amplification substrate of FIG.

  In the figure, reference numeral 10 denotes a nucleic acid amplification substrate according to an embodiment of the present invention. The nucleic acid amplification substrate 10 has a substantially rectangular plate shape, and the size thereof is about 30 × 40 mm to 70 × 100 mm.

The nucleic acid amplification substrate 10 is formed by laminating a first layer 11 made of a glass plate and a second layer 12 made of silicon rubber. The first layer 11 is a layer provided mainly to ensure the overall rigidity, and a material such as quartz or acrylic resin can be used in addition to glass.
The second layer 12 is a layer provided to form a void at the interface with the first layer 11, and a material capable of forming fine grooves and recesses is selected. An example of silicone rubber is PDMS (Polydimethylsiloxane). In addition to silicon rubber, various materials such as plastic such as polymethyl methacrylate (PMMA), glass, and quartz can be used.
When the sample is optically detected, the first layer 11 and the second layer 12 are desirably transparent or light transmissive.

In the present embodiment, the glass plate as the first layer 11 is smooth on both the front and back surfaces as shown in FIGS. On the other hand, the silicon rubber as the second layer 12 is provided with fine grooves 5 on the contact surface side with the first layer 11 as shown in FIGS. A gap 6 is formed between them.
Further, the second layer 12 is provided with a plurality of through holes as shown in FIGS. 1 and 2, and the gap and the outside communicate with each other through the through holes.

  The inside of the gap and the opening are both subjected to hydrophilic treatment, and the surface thereof is hydrophilic.

  The thickness of the first layer 11 is about 0.2 to 5.0 mm. When the thickness of the first layer 11 is less than 0.2 mm, the overall rigidity is low, and it is not suitable for heating by being sandwiched between heat blocks. Conversely, if the thickness of the first layer 11 exceeds 5.0 mm, it is difficult for heat to be transferred from the heat block to the inside. The most preferred range for the thickness of the first layer is about 0.2 to 0.6 mm.

The thickness of the second layer 12 is not uniform, and an area D provided with a reaction liquid reservoir 20 to be described later is thinner than other areas as shown in FIG. Specifically, the thickness of the reaction liquid reservoir 20 is 0.2 to 1.0 mm, and the thickness of other parts is 0.6 to 3 mm.
When the thickness of the portion corresponding to the reaction liquid reservoir 20 of the second layer is less than 0.2 mm, the overall rigidity is low and the deflection is large, so that it is not suitable for heating with a heat block. If the thickness of the part exceeds 1.0 mm, it is difficult for heat to be transferred from the heat block to the inside. The most recommended range from this point is about 0.3 mm to 0.6 mm.
The electrophoretic part 21 to be described later is preferably not affected by heat from the outside, and the thickness of the second layer is designed to be thicker than the reaction liquid reservoir 20. In addition, it is desirable to have a certain thickness in terms of attaching electrodes to the electrophoresis unit 21.

  In the nucleic acid amplification substrate 10 of the present embodiment, four systems of nucleic acid amplification / separation channels 15, 16, 17, and 18 are formed as shown in FIG.

  Since the four systems of nucleic acid amplification / separation channels 15, 16, 17, and 18 have substantially the same shape, the nucleic acid amplification / separation channel 18 illustrated at the bottom of FIG. Will be described.

The nucleic acid amplification / separation flow path 18 has a reaction liquid reservoir 20 and an electrophoresis part 21 as shown in FIGS. 1 and 3, and a reaction liquid reciprocating flow path 22, a reaction liquid suction path 23, and a nucleic acid supply around these. A path 25 is provided.
Further, the nucleic acid amplification / separation flow path 18 includes a reaction liquid introduction opening (reaction liquid introduction section) 30, a suction opening 31, a nucleic acid supply path end opening 33, a migration section start end opening 35, and a migration section end opening 36. Is provided. Each of the openings 30, 31, 33, 35, 36 is configured by a through hole provided in the second layer 12, and the gap 6 formed between the first layer 11 and the second layer 12 and the outside It communicates with.

Hereinafter, description will be made sequentially.
The reaction liquid reservoir 20 is a gap formed for the purpose of storing the reaction liquid inside. In this embodiment, the reaction liquid reservoir 20 is formed by connecting the flow path 40 bent in an S-shaped curve in a dense state. In other words, it can be said that the reaction liquid reservoir 20 is provided with the partition wall 7 (FIG. 5) in a space having a substantially square shape when viewed from the front, and provided with a zigzag labyrinth-like flow path. The flow paths in the reaction liquid reservoir 20 are in series. That is, in the reaction liquid reservoir 20, eight straight paths 41 having substantially the same length are arranged in parallel, and the ends of the adjacent straight paths 41 are connected by the curved path 43, so that they are in series as a whole. ing.
The curved path 43 is a circular flow path as shown in FIG. The curved path may be a rectangular channel as shown in FIG. 3C, but bubbles are likely to remain at the corners of the rectangular channel, so a circle as shown in FIG. It is recommended to use a uniform flow path.

Since the reaction liquid reservoir 20 is a part formed to store the reaction liquid inside as described above, the internal flow path is larger in both width and depth than the flow paths of other parts. .
That is, the flow path in the reaction liquid reservoir 20 is constituted by the groove formed at the interface of the second layer 12 as described above, and the width of the groove is 100 μm to 500 μm, more preferably 250 to 450 μm. It is.
The depth of the groove of the reaction liquid reservoir 20 is 100 to 300 μm. In addition, the depth of the groove | channel in another site | part is about 10-50 micrometers.
The cross-sectional area of the groove of the reaction liquid reservoir 20 (the cross-sectional area of the flow path) is 0.01 to 0.15 square millimeters, which is 3 to 50 times larger than the grooves in other parts.
The total volume of the reaction liquid reservoir 20 is about 3 to 10 ml.

  One end side of the reaction liquid reservoir 20 communicates with a reaction liquid introduction opening (reaction liquid introduction part) 30 via a reaction liquid reciprocating flow path 22. The reaction solution introduction opening 30 is an opening provided near the center of the nucleic acid amplification substrate 10. The reaction liquid reciprocating flow path 22 connecting the reaction liquid introduction opening 30 and the reaction liquid reservoir 20 is based on a straight line, and there is no “U” path or “S” path.

The other end side of the reaction liquid reservoir 20 communicates with the suction opening 31 via the reaction liquid suction path 23. The suction opening 31 is also an opening provided in the nucleic acid amplification substrate 10. The reaction liquid suction path 23 is based on a curved path, and is a flow path bent in an S-shaped curve. The curved path of the reaction solution suction path 23 is for the purpose of increasing the flow path resistance, and has a square shape as shown in FIG.
The curved portion of the reaction liquid suction path 23 functions as “movement restraining means”.

The electrophoresis part 21 is a flow path from the migration part start side opening 35 to the migration part termination side opening 36, and intersects the nucleic acid supply path 25 in the middle part. During the period from the migration part start side opening 35 to the intersection 50, the migration preparation path 51 functions simply as a current path.
The migration preparation path 51 has a plurality of curved paths and a considerable amount of distance is secured.

Between the crossing part 50 and the migration part terminal side opening 36 is a migration path 52, which is a part where gels are filled and nucleic acids such as amplified DNA are actually migrated and separated as described later.
The migration path 52 is partially folded in a “<” shape, but basically has a straight line as a basis.

The nucleic acid supply path 25 connects the reaction solution introduction opening 30 and the nucleic acid supply path end side opening 33, and intersects the electrophoretic unit 21 at the intermediate portion as described above.
Since the reaction solution introduction opening 30 is provided in the vicinity of the central portion of the nucleic acid amplification substrate 10 as described above, the reaction solution introduction opening 30 becomes a flow path extending in the direction returning to the reaction solution reservoir 20 side while reaching the intersection 50. Further, in the flow path from the reaction solution introduction opening 30 to the intersection 50, a portion bent in a “U” shape is provided. The portion bent in the “U” shape is one in which the channels are arranged in parallel, but there is a considerable distance between the channels arranged in parallel, and the pressure loss at the portion is small. In other words, the above-mentioned “U” -shaped portion is not provided to increase the flow resistance, but is provided to ensure a distance from the reaction solution introduction opening 30 to the intersection 50 at a predetermined length or more. It is what was done.
In this embodiment, the layout of the flow path from the intersection 50 to the nucleic acid supply path end opening 33 is symmetrical to the layout of the flow path from the reaction solution introduction opening 30 to the intersection 50.

  In the present embodiment, as described above, the nucleic acid amplification / separation channels 15, 16, 17, and 18 have four systems, and the layout of each channel is slightly different. , 17 and 18 have the same length of the flow path of the electrophoresis unit 21. That is, the distance of each of the nucleic acid amplification / separation channels 15, 16, 17, and 18 from the migration unit start side opening 35 to the migration unit termination side opening 36 is the same. Further, the distance from the migration part start end side opening 35 to the intersection part 50 and the distance from the intersection part 50 to the migration part end side opening 36 are also different among the nucleic acid amplification / separation channels 15, 16, 17, 18. There is no.

  The same applies to the nucleic acid supply path 25, and the lengths of the reaction solution introduction opening 30 and the nucleic acid supply path end opening 33 are the same in any of the nucleic acid amplification / separation flow paths 15, 16, 17, and 18. The length from the reaction solution introduction opening 30 to the intersection 50 and the length from the intersection 50 to the nucleic acid supply channel end opening 33 are also between the nucleic acid amplification / separation channels 15, 16, 17, 18. There is no difference.

  In the present embodiment, as shown in FIG. 1, the area A of the reaction liquid reservoir 20 that performs the nucleic acid amplification reaction and the area B that performs electrophoresis are separated, and there is a considerable distance between them. This distance C is a distance at which the heat of the reaction liquid reservoir 20 is not transmitted to the electrophoresis unit 21, and is not less than three times the plate thickness (thickness of the thickest portion) of the nucleic acid amplification substrate 10, or a first made of a glass plate. The thickness of the layer 11 is 5 times or more.

The electrophoresis unit 21 and the nucleic acid supply channel 25 are filled with a gel made of an aqueous polymer generally used for electrophoresis of nucleic acids. Examples of the gel include agarose, polyacrylamide, polydimethylacrylamide, polymethoxycellulose and the like.
The concentration of the gel is appropriately selected depending on the size of the nucleic acid to be separated. Taking the case where the nucleic acid is DNA as an example, about 5% of polydimethylacrylamide can be used if the DNA has a size of about several tens to several hundred base pairs. In the case of DNA exceeding 1000 base pairs, an agarose gel of about 0.5 to 2.5% can be used.
The gel generally contains an intercalator that emits fluorescence like ethidium bromide in advance. By doing so, the nucleic acid such as DNA subjected to electrophoresis is stained with ethidium bromide or the like simultaneously with the electrophoresis, and the nucleic acid stained with the fluorescence detector can be detected immediately.

It is desirable to fill the gel into the electrophoresis unit 21 and the nucleic acid supply path 25 immediately before using the nucleic acid amplification substrate 10.
The gel is filled by press-fitting from the migration unit end side opening 36.

Next, the function of the nucleic acid amplification substrate 10 of this embodiment will be described.
The nucleic acid amplification substrate 10 of this embodiment is mounted on an apparatus as shown in FIG. 7 and performs a predetermined inspection.
Here, FIG. 7 is a perspective view of a temperature control apparatus and an electrophoresis apparatus that perform nucleic acid amplification and separation by mounting the nucleic acid amplification substrate of FIG. FIG. 8 is an explanatory diagram showing the behavior of the reaction liquid when the reaction liquid is sent from the reaction liquid inlet to the reaction liquid reservoir. FIG. 9 is an explanatory diagram showing the behavior of the reaction liquid when returning the reaction liquid from the reaction liquid reservoir to the reaction liquid inlet side. FIG. 10 is an explanatory diagram showing the behavior of the reaction solution when separating nucleic acids.
The apparatus shown in FIG. 7 is a combination of a temperature control apparatus 80 for performing a nucleic acid amplification reaction and an electrophoresis / detection apparatus 81.

The temperature control device 80 includes three sets of heat blocks 55, 56, 57, a substrate feed mechanism 58, and a reaction liquid injection / suction mechanism 60.
The three sets of heat blocks 55, 56, and 57 have upper pieces 55a, 56a, and 57a and lower pieces 55b, 56b, and 57b, respectively. Can be pinched. The positions where the heat blocks 55, 56, and 57 are sandwiched are only the region of the reaction liquid reservoir 20 of the nucleic acid amplification substrate 10, and the electrophoresis unit 21 is not sandwiched.

The three sets of heat blocks 55, 56, and 57 are temperature-adjusted to a predetermined temperature in advance. Taking the case of performing PCR as a nucleic acid amplification reaction as an example, the heat block 55 at the right end is near the saturation temperature (95 ° C.), the heat block 57 at the left end is near the annealing temperature (55 ° C.), The temperature of the heat block 56 is adjusted in the vicinity of each reaction temperature (72 ° C.) for double-strand extension.
Of the three sets of heat blocks 55, 56, and 57, when performing PCR, the heat block 55 is adjusted to the temperature near the saturation temperature, and the heat block 56 is adjusted to the temperature near each reaction temperature of double-strand extension. Is a built-in electric heater. On the other hand, the heat block 57 whose temperature is adjusted near the annealing temperature has a cooling function by an air cooling fan in addition to the electric heater.

  A sheet having excellent thermal conductivity is bonded to the contact surface of each heat block with respect to the nucleic acid amplification substrate 10. For example, a sheet made of silicon is used as the sheet.

  The substrate feed mechanism 58 holds the nucleic acid amplification substrate 10 and moves it horizontally, and sequentially holds the nucleic acid amplification substrate 10 between the heat blocks. The substrate feeding mechanism 58 includes a mounting base 61 for the nucleic acid amplification substrate 10. The mounting table 61 is provided with a substrate holder 62. The substrate holder 62 presses the nucleic acid amplification substrate 10 with a leaf spring and has a function of preventing the nucleic acid amplification substrate 10 from vibrating.

  The reaction solution injection / suction mechanism 60 includes a small pressurizing device and a vacuum device.

  Although the inside of the electrophoresis / detection device 81 is not shown, it incorporates an electrophoresis device and a fluorescence detection device. The electrophoresis apparatus includes an amplified nucleic acid migration electrode and an amplified nucleic acid electrophoresis electrode.

The nucleic acid amplification substrate 10 described above is mounted on the substrate feed mechanism 58 and is first transferred to the position of the reaction liquid injection / suction mechanism 60.
In the reaction liquid injection / suction mechanism 60, the reaction liquid is injected into the reaction liquid introduction opening 30 as shown in FIG.
This reaction solution contains all components necessary for the nucleic acid amplification reaction. When the nucleic acid amplification reaction is PCR, a mixture of template DNA, primer, deoxynucleotide triphosphates, heat-resistant DNA polymerase, etc. is used as a reaction solution. In this case, the primer may be preliminarily fluorescently labeled. When the primer is fluorescently labeled in advance, the amplified DNA is already fluorescently labeled, so there is no need to add an intercalator to the gel of the electrophoresis unit 21.

In the present embodiment, the reaction solution injected into the reaction solution introduction opening 30 is drawn into the reaction solution reciprocating flow path 22 by a capillary phenomenon and filled into the reaction solution reservoir 20. At this time, since the reaction solution reciprocating flow path 22 and the reaction solution reservoir 20 are hydrophilic, the sample is easily adapted to the surface, and the sample can be easily introduced into the reaction solution introduction opening 30. Furthermore, heat transfer from the substrate to the sample is good in the reaction liquid reservoir 20.
A vacuum source is connected to the suction opening 31 as necessary. As a result, the reaction liquid injected into the reaction liquid introduction opening 30 is sucked by the negative pressure of the vacuum source, flows through the reaction liquid reciprocating flow path 22 and enters the reaction liquid reservoir 20 as shown in FIG.

Then, a nucleic acid amplification reaction such as PCR is performed in the nucleic acid amplification substrate 10.
Specifically, the substrate feeding mechanism 58 is operated, the nucleic acid amplification substrate 10 is transferred to the rightmost heat block 55, and the reaction liquid reservoir of the nucleic acid amplification substrate 10 with the upper and lower pieces 55a and 55b of the upper and lower heat blocks. 20 is sandwiched.
Hereinafter, a case where PCR is performed will be described as an example. First, the temperature of the heat block 55 is adjusted to be close to the saturation temperature. The nucleic acid amplification substrate 10 is thin, and particularly the reaction liquid reservoir 20 sandwiched between the heat blocks 55 is thin. For this reason, the reaction liquid in the reaction liquid reservoir 20 reaches the dynalysis temperature within a few seconds, and the dynalysis reaction proceeds rapidly.

  In the nucleic acid amplification substrate 10 of the present embodiment, the reaction liquid reservoir 20 is formed in the gap 6 in the nucleic acid amplification substrate 10, and the upper surface side and the lower surface side are completely sealed. Therefore, even if the nucleic acid amplification substrate 10 is sandwiched between the heat blocks 55, the reaction solution does not leak from the reaction solution reservoir 20, and the heat block 55 is not contaminated.

  For several seconds, the reaction solution reservoir 20 is kept sandwiched by the heat block 55 and maintained at the dynaration temperature, and then the nucleic acid amplification substrate 10 is moved to carry the nucleic acid amplification substrate 10 to the position of the heat block 57 provided at the left end. At this time, the nucleic acid amplification substrate 10 is subjected to considerable vibration and impact, but the reaction solution does not leak from the reaction solution reservoir 20.

Then, the nucleic acid amplification substrate 10 is sandwiched between the heat blocks 57 provided at the left end. Also in this case, only the reaction liquid reservoir 20 is sandwiched between the heat blocks 57, and the reaction liquid in the reaction liquid reservoir 20 immediately reaches the annealing temperature.
Also in this case, the reaction solution does not leak from the reaction solution reservoir 20 and the heat block 57 is not contaminated.

  When the annealing is completed, the nucleic acid amplification substrate 10 is moved again, and the nucleic acid amplification substrate 10 is sandwiched by the central heat block 56, and a double-strand elongation reaction is performed. Also in this case, the reaction solution does not leak from the reaction solution reservoir 20 and the heat block 56 is not contaminated.

When the double-strand extension reaction is completed, the nucleic acid amplification substrate 10 is moved by the substrate feeding mechanism 58, the nucleic acid amplification substrate 10 is sandwiched by the first heat block 55, and the dialysis reaction is performed again.
Thereafter, the same series of steps as described above are repeated about 30 times, and the target DNA is amplified by the PCR method.

  Thus, in this embodiment, the reaction liquid in the reaction liquid reservoir 20 is repeatedly heated and cooled, and the internal reaction liquid is repeatedly expanded and contracted. Moreover, in this embodiment, since the reaction liquid storage part 20 is pinched | interposed by the heat blocks 55,56,57, the wall of the front and back side of the reaction liquid storage part 20 is compressed repeatedly.

However, in the nucleic acid amplification substrate 10 of this embodiment, the reaction solution does not flow out from the reaction solution reservoir 20.
That is, in the nucleic acid amplification substrate 10 of the present embodiment, the partition wall 7 (FIG. 5) is provided inside the reaction liquid reservoir 20 to form a zigzag flow path, and the overall flow path resistance is high. The movement of the reaction solution is suppressed. Furthermore, in the present embodiment, the reaction liquid suction path 23 is provided in the vicinity of the reaction liquid reservoir 20, but the reaction liquid suction path 23 has a flow path based on a curved path, and the flow path is also “moving”. It functions as a “suppression means”.
Therefore, in the nucleic acid amplification substrate 10 of this embodiment, there is little movement of the reaction solution inside and outside the reaction solution reservoir 20, and most of the reaction solution remains in the reaction solution reservoir 20.

Furthermore, in this embodiment, as described above, the flow path is formed inside the reaction liquid reservoir 20, and the tip of the partition wall 7 of the flow path is in contact with the surface of the first layer 11 as shown in FIG. . Therefore, when the reaction liquid reservoir 20 receives a compressive force, the compressive force is borne by the partition wall 7 of the flow path, and the surface side deflection of the reaction liquid reservoir 20 is small.
That is, in this embodiment, the partition wall 7 of the flow path functions as a compression resistant part, and the surface side of the reaction liquid reservoir 20 is not bent.
Therefore, in this embodiment, the pressure applied to the reaction liquid reservoir 20 is small, and the force itself for discharging the reaction liquid from the reaction liquid reservoir 20 is small.
Therefore, in the nucleic acid amplification substrate 10 of the present embodiment, most of the reaction solution reacts even though the reaction solution in the reaction solution reservoir 20 repeats heating and cooling and the reaction solution reservoir 20 is repeatedly compressed. It remains in the liquid reservoir 20. Conversely, the nucleic acid amplification substrate 10 of the present embodiment can perform a nucleic acid amplification reaction by a PCR method or the like with a small number of samples.

When the nucleic acid amplification is completed by the nucleic acid amplification reaction, the nucleic acid amplification substrate 10 is transferred again to the position of the reaction liquid injection / suction mechanism 60 by the substrate feeding mechanism 58.
Then, as shown in FIG. 9A, the pressure source P is connected to the suction opening 31 and the side of the suction opening 31 is set to a high pressure atmosphere to push out the reaction liquid in the reaction liquid reservoir 20. As shown in FIG. 9A, the reaction liquid returns to the reaction liquid reciprocating flow path 22 and returns to the reaction liquid introduction opening 30 (FIG. 9B).

In this state, the substrate transfer mechanism 59 transfers the nucleic acid amplification substrate 10 to the electrophoresis / detection device 81.
In the electrophoresis / detection device 81, electrodes are first connected to the reaction solution introduction opening 30 and the nucleic acid supply path end opening 33 as shown in FIG. A direct current is applied with the reaction solution introduction opening 30 side being negative.

As a result, the nucleic acid in the reaction solution introduction opening 30 moves to the nucleic acid supply path end opening 33 side.
Then, as shown in FIG. 10B, electrodes are connected to the migration unit start side opening 35 and the migration unit end side opening 36, the migration unit termination side opening 36 is positive, and the migration unit start side opening 35 is negative. Is energized.
As a result, the nucleic acid that has reached the intersection 50 is attracted toward the migration unit termination side opening 36, and the nucleic acid is separated in the migration path 52 according to the molecular weight. Then, fluorescence analysis is performed by a fluorescence detection device (not shown).

  In the nucleic acid amplification substrate 10 described above, an example in which a zigzag channel is formed inside the reaction liquid reservoir 20 is taken as an example. When a structure in which a flow path is formed inside is employed as in the present embodiment, the flow resistance of the reaction liquid reservoir 20 is increased as described above, and the deflection on the surface side of the reaction liquid reservoir 20 is small. The reaction liquid in the reaction liquid reservoir 20 is difficult to disappear and is a recommended structure.

In this embodiment, the zigzag channel has a constant channel width. However, like the reaction solution reservoir 70 shown in FIG. 11, the channel width is partially narrowed to prevent the reaction solution from moving. Also good.
FIG. 11 is a front view of the reaction liquid reservoir of the nucleic acid amplification substrate according to another embodiment of the present invention.

Further, like the reaction liquid reservoir 71 shown in FIG. 12, a configuration in which a columnar portion 72 is provided inside, and a compressive force is borne by the columnar portion 72 to suppress the deflection on the surface side of the reaction liquid reservoir 20 is also conceivable. . FIG. 12 is a front view of a nucleic acid amplification substrate according to still another embodiment of the present invention.
Further, the present invention does not deny a reaction liquid reservoir having a single chamber as shown in FIGS. 13 and 14 and having a hollow inside.

  In the nucleic acid amplification substrate 10 of the present embodiment, four systems of nucleic acid amplification / separation channels 15, 16, 17, and 18 are provided. However, the number of nucleic acid amplification / separation channels is arbitrary, and one system has five or more systems. But you can.

Next, examples performed for confirming the effects of the present invention will be described.
The inventors made a prototype of the nucleic acid amplification substrate 10 shown in FIG. 1 and conducted an experiment by PCR. The template DNA used in the experiment is a 200 base pair DNA fragment obtained from the porcine genome and is a PCR product amplified by a conventional PCR apparatus. In the experiment, this PCR product was diluted and used.
As the primers, the same forward and reverse primers as those used for obtaining the template DNA described above were used.
As other reagents such as an enzyme, a substrate, and a buffer, a kit containing “TaKaRa Taq” manufactured by Takara Bio Inc. was used.

The reaction solution used for PCR was about 8 to 10 ml, supplied to the reaction solution introduction opening 30, sucked from the suction opening 31, and led to the reaction solution reservoir 20.
In addition, the nucleic acid amplification substrate 10 made experimentally had a volume of the reaction liquid reservoir 20 of about 5 ml, and the remaining amount remained in the suction opening 31.
Then, PCR and electrophoresis were performed using the apparatus shown in FIG.
The heat block 55 of the temperature control device 80 was temperature-controlled at 98.5 ° C. in order to set the reaction liquid reservoir 20 to the saturation temperature.
The heat block 57 was adjusted to 53.0 ° C. in order to set the reaction liquid reservoir 20 to the annealing temperature.
Furthermore, the temperature of the heat block 56 was adjusted to 74.5 ° C. in order to set the reaction liquid reservoir 20 to a reaction temperature for double-strand elongation.

Then, the nucleic acid amplification substrate 10 is sandwiched by the heat block 55 for 5 seconds, followed by the annealing reaction, followed by annealing for 5 seconds by the heat block 57, followed by annealing for 5 seconds by the heat block 56. An elongation reaction was performed. This was repeated 30 times.
The time required for the above PCR method was about 10 minutes.
Then, when the nucleic acid amplification substrate 10 was transferred to the electrophoresis / detection device 81 and a DNA electrophoresis experiment was performed, a peak that apparently resulted from amplification of the template DNA appeared.
Therefore, it was demonstrated that DNA can be amplified by performing PCR using the nucleic acid amplification substrate 10 of this example.

It is a front view of the nucleic acid amplification substrate of an embodiment of the present invention. It is a perspective view of the nucleic acid amplification substrate of FIG. FIG. 2 is a front view of two systems of nucleic acid amplification / separation flow paths of the nucleic acid amplification substrate of FIG. 1. FIG. 2 is an exploded perspective view of a first layer and a second layer around a reaction liquid reservoir of the nucleic acid amplification substrate of FIG. 1. FIG. 2 is an enlarged cross-sectional view of the periphery of a reaction liquid reservoir of the nucleic acid amplification substrate of FIG. FIG. 2 is an enlarged perspective view of the vicinity of a reaction solution inlet of the nucleic acid amplification substrate of FIG. FIG. 2 is a perspective view of a temperature control apparatus and an electrophoresis apparatus that perform nucleic acid amplification and separation by mounting the nucleic acid amplification substrate of FIG. 1. It is explanatory drawing which shows the behavior of the reaction liquid at the time of sending a reaction liquid from the reaction liquid inlet to the reaction liquid reservoir. It is explanatory drawing which shows the behavior of the reaction liquid at the time of returning a reaction liquid from the reaction liquid reservoir to the reaction liquid inlet side. It is explanatory drawing which shows the behavior of the reaction liquid at the time of isolate | separating a nucleic acid. It is a front view of the reaction liquid storage part of the nucleic acid amplification substrate in other embodiment of this invention. It is a front view of the nucleic acid amplification substrate in further another embodiment of the present invention. It is a front view of the nucleic acid amplification substrate in further another embodiment of the present invention. It is an AA expanded sectional perspective view of FIG. It is explanatory drawing which shows the behavior at the time of pinching the front and back of the nucleic acid amplification substrate shown in FIG. 13 with a heat block.

Explanation of symbols

1, 20, 70, 71 Reaction liquid reservoir 2,10 Nucleic acid amplification substrate 5 Groove 6 Void 7 Bulkhead (compression resistant part)
11 First layer
12 Second layer (layer)
21 Electrophoresis part 22 Reaction solution reciprocating flow path (Reaction liquid flow path)
23 Reaction liquid suction path (movement suppression means)
30 Reaction liquid introduction opening (Reaction liquid introduction part)
41 Straight path
43 Curve 72 Columnar part (compression resistant part)

Claims (10)

  A reaction liquid reservoir is formed on a part of the substrate, the reaction liquid reservoir is filled with a reaction liquid containing components necessary for the nucleic acid amplification reaction, and the temperature of the reaction liquid reservoir is adjusted to adjust the temperature in the reaction liquid reservoir. In a nucleic acid amplification substrate that performs a nucleic acid amplification reaction and amplifies nucleic acid, the reaction liquid reservoir is constituted by a gap provided in the substrate, and the nucleic acid amplified in the reaction liquid reservoir is separated by electrophoresis. A nucleic acid amplification substrate comprising an electrophoresis portion that performs the above-described electrophoresis.   A reaction liquid introduction part for introducing a reaction liquid and a reaction liquid flow path formed by a gap provided inside the substrate and connecting the reaction liquid introduction part and the reaction liquid reservoir, and the reaction liquid is introduced into the reaction liquid introduction part The nucleic acid amplification substrate according to claim 1, further filled in a reaction liquid reservoir through a reaction liquid channel.   The substrate is formed by laminating two or more layers, and a groove is provided on one of the two laminated layers, and the gap is formed inside the substrate by the groove. The nucleic acid amplification substrate according to claim 1 or 2.   The nucleic acid amplification substrate according to any one of claims 1 to 3, wherein a movement suppressing means for suppressing movement of the reaction liquid is provided in the reaction liquid reservoir or in the vicinity of the inlet / outlet of the reaction liquid reservoir.   The nucleic acid amplification substrate according to any one of claims 1 to 4, wherein a flow path including a plurality of curved paths is formed in the reaction liquid reservoir or in the vicinity of the inlet / outlet of the reaction liquid reservoir.   The nucleic acid amplification substrate according to any one of claims 1 to 4, wherein a serial flow path including a plurality of curved paths is formed in the reaction liquid reservoir.   7. A compression-resistant part is provided in the reaction liquid reservoir, and when the reaction liquid reservoir is compressed in the front and back direction, the compression force is borne by the compression-resistant part. The nucleic acid amplification substrate as described.   The nucleic acid according to any one of claims 1 to 7, wherein the reaction liquid storage part and the electrophoresis part are separated, and the distance between them is at least three times the thickness of the thickest part of the substrate. Amplification board.   The nucleic acid amplification substrate according to any one of claims 1 to 8, wherein the thickness of the reaction liquid reservoir is smaller than the thickness of the electrophoresis part.   The nucleic acid amplification substrate according to any one of claims 1 to 9, wherein the region from the reaction solution introduction part to the reaction solution reservoir part is hydrophilic.

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