JP6979333B2 - DNA sequencer - Google Patents

Description

The present invention relates to a DNA sequencer.

A DNA sequencer is known as a device for analyzing a base sequence of DNA. The DNA sequencer is a device that denatures a DNA fragment into a single strand, sequentially extends a nucleic acid labeled with a fluorescence label using this as a type, and sequentially analyzes the base sequence of DNA by fluorescence observation with a microscope. To perform analysis, prepare a plate made of a partially or wholly transparent material, for example, a flow cell having a flow path in a glass plate, and in the flow cell, a DNA fragment that has been denatured into a single strand. Generate multiple colonies of clones. A reagent containing a fluorescent dye that makes it possible to distinguish four types of nucleotides (A, T, C, G) constituting DNA and a nucleic acid to which a molecule that inhibits the elongation of the next base is added to this flow path. , The fluorescent dye and the reagent that removes the molecule that inhibits elongation are alternately flowed. Multiple colonies with a size of several μm of single-stranded DNA fragments are provided in the flow cell flow path, and the process of restoring the single-stranded DNA fragments to double strands is observed with a fluorescence microscope. By going through this, the DNA base sequence can be read sequentially.

Observation with a fluorescence microscope in a DNA sequencer is performed while appropriately selecting a plurality of types of reagents with a switching valve and alternately sending them to a pipe.
In such a DNA sequencer, when the vibration of the switching valve is transmitted to the stage on which the flow cell is placed, the flow cell also vibrates, which causes a problem that the fluorescence microscope image of DNA may be blurred. Further, since the reagent is expensive, there is also a demand to reduce the amount of the reagent and reduce the running cost of the DNA sequencer.

Japanese Unexamined Patent Publication No. 2012-152149

The present invention provides a DNA sequencing method that prevents vibration of a flow cell, captures a clear DNA fluorescence microscope image, reduces the amount of reagents, and reduces running costs.

In order to solve the above problems, the DNA sequencer according to the first aspect of the present invention is configured to be able to mount a flow cell provided with a flow path, and has a stage that can move along the direction of the flow path and the above. A liquid feed system that supplies reagents to the flow path, a switching valve configured to selectively deliver a plurality of reagents to the liquid feed system, and a switching valve mounted and moved along the direction of the flow path. It includes a valve unit that can be configured and a movement control mechanism that moves the valve unit in accordance with the movement of the stage.

Further, in the DNA sequencer according to the second aspect of the present invention, a flow cell provided with a flow path is configured to be mountable, a stage movable along the direction of the flow path, and a reagent are supplied to the flow path. A liquid feeding system, a switching valve configured to selectively deliver a plurality of reagents to the liquid feeding system, and a valve on which the switching valve is placed and configured to be movable along the direction of the flow path. It includes a unit and a vibration isolation mechanism provided between the stage and the valve unit.

According to the DNA sequencer according to the first aspect of the present invention, the switching valve is mounted on the valve unit and separated from the stage on which the flow cell is mounted, so that the vibration of the switching valve is prevented from being transmitted to the stage. This makes it possible to prevent vibration of the flow cell and capture a clear DNA fluorescence microscope image. In addition, since the valve unit on which the switching valve is mounted is moved by the movement control mechanism following the movement of the stage on which the flow cell is mounted, the two can always be kept close to each other, and the piping between the two can be shortened. can do. Therefore, it is possible to save expensive reagents and reduce running costs.

Further, according to the DNA sequencer according to the second aspect of the present invention, the switching valve is mounted on the valve unit and separated from the stage on which the flow cell is mounted, so that the vibration of the switching valve is transmitted to the stage. Moreover, a vibration isolation mechanism is provided between the stage and the valve unit. This makes it possible to prevent vibration of the flow cell and capture a clear DNA fluorescence microscope image. Since the vibration is suppressed, the stage and the valve unit can be arranged close to each other by that amount, and as a result, the piping between the two can be shortened. Therefore, it is possible to save expensive reagents and reduce running costs.

A schematic configuration example of the DNA sequencer according to the first embodiment is shown. It is a schematic diagram of the flow path of the reagent including the switching valve 23. An example of the structure of the flow cell FC will be described. A modification of the first embodiment is shown. A schematic configuration example of the DNA sequencer according to the second embodiment is shown. A schematic configuration example of the DNA sequencer according to the third embodiment is shown. A schematic configuration example of the DNA sequencer according to the fourth embodiment is shown. A modification of the fourth embodiment is shown. A schematic configuration example of the DNA sequencer according to the fifth embodiment is shown.

Hereinafter, the present embodiment will be described with reference to the accompanying drawings. In the attached drawings, functionally the same elements may be displayed with the same number. The accompanying drawings show embodiments and implementation examples in accordance with the principles of the present disclosure, but these are for the purpose of understanding the present disclosure and are never used for the limited interpretation of the present disclosure. is not it. The description of the present specification is merely a typical example, and does not limit the scope of claims or application examples of the present disclosure in any sense.

In this embodiment, the description is given in sufficient detail for those skilled in the art to implement the present disclosure, but other implementations and embodiments are also possible and do not deviate from the scope and spirit of the technical idea of the present disclosure. It is necessary to understand that it is possible to change the structure and structure and replace various elements. Therefore, the following description should not be construed as limited to this.

[First Embodiment]
First, the DNA sequencer according to the first embodiment will be described with reference to FIG.
As shown in FIG. 1, the DNA sequencer 1 (DNA sequencer device) includes a stage base 100 and a valve unit base 200.
The stage base 100 includes a flat plate 101 and a support column 102 connected to the four lower corners of the flat plate 101 and supporting the flat plate 101 from below. A vibration isolation mechanism 103 for suppressing vibration from others is provided between the support column 102 and the flat plate 101. As the anti-vibration mechanism 103, for example, a spring, rubber, a damper, or the like can be used. Regardless of the method, the method does not matter as long as the vibration during the operation of the switching bulb outside the device and inside the device is attenuated and the image pickup is not affected. Further, the position of the vibration isolator 103 is not limited to the space between the support column 102 and the flat plate 101, and may be arranged at a position where vibration propagation can be prevented.

A stage 13 is placed on the upper surface side of the flat plate 101 via a moving rail 14. The moving rail 14 is configured to be able to move the stage 13 in one direction along the plane of the stage 13, for example, along the X direction in FIG. 1 (direction along the flow path of the flow cell FC). The stage 13 is driven by the servomotor M1.

An imaging system 15 is provided above the stage 13 (Z direction). The image pickup system 15 is supported by, for example, an image pickup system mount 16 extending from the stage base 100. The imaging system 15 includes a camera (not shown), which captures the flow cell FC mounted on the stage 13. The image pickup system 15 is controlled based on a control signal from the control unit 30, and is controlled to emit illumination light, take an image, and focus. The focal position data of the imaging system 15 is stored in the memory 40 (storage means) during the past focusing operation, and is read out from the memory 40 and used at the start of the next analysis. When the focal position is read out, it moves to the focal position of the imaging system 15 and the flow cell FC is imaged.

The stage 13 may be provided with a heating device (not shown) for heat-treating the flow cell FC in order to promote the reaction between the DNA colony and the reagent. Further, the stage 13 may be configured so that a plurality of flow cells FC can be arranged at the same time.

The valve unit base 200 is arranged below the stage base 100, preferably directly below. The valve unit base 200 is configured as a mounting portion independent of the stage base 100, and the valve unit 21 is mounted on the mounting portion. The valve unit base 200 includes a flat plate 201 as a support surface and a support plate 202 that supports both sides of the flat plate 201 on the lower surface side. A valve unit 21 is mounted on the upper surface side of the flat plate 201 of the valve unit base 200 via a moving rail 22, and the valve unit 21 is movable on the valve unit base 200 in the X direction of FIG. ing.

A switching valve 23 is mounted on the upper surface of the valve unit 21. The switching valve 23 causes vibration by the switching operation described later, but the switching valve 23 is mounted on the valve unit base 200, which is different from the stage base 100 on which the flow cell FC is mounted, via the valve unit 21. Moreover, a vibration isolator 103 is provided between the stage 13 and the valve unit 21. Therefore, it is prevented that vibration is transmitted between the stage 13 and the valve unit 21, and there is no problem even if the stage 13 and the valve unit 21 are arranged close to each other.

FIG. 2 is a schematic view of the flow path of the reagent including the switching valve 23. The switching valve 23 is connected to a plurality of reagent tanks 25 via a pipe 29. Further, the switching valve 23 is connected to the flow cell FC arranged on the stage 13 via a pipe 24 and a manifold MF that provides a path for the reagent. The manifold MF is not shown in FIG. The pipe 24 may be made of a flexible material, for example a resinous tube. The pipe 24 and the manifold MF form a liquid feeding system that supplies reagents to the flow path of the flow cell FC.
The switching valve 23 has a function of selectively delivering which reagent from the plurality of reagent tanks 25 to the liquid feeding system and flowing it to the flow path 303 of the flow cell FC. It should be noted that some reagent tanks 25 can be connected to the manifold MF via a single valve 23'without passing through the switching valve 23. One end of the flow path of the flow cell FC is connected to the manifold MF, while the other end of the flow path is connected to the waste liquid tank 27 via the pipe 26. Valves B1 and B2 and a pump 28 are connected to the pipe 26. The valves B1 and B2 are opened when the reagent remaining in the flow cell FC or the like is discharged, and the pump 28 is driven and sucks when the valves B1 and B2 are in the open state.

FIG. 3 describes an example of the structure of the flow cell FC. The flow cell FC includes, for example, an upper plate 301 and a lower plate 302. The upper plate 301, which is the observation surface of the flow cell FC, is made of a transparent material and is configured to cover the lower plate 302. Since the upper plate 301 is a transparent material, the flow path formed in the lower plate 302 can be grasped via the upper plate 301. The upper plate 301 may be made of, for example, highly transparent glass, quartz, or plastic.

The lower plate 302 includes a flow path 303, an injection port 304, and an discharge port 305. The injection port 304 is an opening connected to the pipe 24 via the manifold MF, and the reagent can be injected into the flow path 303 through the injection port 304. Further, the discharge port 305 is an opening connected to the waste liquid tank 27 via the pipe 26, and the reagent is discharged from the flow path 303 through the discharge port 305. Since the flow cell FC may improve the reaction efficiency between the DNA colony and the reagent by heating on the stage 13, the upper plate 301 and the lower plate 302 are required to have heat resistance of at least 100 ° C. The flow path 303 of the flow cell FC shows an example of digging into the lower plate 302 by etching. However, by providing an adhesive layer on the surface of the lower plate 302, for example, and applying the flow path pattern processing to the adhesive layer. , It is also possible to form the flow path 303.

As described above, in the present embodiment, the stage 13 on which the flow cell FC is mounted and the valve unit 21 on which the switching valve 23 is mounted are mounted on different mounting portions (100, 200), and are placed between the two. By providing the vibration isolation mechanism 103, the vibration of the switching valve 23 is prevented from being transmitted to the flow cell FC. Therefore, since the stage 13 and the valve unit 21 can be arranged close to each other, the length of the pipe 24 connecting the switching valve 23 and the manifold MF can be shortened. When the length of the pipe 24 is shortened, the amount of the reagent used can be reduced, and the running cost of the DNA sequencer can be reduced.

In order to make it possible to further reduce the length of the pipe 24, in the present embodiment, as shown in FIG. 1, a movement control mechanism 41 for moving the valve unit 21 following the movement of the stage 13 is provided. ing. As an example, the movement control mechanism 41 includes a first regulating member 41a provided at both ends of the stage 13 and a second regulating member 41b attached to both ends of the valve unit 21. The first regulating member 41a and the second regulating member 41b are separated from each other (non-contact state) when the stage 13 is in the reference position (eg, near the center of the moving rail 14). However, when the stage 13 is moved beyond a certain range, for example, outside the measurement range (or out of the field of view) of the flow cell FC by the imaging system 15, either the left or right first regulating member 41a is second regulated. It is in contact with the member 41b. Then, following the movement of the stage 13 outside the measurement range, the valve unit 21 also moves in the same direction. In this way, in the movement control mechanism 41, the valve unit 21 moves due to the action applied to the first regulating member 41a to the second regulating member 41b, so that the piping 24 can be shortened. Here, the case where the stage 13 is moved out of the measurement range of the flow cell FC is, for example, a case where the flow cell FC is replaced, a case where the origin setting operation of the stage 13, a cleaning / maintenance operation, or the like is performed.

As described above, when the movement of the stage 13 is within a certain range, the stage 13 can move independently of the valve unit 21, while when the movement of the stage 13 exceeds a certain range, the movement control mechanism 41 causes the stage 13 to move independently. The valve unit 21 moves following the movement of the stage 13. In other words, the movement control mechanism 41 of the first embodiment is given a certain "play", and the valve unit 21 does not move when the stage 13 is moved within the range of the "play". ..

The setting of the amount of "play" can be appropriately selected according to the design purpose of the device.
If the design is such that the first regulating member 41a and the second regulating member 41b do not come into contact with each other during the measurement, increase the "play", for example, between the left and right first regulating members 41a and the second regulating member 41b. The total of the distances D1 and D2 (see FIG. 1) is set to be larger than the moving distance Ds of the stage being measured. Such a design is preferable in order to avoid vibration during measurement as much as possible.

On the other hand, from the viewpoint of making the pipe 24 as short as possible, it is preferable to make the "play" small, and for example, the total of D1 and D2 may be made smaller than the moving distance Ds. Which design concept to adopt can be arbitrarily selected.

As shown in FIG. 4, it is also possible to provide the first regulating member 41a and / or the second regulating member 41b with a cushioning material 42 that absorbs the impact when they come into contact with each other.

Next, the operation of the DNA sequencer of the first embodiment will be described.
First, the flow cell FC is fixed to the stage 13. When the DNA analysis is started, a plurality of reagents are sequentially flowed from the reagent tank 25 via the switching valve 23 or the like toward the flow path 303 in which the DNA colonies are arranged in a predetermined order. In the present embodiment, the reagent is sucked up from the reagent tank 25 by the pump 28, passes through the pipe 29, the switching valve 23, and the pipe 24, and is sent to the flow cell FC. The reagent discharged from the flow cell FC is accumulated in the waste liquid tank 27. By the switching operation by the switching valve 23, the types of reagents flowing through the flow cell FC are sequentially switched.

As described above, the flow cell FC may be heated by a heating device (not shown) provided on the stage 13. During the heat treatment of any of the flow cell FCs, another flow cell FC may be imaged. This makes it possible to improve the efficiency of measurement.

Even if vibration is generated by the switching operation of the switching valve 23, the propagation of the vibration to the flow cell FC is suppressed by the vibration isolation mechanism 103. Further, the valve unit 21 follows the movement of the stage 13 by the movement control mechanism 41.

[effect]
The effect of the first embodiment will be described.
Next-generation DNA sequencers are very expensive in reagents used for analysis. On the other hand, for analysis, it is necessary to replace the reagent in the flow path of the flow cell many times, and it is a problem to reduce the consumption of the reagent.

It is the pipe 24 between the switching valve 23 and the flow cell FC that determines the consumption of expensive reagents. This pipe 24 is a pipe in which a plurality of pipes 29 merge from the switching valve 23, and after using one reagent, when another reagent is used, it is necessary to completely replace the pipe 24. Waste reagents. That is, the internal volume of the pipe 24, the internal volume of the flow path of the manifold MF, and the internal volume of the flow path 303 of the flow cell FC are the amounts of the reagents to be replaced at one time. The anti-vibration mechanism 103 and the movement control mechanism 41 can shorten the length of the pipe 24 as much as possible.

In this first embodiment, the switching valve 23 vibrates due to the operation of the switching valve 23 during imaging. However, the stage 13 and the valve unit 21 are separately arranged in the stage base 100 and the valve unit base 200, respectively, and the anti-vibration mechanism 103 is further provided in the stage base 100 that supports the stage 13. Therefore, the vibration of the switching valve 23 is suppressed from propagating to the flow cell FC, and does not affect the image pickup. Therefore, the stage base 100 and the valve unit base 200 can be arranged close to each other, preferably downward, and the pipe 24 can be shortened by that amount. The valve unit base 200 may be arranged directly below the stage base 100. Even if it is arranged directly below, the vibration of the switching valve 23 is suppressed from propagating to the flow cell FC due to the action of the vibration isolation mechanism 103.

The anti-vibration mechanism 103 is effective by itself for shortening the piping 24, but in the first embodiment, a movement control mechanism 41 may be provided in addition to the anti-vibration mechanism 103. .. Since the valve unit 21 moves following the movement of the stage 13 by the movement control mechanism 41, the length of the pipe 24 can be further shortened.

[Second Embodiment]
Next, the DNA sequencer according to the second embodiment will be described with reference to FIG.
The same components as those in the first embodiment are designated by the same reference numerals as those in FIG. 5 in FIG. 5, and detailed description thereof will be omitted below. This second embodiment is different from the first embodiment in that it includes a centering mechanism 51 for returning (centering) the valve unit 21 to, for example, a reference position. Since other points are the same as those of the first embodiment, the centering mechanism 51 will be described below.

As shown in FIG. 5, the centering mechanism 51 has a spring receiver 52 provided on both sides of the valve unit 21 and a spring 53 connected between the spring receiver 52 and the valve unit 21. Due to the elastic force of the spring 53, the valve unit 21 is configured to be restored to its reference position when it is not affected by the movement control mechanism 41.

According to this second embodiment, the same effect as that of the first embodiment can be obtained. In addition, the valve unit 21 is returned to the reference position when the movement control mechanism 41 does not act. Therefore, the length of the pipe 24 can be further reduced as compared with the first embodiment.

[Third Embodiment]
Next, the DNA sequencer according to the third embodiment will be described with reference to FIG.
The same components as those in the first embodiment are designated by the same reference numerals as those in FIG. 6 in FIG. 6, and detailed description thereof will be omitted below. In this third embodiment, the shape of the movement control mechanism 41 is different from that of the first embodiment. In this third embodiment, the movement control mechanism 41 is composed of the band member 41d. The band member 41d is made of a flexible member such as rubber, and usually has a predetermined slack, one end of which is the stage 13 and the other end of which is the valve unit 21. It is connected.

When the stage 13 is within a predetermined range, the valve unit 21 does not follow the stage 13 due to the presence of the slack of the band member 41d. However, when it is moved beyond a predetermined range, the slack of the band member 41d disappears, and the valve unit 21 is pulled by the band member 41d and moves following the stage 13. As described above, the same effect as that of the above-described embodiment can be obtained by the configuration of the third embodiment. In addition, in FIG. 3, the centering mechanism 51 may be omitted.

[Fourth Embodiment]
The DNA sequencer according to the fourth embodiment will be described with reference to FIG. 7. In this fourth embodiment, the movement control mechanism 41 is different from the above-described embodiment, and other than that, it is the same as the above-mentioned embodiment. As shown in FIG. 7, in the fourth embodiment, the permanent magnet 44 is arranged between the first regulating member 41a and the second regulating member 41b. The two permanent magnets 44 are arranged so that, for example, the same poles (for example, N poles) face each other. As a result, when the first regulating member 41a approaches the second regulating member 41b, the valve unit 21 is moved by the repulsive force of the permanent magnet 44. As a result, it is possible to prevent the first regulating member 41a and the second regulating member 41b from coming into contact with each other, and it is possible to suppress the propagation of vibration to the flow cell FC. It is also possible to use an electromagnet instead of the permanent magnet 44.

FIG. 8 shows a modified example of the fourth embodiment. In the example of FIG. 8, the permanent magnets 45 and 46 constituting the movement control mechanism 41 are arranged on the back surface of the stage 13 and the front surface of the valve unit 21, respectively, so that the opposite poles face each other. When the stage 13 moves, the valve unit 21 moves following the movement of the stage 13 due to the attractive force between the magnets 45 and 46. Therefore, the same effect as the configuration of FIG. 7 can be obtained.

[Fifth Embodiment]
The DNA sequencer according to the fifth embodiment will be described with reference to FIG. In this fifth embodiment, the movement control mechanism 41 is different from the above-described embodiment, and other than that, it is the same as the above-mentioned embodiment. In the above-described embodiment, while the stage 13 is moved by the driving force of the servomotor M1, the valve unit 21 is not provided with an independent drive source, and the movement is mechanical movement control mechanism 41 (first). 1 regulated member 41a, second regulated member 41b, etc.).

On the other hand, in the fifth embodiment, a servomotor M2 separate from the servomotor M1 is provided for driving the valve unit 21, and the servomotor M2 follows the movement of the stage 13 of the valve unit 21. The movement control is performed. That is, when the servomotor M1 moves the stage 13 by a predetermined distance, a control signal corresponding to the moving distance is also given to the motor M2 from the control unit 30, thereby performing follow-up control. Will be. The follow-up control at this time may be given a certain "play" as in the first embodiment, or may be set to follow-up control without "play".

1 ... Sequencer, 13 ... Stage, 14 ... Moving rail, 15 ... Imaging system, 16 ... Imaging system mount, 21 ... Valve unit, 22 ... Moving rail, 23 ... Switching valve, 23'... Valve, 24 ... Piping, 25 ... Reagent tank, 26 ... Piping, 27 ... Waste liquid tank, 28 ... Pump, 29 ... Piping, 30 ... Control unit, 34 ... Piping, 40 ... Imaging system, 40 ... Memory, 41 ... Movement control mechanism, 41a ... First regulatory member , 41b ... Second regulation member, 41d ... Band member, 42 ... Cushioning material, 44, 45, 46 ... Permanent magnet, 51 ... Centering mechanism, 100 ... Stage base, 101 ... Flat plate, 102 ... Support, 103 ... Anti-vibration mechanism , 200 ... Valve unit base, 201 ... Flat plate, 202 ... Support plate, 301 ... Upper plate, 302 ... Lower plate, 303 ... Flow path, 304 ... Injection port, 305 ... Discharge port.

Claims (10)

A stage that is configured to be able to mount a flow cell provided with a flow path and can move along the direction of the flow path,
An imaging system that captures a DNA fluorescence microscope image of the flow cell placed on the stage,
A liquid feeding system that supplies reagents to the flow path, and
A switching valve configured to selectively deliver multiple reagents to the liquid delivery system,
A valve unit on which the switching valve is placed and configured to be movable along the direction of the flow path, and a valve unit.
A DNA sequencer device provided with a movement control mechanism for moving the valve unit following the movement of the stage. In the DNA sequencer device of claim 1,
An anti-vibration mechanism is further provided between the stage and the valve unit.
A DNA sequencer device characterized by this. In the DNA sequencer device of claim 1,
Further provided with a storage means for storing the focal position of the imaging system,
A DNA sequencer apparatus characterized by moving to the focal position of the imaging system and imaging the flow cell at the start of analysis. In the DNA sequencer device of claim 1,
The movement control mechanism is
When the movement of the stage is within the first range, the stage can be moved independently of the valve unit, while when the movement of the stage exceeds the first range, the valve unit is moved. Move according to the movement of the stage,
A DNA sequencer device characterized by this. In the DNA sequencer device of claim 1,
The movement control mechanism is
The first regulatory member attached to the stage and
A second regulatory member attached to the valve unit is provided.
The DNA sequencer device according to claim 1, wherein when the stage moves, the valve unit moves due to an action given from the first regulating member to the second regulating member. In the DNA sequencer apparatus of claim 5,
The first regulating member and the second regulating member are brought into a non-contact state when the movement of the stage is within the first range, and when the movement of the stage exceeds the first range, the said. A DNA sequencer device characterized in that the first regulating member and the second regulating member are in contact with each other. In the DNA sequencer device of claim 1,
The movement control mechanism is a DNA sequencer device characterized in that the valve unit is configured to be movable according to the movement of the stage by a magnetic force. A stage that is configured to be able to mount a flow cell provided with a flow path and can move along the direction of the flow path,
An imaging system that captures a DNA fluorescence microscope image of the flow cell placed on the stage,
A liquid feeding system that supplies reagents to the flow path, and
A switching valve configured to selectively deliver multiple reagents to the liquid delivery system,
A valve unit on which the switching valve is placed and configured to be movable along the direction of the flow path, and a valve unit.
A DNA sequencer device provided with an anti-vibration mechanism provided between the stage and the valve unit. In the DNA sequencer apparatus of claim 8,
The valve unit is a DNA sequencer device characterized in that it is arranged below the stage. In the DNA sequencer apparatus of claim 9,
Further provided with a storage means for storing the focal position of the imaging system,
A DNA sequencer apparatus characterized by moving to the focal position of the imaging system and imaging the flow cell at the start of analysis.

Images