Patent Application
20100144541
1. Field of the Invention
The present invention relates to a DNA-array-equipped cartridge, an analyzer, and a method for using the DNA-array-equipped cartridge.
2. Description of the Related Art
Conventionally, a DNA array in which DNA probes are circularly arranged is known. For example, in a DNA array disclosed in Patent Document 1, a plurality of DNA probes are concentrically arranged on a disk-shaped substrate. When the DNA array is rotated once, a DNA array reader detects light incident from each of DNA probes arranged in a circle.
[Patent Document 1] Japanese Unexamined Patent Application Publication No. 2001-238674
However, in the technique disclosed in Patent Document 1, before the DNA array reader detects light incident from DNA probes, it is necessary to use a different apparatus to prepare target DNA, carry out a hybridization reaction between the target DNA and the DNA probes, etc. For example, the process from preparation of the target DNA to detection of light incident from the DNA probes subjected to the hybridization reaction involves transporting the DNA array from one apparatus to another.
The present invention has been made in view of the problems described above. A primary object of the present invention is to make it possible to relatively easily carry out the process from preparation of target DNA to detection of light incident from DNA probes at a light detector.
The present invention adopts the following means to achieve the object described above.
A DNA-array-equipped cartridge of the present invention includes a housing rotatable about a center axis;
a plurality of fluid containing spaces formed inside the housing and including a plurality of reagent containing spaces and a DNA array space, the reagent containing spaces holding fluids for preparation of target DNA, the DNA array space formed in a circumferential shape coaxial with the center axis and having a plurality of DNA probes spotted along the circumferential shape; and a plurality of openings communicating with the corresponding fluid containing spaces, formed on an upper side of the housing, and arranged side-by-side along a circumference coaxial with the center axis, wherein rotating the housing allows the plurality of openings to sequentially face a position setting a fluid port of a reaction tank independent of the housing, and allows the plurality of DNA probes to sequentially face a position setting a light detector independent of the housing.
In the DNA-array-equipped cartridge described above, when the housing is rotated to allow the openings of the reagent spaces to sequentially face the fluid port of the reaction tank, the rotation of the housing is temporarily stopped in a state where the opening of each of the reagent spaces faces the reaction tank, so that fluid is transported between the reaction tank and the reagent space. Thus, the target DNA can be prepare and eventually stored in the reaction tank. Next, when the housing is rotated to allow the opening of the DNA array space to face the fluid port of the reaction tank, the target DNA in the reaction tank can flow into the DNA array space and the target DNA can react with each of the DNA probes. Next, when the housing is rotated, light incident from each of the DNA probes subjected to the reaction can be detected by the light detector. Thus, it is possible to relatively easily carry out the process from preparation of the target DNA to detection of light incident form the DNA probes at the light detector.
In the DNA-array-equipped cartridge of the invention, the housing may be formed in a substantially disk-like shape. With this arrangement, the cartridge body is easily rotatable.
In the DNA-array-equipped cartridge of the present invention, the plurality of DNA probes may be spotted along a plurality of circumferential shapes coaxial with the center axis and having different diameters. With this arrangement, it is possible to spot a larger number of DNA probes.
The DNA-array-equipped cartridge of the present invention may further include a circular valve coaxial with the center axis of the housing, unrotatably secured, capable of supporting the reaction tank on an upper side of the circular valve, and having a through hole extending vertically therethrough from the fluid port of the reaction tank, wherein rotating the housing allows the plurality of openings to sequentially face the through hole of the circular valve. With this arrangement, with a relatively simple structure, any one of the fluid containing spaces can communicate with the reaction tank.
In the present invention, the DNA-array-equipped cartridge may further include a light guide configured to the position setting the guide light to the light detector, the light being incident from the DNA probe facing the position setting the light detector. With this arrangement, light incident from each of the DNA probes can be efficiently guided to the position setting the light detector.
In the DNA-array-equipped cartridge including the circular valve of the present invention, the circular valve may include a light guide configured to guide light to the position setting the light detector, the light being incident from the DNA probe facing the position setting the light detector. With this arrangement, the structure becomes simpler than the case where the circular valve and the light guide are formed separately.
In the DNA-array-equipped cartridge including the light guide, the light guide may be a lens configured to collimate and guide light to a position setting the light detector, the light being incident from the DNA probe facing the light detector. With this arrangement, light incident from each of the DNA probes can be more efficiently guided to the position setting the light detector.
In the present invention, the DNA-array-equipped cartridge may further include a highly thermal-conductive member disposed opposite a position setting the light detector with respect to the DNA array space and made of carbon-containing resin or metal. The highly thermal-conductive member made of carbon-containing resin or metal having relatively high thermal conductivity. Therefore, for a hybridization reaction between target DNA and the DNA probe 53a, it is possible to reduce variations in temperature among the spotted DNA probes. Also, an error in light detection due to disturbance can be prevented from occurring. The DNA-array-equipped cartridge including the highly thermal-conductive member may further include a low-reflection ring disposed on the same side as the light detector with respect to the DNA array space, the low-reflection ring having a through portion communicating with the light detector and made of carbon-containing resin or metal. With this arrangement, the error in light detection due to disturbance can be further reliably prevented from occurring.
In the DNA-array-equipped cartridge of the present invention, the plurality of fluid containing spaces may include a column containing space and a waste liquid tank, the column containing space containing a column for purification of the target DNA, the waste liquid tank communicating with an upper part of the column containing space. Also, the plurality of openings may include first and second openings communicating with the column containing space, the first opening communicating with a lower part of the column, the second opening communicating with an upper part of the column. In this case, the second opening is closed, so that the solution containing the target DNA flows through the first opening, passes through the column from the lower side to the upper side, and flows into the waste liquid tank. Hence, the target DNA is absorbed to the column. Then, the first opening is closed, so that the wash liquid flows through the second opening, passes through the upper part of the column, and flows into the waste liquid tank. Thus, the channel from the upper part of the column to the waste liquid tank can be washed. The channel is a space where eluate collects in, which will be described later. Thus, washing the channel can prevent the eluate from being contaminated. Then, the second opening is closed, so that the eluate flows through the first opening but stops at a position in the channel before the eluate reaches the waste liquid tank. Thus, the DNA probes separated from the column is eluted into the eluate. Then, the first opening is closed, so that the eluate is drawn out through the second opening and the eluate is recovered. The eluate can be recovered through the second opening without passing through the column. Thus, recovery loss can be decreased as compared with the arrangement, in which the eluate is recovered through the column.
In the DNA-array-equipped cartridge of the present invention, labeled markers may be spotted at at least two predetermined positions in the DNA array space. With this arrangement, for example, when the DNA array is not horizontal but is inclined, the fluorescence intensities of the labeled markers may vary depending on the inclinations thereof. Hence, correction coefficients can be calculated respectively for the spotted positions of the DNA probes on the basis of the variation amounts of the fluorescence intensities of the labeled markers, and the fluorescence intensities of the DNA probes can be corrected respectively with the correction coefficients.
In the present invention, an analyzer includes a holder for holding the DNA-array-equipped cartridge according to any one of claims 1 to 11; a rotator for rotating, about the center axis, the housing of the DNA-array-equipped cartridge held by the holder; the reaction tank; the light detector; and a liquid transporter for transporting, through the corresponding openings, fluid held in the fluid containing spaces to the reaction tank, and fluid held in the reaction tank to the fluid containing spaces, wherein when the housing of the DNA-array-equipped cartridge held by the holder is rotated by the rotator, the plurality of openings of the DNA-array-equipped cartridge sequentially face the fluid port of the reaction tank, and the plurality of DNA probes sequentially face the light detector.
In the analyzer described above, when the housing is rotated to allow the openings of the reagent spaces to sequentially face the fluid port of the reaction tank, the rotation of the housing is temporarily stopped in a state where the opening of each of the reagent spaces faces the reaction tank, so that fluid is transported between the reaction tank and the reagent space. Thus, the target DNA can be prepare and eventually stored in the reaction tank. Next, when the housing is rotated to allow the opening of the DNA array space to face the fluid port of the reaction tank, the target DNA in the reaction tank can flow into the DNA array space and the target DNA can react with each of the DNA probes. Next, when the housing is rotated, light incident from each of the DNA probes subjected to the reaction can be detected by the light detector. Thus, it is possible to relatively easily carry out the process from preparation of the target DNA to detection of light incident form the DNA probes at the light detector.
A method for using the DNA-array-equipped cartridge in the present invention, the method includes the steps of:
(a) preparing the DNA-array-equipped cartridge in which fluids for preparation of the target DNA are held in the reagent containing spaces;
(b) preparing the reaction tank independent of the housing of the DNA-array-equipped cartridge and holding a sample from which the target DNA is prepared;
(c) rotating the housing to allow the openings of the reagent spaces to sequentially face the fluid port of the reaction tank, temporarily stopping the rotation of the housing in a state where the opening of each of the reagent spaces faces the reaction tank, transporting fluid between the reaction tank and the reagent space to prepare the target DNA, and eventually storing the target DNA in the reaction tank;
(d) rotating the housing to allow the opening of the DNA array space to face the fluid port of the reaction tank, causing the target DNA in the reaction tank to flow into the DNA array space, and causing the target DNA to react with each of the DNA probes; and
(e) rotating the housing and detecting light incident from each of the DNA probes subjected to the reaction by means of the light detector independent of the housing.
With the method for using the DNA-array-equipped cartridge described above, it is possible to relatively easily carry out the process from preparation of the target DNA to detection of light incident from the DNA probes at the light detector.
The best mode for carrying out the present invention will now be described with reference to the drawings.
As illustrated in
As illustrated in
The circular valve 51 is a circular member coaxial with a center axis 59 of the cartridge body 54. The circular valve 51 is provided with a condenser lens 57. The circular valve 51 is supported by the center pin 55 inserted through the center thereof. The circular valve 51 includes a block 51b at the top. The block 51b has upright walls 51c and 51c parallel to each other and a notch 51d. A retainer 84 (see
In the ring array 53, the plurality of DNA probes 53a are spotted along the circumference coaxial with the center axis 59 of the cartridge body 54.
As illustrated in
The cartridge body 54 is a disk-like member made of cyclo-olefin copolymer, and is composed of four disk-like layers: a first layer 54a, a second layer 54b, a third layer 54c, and a fourth layer 54d.
The liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 each are a space narrowed at both ends. Of these liquid containers, the liquid containers 304, 308, 309, 315, 316, 318, 319, 321, and 323 each are configured to hold a large amount of liquid and are formed as a space extending from the second layer 54b to the third layer 54c, while the liquid containers 302, 303, 311, 317, 320, and 325 each are configured to hold a small amount of liquid and are formed only in one of the second layer 54b and the third layer 54c. The liquid containers 302 to 304, 308, 309, 311, 315, 316, 318, 319, 321, 323, and 325 are connected, at their respective one ends adjacent to the center of the cartridge body 54, to the distribution ports 302a to 304a, 308a, 309a, 311a, 315a, 316a, 318a, 319a, 321a, 323a, and 325a, respectively, through channels formed in the lower surface of the third layer 54c and connected to the corresponding liquid containers, and further through vertical channels in the third layer 54c and the second layer 54b. The liquid containers 317 and 320 are connected, at their respective one ends adjacent to the center of the cartridge body 54, to the distribution ports 317a and 320a, respectively, through vertical channels formed in the third layer 54c and further through radial channels connected to the vertical channels. The liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 are connected, at their respective other ends remote from the center of the cartridge 50, to the outside-air distribution portions 326. A detailed description of the outside-air distribution portions 326 will be given later.
The distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a are openings communicating with the liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325, respectively. The distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a are used to supply liquids from the corresponding liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325, and formed in the upper surface of the third layer 54c. The distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a are arranged along a circumference coaxial with a rotation axis about which the cartridge body 54 is rotated by the rotating mechanism 32. That is, the distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a are arranged along a circumference coaxial with the center axis 59 of the cartridge body 54. By a differential pressure applied to liquid held in one of the liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 connected to the distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a, respectively, the liquid held in the liquid container can be supplied to the reaction tank 30.
The outside-air distribution portion 326 is a general term used to refer to any of outside-air distribution channels 302c, 303c, 309c, 311c, and 325c formed in the lower surface of the third layer 54c and radially extending outward from the respective one ends of the liquid containers 302, 303, 309, 311, and 325 remote from the center of the cartridge body 54; outside-air distribution channels 317c and 320c formed in the lower surface of the second layer 54b and radially extending outward from the respective one ends of the liquid containers 317 and 320 remote from the center of the cartridge body 54; and air vents 302d to 304d, 308d, 309d, 311d, 315d to 321d, 323d, and 325d vertically formed in the first layer 54a. Of the air vents 302d to 304d, 308d, 309d, 311d, 315d to 321d, 323d, and 325d, the air vents 302d, 303d, 309d, 311d, and 325d allow the corresponding liquid containers 302, 303, 309, 311, and 325 to communicate with the outside air, through the corresponding outside-air distribution channels 302c, 303c, 309c, 311c, and 325c and further through the corresponding channels vertically formed in the second layer 54b and the third layer 54c. The air vents 317d and 320d allow the corresponding liquid containers 317 and 320 to communicate with the outside air, through the corresponding outside-air distribution channels 317c and 320c and further through the corresponding channels vertically formed in the second layer 54b. The air vents 304d, 308d, 315d, 316d, 318d, 319d, 321d, and 323d allow the corresponding liquid containers 304, 308, 315, 316, 318, 319, 321, and 323 to directly communicate with the outside air.
As illustrated in
The column containing space 306 is provided between the combined distribution port 306a and the diffusion channel 327f, and includes a column. A ceramic column (e.g., silica gel column) is used here. When the pump 34 is actuated to increase pressure in the reaction tank 30, liquid held in the reaction tank 30 is distributed to the column containing space 306 and allowed to collect in the diffusion channel 327f. If further pressure is applied, the liquid collecting in the diffusion channel 327f is stored in the waste liquid tank 327. If the applied pressure is reduced, the liquid passes through the column containing space 306 again and is stored in the reaction tank 30. Filling the column of the column containing space 306 is effected by covering the lower surface of the fourth layer 54d after filling the column from the lower surface of the fourth layer 54d through the filling opening 341. Thus, replacement of the column in the column containing space 306 is effected by uncovering the lower surface of the fourth layer 54d, if necessary.
The combined distribution port 306a and the channel inlet 53c of the ring array 53 are openings that communicate with the waste liquid tanks 327 and 328, respectively, and through which liquids are eventually stored in the waste liquid tanks 327 and 328. The combined distribution port 306a is provided in the upper surface of the third layer 54c, and the channel inlet 53c is provided in the upper surface of the ring array 53 (see
The closed ports 301a, 305a, 307a, 312a, 322a, and 324a are non-hole portions of the third layer 54c, and their positions are defined by the linked packing member 52 (see
The closed channel 310 is formed as a groove in the third layer 54c. The closed channel 310 is connected to the injection port 310a through a radially extending channel formed in the third layer 54c and a vertical channel connected to this radially extending channel. Unlike in the case of the liquid containers described above, one end of the closed channel 310 remote from the center of the cartridge body 54 is not connected to any of the outside-air distribution portions 326. Therefore, when the closed channel 310 does not communicate with the reaction tank 30, the injection port 310a is closed by the lower surface of the circular valve 51, so that the closed channel 310 becomes a closed space.
The injection port 310a is an opening communicating with the closed channel 310 and provided in the upper surface of the third layer 54c. The injection port 310a is used to store liquid in the closed channel 310 or supply liquid held in the closed channel 310 to the reaction tank 30. The injection port 310a and the other ports are arranged along the circumference coaxial with the rotation axis about which the cartridge body 54 is rotated by the rotating mechanism 32 (see
The cartridge holding mechanism 80 is a mechanism to which the cartridge 50 is attached.
As illustrated in
The reaction-tank securing unit 36 is formed by applying electroless nickel plating to a copper member. The reaction-tank securing unit 36 is secured to the center of the upright wall portion 92b of the supporting member 92. At a position above the cartridge 50 placed on the rotating stage 38, the reaction-tank securing unit 36 removably secures the reaction tank 30. The Peltier device 36a for the reaction tank 30 is provided inside the reaction-tank securing unit 36. By regulating the temperature of the reaction-tank securing unit 36, the Peltier device 36a can regulate the temperature of the reaction tank 30 at a constant level. The material used to form the reaction-tank securing unit 36 may be an anodized aluminum.
The reaction tank 30 is made of polypropylene. As illustrated in
The pump 34 is a so-called tube pump that applies pressure, by squeezing its tube with rollers, to a component connected to the tube. As illustrated in
The light detecting unit 60 includes the optical fiber 62 that transmits light incident from each of the DNA probes 53a, and a light detecting module 64 that converts light input through the optical fiber 62 into an electric signal. The optical fiber 62 is secured by the retainer 84 (see
The controller 40 is configured as a microprocessor centered on a CPU 42. The controller 40 includes a flash ROM 43 that stores various processing programs, and a RAM 44 that temporarily stores or saves data. The controller 40 outputs a control signal to the pump 34, a control signal to the motor 37, a control signal to the light detecting unit 60, and supply voltages to the Peltier device 36a for the reaction tank and the Peltier device 38a for the cartridge. The controller 40 inputs a detection signal from the light detecting unit 60.
A cross section of the cartridge 50 attached to the cartridge holding mechanism 80 is illustrated in
In the analyzer 90 configured as described above, the cartridge 50 in which the ring array 53 is mounted on the cartridge body 54 in advance is used. In the cartridge 50, desired amounts of liquids including reagents used in predetermined reactions are separately stored in appropriate liquid containers. To sequentially supply liquids from the liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 to the reaction tank 30 for predetermined reactions in the reaction tank 30, and transport the liquids after the reactions to the waste liquid tanks 327 and 328, the motor 37 rotates the cartridge body 54 to allow the different ports of the cartridge body 54 to be sequentially connected to the reaction tank 30. In particular, purification of a reaction product is effected by adsorbing the reaction product to a column and discharging waste liquid to the waste liquid tank 327, eluting the reaction product adsorbed to the column with liquid held in any of the liquid containers, allowing the eluted reaction product to temporarily collect in the diffusion channel 327f, and supplying the eluted reaction product to the reaction tank 30. Since the reaction tank 30 of the analyzer 90 is provided outside the cartridge 50, changes in temperature in the reaction tank 30 are not easily transmitted to the cartridge 50. Therefore, temperatures in the reaction tank 30 and the cartridge 50 can be kept at different levels (e.g., a reaction temperature and a storage temperature). A motor (not shown) that rotates a magnet attached thereto is provided beside the reaction-tank securing unit 36, and a rotor including a magnet is provided inside the reaction tank 30. When the motor rotates the magnet attached thereto, the rotor rotates to stir liquid in the reaction tank 30.
Next, an operation of the analyzer 90 will be described. In particular, a description will be given about a process in which rice genomic DNA, which is a sample, is amplified, prepared, and subjected to reaction with each of the DNA probes 53a formed in the ring array 53 and thus, light incident from each of the DNA probes 53a is detected.
First, amplification and preparation of genomic DNA will be described with reference to
Next, the CPU 42 allows the distribution port 303a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid held in the liquid container 303 to be drawn out (step S1110). Next, the CPU 42 rotates the cartridge body 54 to allow the closed port 305a to be connected to the reaction tank 30, and performs stirring for 15 minutes to allow a reaction to occur in the reaction tank 30 while keeping the temperature therein at 95° C. Then, the CPU 42 performs 40 cycles, each involving stirring for 1 minute in the reaction tank 30 kept at a temperature of 95° C., stirring for 1 minute and 30 seconds at a temperature of 66° C., and stirring for 30 seconds at a temperature of 72° C. Last, the CPU 42 performs stirring for 10 minutes at a temperature of 72° C. to allow a reaction to occur (step S1120). The term “stirring” means to mix solutions in the reaction tank 30 by causing the motor 72 to rotate the rotor 47 placed in the reaction tank 30. Next, the CPU 42 allows the distribution port 304a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid (adsorption buffer (3.8 mol/L, ammonium sulfate)) held in the liquid container 304 to be drawn out (step S1130). Next, the CPU 42 allows the combined distribution port 306a to communicate with the reaction tank 30, and actuates the pump 34 to distribute the mixed solution in the reaction tank 30 to the column containing space 306 (step S1140). When the mixed solution flows, through the combined distribution port 306a (see
Next, the CPU 42 allows the distribution port 323a to communicate with the reaction tank 30, actuates the pump 34 to allow liquid (first wash buffer (1.9 mol/L, ammonium sulfate)) held in the liquid container 323 to be drawn out, performs stirring for 1 minute while keeping the temperature in the reaction tank 30 at 25° C., and washes the inside of the reaction tank 30 (step S1150). The inside of the reaction tank 30 is washed to prevent salt precipitation. Next, the CPU 42 actuates the pump 34 to store, in the liquid container 323, the liquid used for washing the reaction tank 30 (step S1160). Next, the CPU 42 allows the distribution port 308a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid (second wash buffer (pH 6.0, 10 mmol/L, phosphoric acid-ethanol mixture (mixing ratio=1:2.8))) held in the liquid container 308 to be drawn out (step S1170). Next, the CPU 42 allows the combined distribution port 306a to communicate with the reaction tank 30, actuates the pump 34 to distribute the second wash buffer in the reaction tank 30 to the column containing space 306, and thereby washes the column (step S1180). Next, the CPU 42 allows the distribution port 309a to communicate with the reaction tank 30, actuates the pump 34 to allow liquid (elution buffer (pH 8.0, 20 mmol/L, tris-hydrogen chloride) held in the liquid container 309 to be drawn out (step S1190). Next, the CPU 42 allows the combined distribution port 306a to communicate with the reaction tank 30, actuates the pump 34 to distribute the elution buffer in the reaction tank 30 to the column containing space 306, and allows the eluate to collect in the diffusion channel 327f, not to flow out to the waste liquid tank 327 (step S1200). Specifically, after distributing the elution buffer to the column containing space 306, the CPU 42 causes the pump 34 (tube pump) to stop squeezing the tube. Since this allows amplified DNA adsorbed to the column to be eluted into the elution buffer, the solution containing the amplified DNA collects in the diffusion channel 327f.
After step S1200, the CPU 42 actuates the pump 34 to allow the elution buffer collecting in the diffusion channel 327f to be drawn back to the reaction tank 30 (step S1210). Next, the CPU 42 allows the injection port 310a to communicate with the reaction tank 30, and actuates the pump 34 to inject the elution buffer in the reaction tank 30 into the closed channel 310 (step S1220). Thus, air in the closed channel 310 is compressed by the injected liquid and increased in pressure. Next, the CPU 42 allows the distribution port 309a to communicate with the reaction tank 30, so as to allow the mixed solution remaining in the reaction tank 30 to be discharged to the liquid container 309 (step S1230). The pressure used in step S1220 to inject the mixed solution into the closed channel 310 remains in the reaction tank 30. Therefore, when the distribution port 309a communicates with the reaction tank 30, the remaining pressure causes the mixed solution in the reaction tank 30 to be discharged to the liquid container 309. Next, the CPU 42 allows the injection port 310a to communicate with the reaction tank 30, and supplies mixed solution injected into the closed channel 310 to the reaction tank 30 (step S1240). Prepared DNA is thus obtained. Since, in step S1240, the mixed solution is discharged to the liquid container 309 by the pressure remaining in the reaction tank 30, the pressure in the reaction tank 30 is reduced. However, the pressure of air in the closed channel 310 remains the same as that used for injection of the mixed solution in step S1220. Therefore, this difference in pressure causes the mixed solution injected into the closed channel 310 to be supplied to the reaction tank 30.
Next, with reference to
Next, the CPU 42 allows the distribution port 315a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid held in the liquid container 315 to be drawn out (step S1340). Next, the CPU 42 allows the channel inlet 53c to communicate with the reaction tank 30, controls the actuation of the pump 34 to allow wash liquid held in the reaction tank 30 to temporarily collect in the reaction channel 53b of the ring array 53, and thus washes the reaction channel 53b while causing the Peltier device 38a to keep the temperature in the reaction channel 53b at 25° C. for 5 minutes. Then, the CPU 42 actuates the pump 34 again to increase air pressure in the reaction tank 30, and allows the wash liquid temporarily collecting in the reaction channel 53b to be discharged to the waste liquid tank 328 (step S1350). Next, the CPU 42 performs processing similar to that of step S1340 and step S1350 using liquid held in the liquid container 316 so as to wash the reaction channel 53b of the ring array 53 (step S1360 and step S1370). Next, the CPU 42 allows the distribution port 317a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid held in the liquid container 317 to be drawn out (step S1380). Next, the CPU 42 allows the channel inlet 53c to communicate with the reaction tank 30, controls the actuation of the pump 34 to allow liquid held in the reaction tank 30 to temporarily collect in the reaction channel 53b of the ring array 53, and causes a chemiluminescent reaction of the DNA probe 53a to occur while keeping the temperature in the reaction channel 53b at 25° C. for 30 minutes. Then, the CPU 42 actuates the pump 34 again to increase air pressure in the reaction tank 30, and allows the liquid temporarily collecting in the reaction channel 53b to be discharged to the waste liquid tank 328 (step S1390). Next, the CPU 42 performs processing similar to that of step S1340 and step S1350 using liquids held in the liquid containers 318 and 319 so as to wash the reaction channel 53b of the ring array 53 (step S1400 to step S1430). Next, the CPU 42 allows the distribution port 320a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid held in the liquid container 320 to be drawn out (step S1440). Next, the CPU 42 allows the channel inlet 53c to communicate with the reaction tank 30, controls the actuation of the pump 34 to allow liquid held in the reaction tank 30 to temporarily collect in the reaction channel 53b of the ring array 53, and causes a pigmentation reaction of the DNA probe 53a to occur while keeping the temperature in the reaction channel 53b at 25° C. for 30 minutes. Then, the CPU 42 actuates the pump 34 again to increase air pressure in the reaction tank 30, and allows the liquid temporarily collecting in the reaction channel 53b to be discharged to the waste liquid tank 328 (step S1450). Next, the CPU 42 allows the distribution port 321a to communicate with the reaction tank 30, and actuates the pump 34 to allow liquid held in the liquid container 321 to be drawn out (step S1460). Next, the CPU 42 allows the channel inlet 53c to communicate with the reaction tank 30, and distributes liquid held in the reaction tank 30 to the reaction channel 53b of the ring array 53 so as to stop the pigmentation reaction of the DNA probe 53a (step S1470). Thus, the pigmented DNA can be obtained in the ring array 53 (step S1480).
Next, a process of detecting light from the DNA probes 53a will be described. The CPU 42 of the controller 40 reads and executes a light detection routine stored in the flash ROM 43.
The correspondence between the components of the present embodiment and the components of the present invention will now be described. The cartridge 50 of the present embodiment corresponds to a DNA-array-equipped cartridge of the present invention. The cartridge body 54 and the ring array 53 of the present embodiment correspond to a housing of the present invention. The liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 and the reaction channel 53b of the present embodiment correspond to fluid containing spaces of the present invention. The liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 of the present embodiment correspond to reagent containing spaces of the present invention. The reaction channel 53b of the present embodiment corresponds to a DNA array space of the present invention. The distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a and the channel inlet 53c of the present embodiment correspond to openings of the present invention. The circular valve 51 of the present embodiment corresponds to a circular valve of the present invention. The condenser lens 57 of the present embodiment corresponds to a light guide of the present invention. The cartridge holding mechanism 80 of the present embodiment corresponds to a holder of the present embodiment. The rotating stage 38 and the motor 37 of the present embodiment correspond to a rotator of the present invention. The collimating lens 62a of the present embodiment corresponds to a light detector of the present invention. The pump 34 of the present embodiment corresponds to a liquid transporter of the present invention.
In the cartridge 50 of the present embodiment described above in detail, when the cartridge body 54 is rotated such that the distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a of the liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325 sequentially face the fluid port 30a of the reaction tank 30, the rotation of the cartridge body 54 is temporarily stopped in a state in which the reaction tank 30 faces each of the distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a, so that fluid is transported between the reaction tank 30 and each of the liquid containers 302 to 304, 308, 309, 311, 315 to 321, 323, and 325. Thus, target DNA can be prepared and eventually stored in the reaction tank 30. When the cartridge body 54 is rotated such that the channel inlet 53c faces the fluid port 30a of the reaction tank 30, it is possible to allow the target DNA in the reaction tank 30 to flow into the reaction channel 53b, and thus to allow the target DNA to react with each of the DNA probes 53a. Next, when the cartridge body 54 is rotated, light incident from each of the DNA probes 53a subjected to the reaction can be detected by the collimating lens 62a of the light detecting unit 60. Thus, it is possible to relatively easily carry out the process from preparation of the target DNA to detection of light incident from each of the DNA probes 53a at the collimating lens 62a.
The cartridge body 54 is easily rotatable since it has a disk-like shape. The cartridge body 54 is provided with the circular valve 51, and rotating the cartridge body 54 allows the distribution ports 302a to 304a, 308a, 309a, 311a, 315a to 321a, 323a, and 325a, the combined distribution port 306a, and the channel inlet 53c to sequentially face the through hole 51a of the circular valve 51. Thus, with a relatively simple structure, any one of the chambers and the reaction channel 53b can communicate with the reaction tank 30. Moreover, since the circular valve 51 has the condenser lens 57, the structure becomes simpler than the case where they are formed separately. Additionally, since the circular valve 51 has the condenser lens 57, light incident from each of the DNA probes 53a can be efficiently guided to the collimating lens 62a serving as a light detector.
As illustrated in
It will be apparent that the present invention is not limited to the embodiments described above, and may be embodied in various forms within the technical scope of the present invention.
For example, in the ring array 53 of the embodiment described above, the plurality of DNA probes 53a are arranged in a row along a circumference. However, as long as it is possible to identify light incident from the DNA probes 53a in each row and to arrange the DNA probes 53a in the reaction channel 53b, the plurality of DNA probes 53a may be arranged in two or more rows along circumferences having different radii. This makes it possible to spot a larger number of DNA probes 53a. For example, the DNA probes 53a may be spotted in two rows along circumferences that are coaxial with the center axis 59 and have different diameters. To accommodate the DNA probes 53a spotted in two rows, two light detecting units 60, each corresponding to the DNA probes 53a in each row, may be provided. At the same time, the condenser lens 57 and the optical fiber 62 are provided at positions opposite relative to one of the DNA probes 53a in each row.
In the ring array 53 of the embodiment described above, the plurality of DNA probes 53a are arranged in a row along a circumference. However, a plurality of DNA probes may be spotted for each of the various DNA probes 53a arranged in a row. For example, two points each may be spotted, as illustrated in
The cartridge body 54 and the ring array 53 are provided as separate units in the embodiment described above, but they may be provided as a single unit.
The analyzer 90 includes the light detecting module 64 in the embodiment described above. Alternatively, the light detecting module 64 may be replaced with an external light detecting module, to which the optical fiber 62 is connected. In this case, the controller 40 transmits and receives control signals and detection signals to and from the external light detecting module.
In the embodiment described above, the analyzer 90 is configured such that, after a pigmentation reaction, light incident from each of the DNA probes 53a is detected through the optical fiber 62 by the light detecting module 64. Alternatively, the analyzer 90 may perform the following process. First, for preparing target DNA, the analyzer 90 fluorescently labels the target DNA and allows the prepared target DNA to be distributed to the reaction channel 53b. Thus, the fluorescently-labeled target DNA is located at a position of one of the plurality of DNA probes 53a, the one having been subjected to hybridization reaction with the target DNA. Next, light for producing fluorescence is applied to the DNA probes 53a. Fluorescence is produced at the position of the DNA probe 53a having been subjected to hybridization reaction with the target DNA, and is detected by the light detecting unit 60. This allows the user to recognize which of the DNA probes 53a has reacted with the target DNA, and thus to identify the target DNA. In this case, the analyzer 90 includes a light emitting unit that applies light for producing fluorescence to the DNA probes 53a. The light detecting module 64 may include the light emitting unit that applies, through the optical fiber 62, light for producing fluorescence to the DNA probes 53a. Specifically, for example, a filter may be provided between the light emitting unit and an end of the optical fiber 62 inside the light detecting module 64. The filter allows light for producing fluorescence, the light being to be incident on the optical fiber 62, to pass through such that the light output from the optical fiber 62 is divided into fluorescence and light for producing fluorescence. The light detecting element is provided at a position at which the resulting fluorescence is received.
Although the cartridge 50 is used in the embodiment described above, a cartridge 150 including a highly thermal-conductive member 58 may be used.
Although the circular valve 51 has the condenser lens 57 in the embodiment described above, the circular valve 51 may be one without the condenser lens 57.
In the embodiment described above, the cartridge body 54 is composed of four layers, that is, the first layer 54a, the second layer 54b, the third layer 54c, and the fourth layer 54d. However, as long as chambers capable of holding liquid and discharging waste liquid are formed therein, the cartridge body 54 does not necessarily need to be composed of four layers. For example, the cartridge body 54 may be composed of three layers or five layers.
Although the cartridge body 54 of the above embodiment has a disk-like shape, the cartridge body 54 may have another shape, such as a rectangular shape or a hexagonal shape.
In the embodiment described above, the DNA preparation routine, the reaction processing routine, and the light detection routine are executed by the controller 40. Alternatively, an operation corresponding to these routines may be manually performed by the operator. In this case, there may be provided, for example, switches used by the operator to control the motor 37, the pump 34, the Peltier device 38a, the Peltier device 36a, and the light detecting unit 60, as well as a storage device for storing detected signals.
In the embodiment described above, the ring array 53 is used to identify a species of rice. However, the ring array 53 may be used for a different reaction. In this case, DNA probes for this different reaction may be formed in the reaction channel 53b. At the same time, the cartridge body 54 may hold liquids for use in this different reaction.
In the embodiment described above, though not described specifically, as illustrated in
In steps S1170 and S1180, the second wash buffer held in the liquid container 308 flows from the combined distribution port 306a, passes through the channel 306b, passes through the column in the column containing space 306 from the lower side to the upper side in the reaction tank 30, passes through the diffusion channel 327f, and then flows into the waste liquid tank 327. Thus, the column is washed. In steps S1190 and S1200, the elution buffer held in the liquid container 309 flows from the combined distribution port 306a, passes through the channel 306b, passes through the column in the column containing space 306 from the lower side to the upper side in the reaction tank 30, and stops in the middle of the diffusion channel 327f (so as not to flow into the waste liquid tank 327). Thus, the DNA absorbed to the column is separated from the column and eluted into the elution buffer. In step S1210, the elution buffer (containing the DNA) in the diffusion channel 327f is drawn back to the reaction tank 30 through the combined distribution port 306a and the elution buffer is recovered.
Alternatively, as illustrated in
In the embodiment described above, the three grooves 342 (
In the embodiment described above, the plurality of DNA probes 53a are spotted along the circumference of the ring array 53. Alternatively, as illustrated in
In the embodiment described above, though not described specifically, when the rotor including the magnet is provided in the reaction tank 30, the arrangement in which a long rotor 75 is used and the longitudinal direction of the rotor 75 is aligned with the vertical direction as illustrated in
In the embodiment described above, although the inner surface of the reaction tank 30 has not been particularly described, vertical grooves 31a to 31e for deaeration are preferably formed in the inner surface of the reaction tank 30 as illustrated in
If necessary, an antifoaming agent may be added to the liquid held in the liquid container of the embodiment described above. With the antifoaming agent, the liquid can be prevented from foaming when the liquid is transported from the liquid container to the reaction tank 30. In particular, when the liquid is highly viscous, the liquid may likely foam. Thus, the antifoaming agent is preferably added.
The present invention contains subject matter related to Japanese Patent Application No. 2008-313336 filed in the Japanese Patent Office on Dec. 9, 2008, and Japanese Patent Application No. 2009-218029 filed in the Japanese Patent Office on Sep. 18, 2009, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-313336 | Dec 2008 | JP | national |
| 2009-218029 | Sep 2009 | JP | national |
