Patent Application
20070264655
Specific embodiments of the present invention will now be described in detail in accordance with the accompanying drawings.
In the following, a nucleic acid examining device according to the present invention will be described with reference to a case where a target nucleic acid is DNA. The target nucleic acid as an object of detection is not restricted to DNA; it may also be RNA.
The nucleic acid examining device of the present invention includes the following components (1) through (5):
(1) a nucleic acid amplification unit for effecting amplification reaction of a target nucleic acid;
(2) a hybridization reaction unit allowing installation of multiple reaction cassettes for effecting hybridization reaction of the target nucleic acid;
(3) a dispenser unit having multiple movable nozzles capable of sucking and discharging the target nucleic acid;
(4) a detector for detecting that the reaction cassettes are set at predetermined positions in the hybridization reaction unit; and
(5) a control section.
In the nucleic acid amplification unit, the target nucleic acid contained in the nucleic acid sample is amplified mainly by the PCR method. When the amount of target nucleic acid extracted is small, or when the amount of target nucleic acid extracted is sufficient but a large amount of substances other than the target nucleic acid are mixed therein and purification of the sample nucleic acid is necessary, it is possible to additionally arrange a DNA purification unit.
The hybridization reaction unit has an installation table on which there is provided a cassette holder allowing installation of multiple reaction cassettes. In this specification, the term “reaction cassette” represents a container having a liquid receiving port allowing dispensing of the nucleic acid sample from outside the container for the purpose of effecting hybridization reaction within the container.
The dispenser unit dispenses a nucleic acid solution containing the target nucleic acid to the reaction cassette. Further, when there are added units for performing the above-mentioned PCR amplification and the step of preparing a hybridization solution, it is also possible to add to the dispenser unit a construction allowing dispensing to a predetermined position of a reagent required in each step or a solution containing the target nucleic acid.
The reaction cassette is detachably installed in a cassette holder. It is possible to use multiple reaction cassettes as needed. The hybridization reaction unit is provided with the cassette holder so as to allow individual installation of multiple reaction cassettes. By using a detector, it is automatically determined whether the reaction cassettes installed in the cassette holder are installed at predetermined positions on the installation table of the hybridization reaction unit.
The necessary reagents for hybridization are respectively prepared in a well plate. While a well plate is naturally suitable for the reagent container, this should not be construed restrictively; it may be a container uniquely designed for the present device.
As the dispenser unit, one with multiple nozzles can be used. By using a dispenser unit having multiple dispensing nozzles, it is possible to dispense reagents and reaction liquids simultaneously to multiple reaction cassettes.
The detector is provided so as to be in contact with the cassette holder, and serves to detect whether the reaction cassettes are set at the predetermined positions in the hybridization reaction unit. It is also possible for this detector to be one capable of detecting whether all the reaction cassettes that can be set in the hybridization reaction unit are set at predetermined attachment positions.
Alternatively, it is also possible to detect whether a specific reaction cassette is set at a predetermined installation position. Further, it is possible for the detector to detect at one time whether a reaction cassette is set at a predetermined position, or to perform detection a number of times corresponding to the predetermined positions for the reaction cassettes, respectively; there are no particular limitations in this regard. The setting as to which reaction cassette, of the reaction cassettes set in the cassette holder, is to be detected, or whether all the reaction cassettes are to be detected, can be effected automatically according to a pre-set program.
In this device, it is possible to implement the amplification step and the hybridization reaction step in parallel. For example, when hybridization reaction is being implemented on one nucleic acid sample, it is possible to implement amplification on another nucleic acid sample.
All the operations described above are controlled by the control section.
In the following, an operation of the device of the present invention will be described with reference as appropriate to the drawings. In this embodiment, description will be made of a case where hybridization reaction is effected after performing nucleic acid amplification processing twice. More specifically, in the example described below, the nucleic acid contained in the nucleic acid sample is amplified by using the PCR method, and then impurities other than the target nucleic acid are removed; and then, the target nucleic acid is amplified again by using the PCR method, and hybridization reaction is effected.
First, the nucleic acid sample standby wells 8 loaded with nucleic acid samples are arranged on the sample stage 7, and a nucleic acid amplification plate 33 to be used, a pipette tip case 35, the hybridization reaction plate 43, and reaction cassettes 45 are respectively set. In each reaction cassette 45, there is provided a reaction chamber, where there is installed a DNA microarray.
The dispenser unit 2 is supported by a dispenser unit guide 6 and a dispenser unit support member 21. The dispenser unit support member 21 is provided with a rail guide (not shown) extending in a Z-direction, enabling the dispenser unit 2 to move in the Z-direction. The dispenser unit 2 is capable of moving along a rail guide (not shown) in an X-direction through a space above the nucleic acid amplification unit 3 and the hybridization reaction unit 4. The dispenser unit 2 is formed by a casing 22 and a pipetting mechanism 24 generating negative pressure in the nozzles, and there are provided nozzle portions 25 allowing attachment of pipette chips to the forward ends of the pipetting mechanism 24. As shown in
On a nucleic acid amplification stage 32, there can be arranged a nucleic acid amplification plate 33 having multiple amplification wells 34, and the pipette chip case 35. As the nucleic acid amplification plate 33, it is possible to employ, for example, a commercially available 96-well plate of polypropylene as shown in
The primer used when executing the PCR method and the magnetic particles and the elution solution used in the purification step may be well-known ones, a detailed description thereof with specific names mentioned will be omitted. Further, the arrangement of the reagents is naturally not restricted to that of the above-described example. To prevent intrusion of impurities, a protective sheet (not shown) may be attached to an upper side of the amplification wells 34. The pipette chip case 35 accommodates twelve pipette chips 36 in a row in a Y-direction. As indicated by dashed lines in
First, the dispenser unit is moved by a drive unit (not shown) to attach the pipette chips 36 to the nozzle portions 25. The dispenser unit is moved such that the nozzle portions 25 and the pipette chips 36 are matched in their positions in the X-direction, and further, the dispenser unit 2 is lowered by the dispenser unit support member 21. As a result, the nozzle portions 25 and the pipette chips 36 are fit-engaged with each other.
The liquid dispensing operation in the present invention is conducted by using the dispenser unit. First, the dispenser unit 2 is driven along the rail guides (not shown) provided on the dispenser unit guide 6 and the dispenser unit support member 21 to bring the forward ends of the pipette chips attached to the nozzle portions 25 into contact with the liquid. The pipetting mechanism 24 is operated to generate negative pressure in the nozzles, thereby sucking the nucleic acid sample into the pipette chips. With the liquid being retained in the pipette chips, the dispenser unit support member 21 is driven again to discharge the retained liquid at desired positions.
By using the dispenser unit 2, the nucleic acid sample is moved from the nucleic acid sample standby wells 8 to the amplification wells 34 in the line A containing the 1st PCR reagent.
The nucleic acid amplification unit 3 includes a thermal cycler (not shown), and a nucleic acid amplification stage 32 capable of temperature adjustment by the thermal cycler. The nucleic acid amplification stage 32 is retained so as to be movable in the Y-direction with respect to a stationary member 31.
The dispenser unit is driven to dispense the nucleic acid sample from the nucleic acid sample standby wells 8 to the amplification wells in the line A. When the 1st PCR reagent and the nucleic acid sample are mixed together in the amplification wells in the line A, a temperature cycle is applied to the amplification wells 34 by using the thermal cycler.
After the completion of the 1st PCR, a purification step for removing impurities other than the target nucleic acid is started. Magnetic particles specifically adsorbing the target nucleic acid are contained in the amplification wells 34 in the line B. Subsequently, the purification step will be described. The nucleic acid having undergone the 1st PCR is moved by using the dispenser unit 2 to the amplification wells 34 in the line B containing magnetic particles, causing the magnetic particles to adsorb the target nucleic acid. The magnetic particles are fixed to the bottom surfaces of the amplification wells 34 by a magnetic force generating unit (not shown), and the remaining solution is removed by the dispenser unit 2 and disposed of through a waste liquid port (not shown). Further, the cleaning liquid stored in the amplification wells 34 in the line C is moved to the line B by the dispenser unit, and the magnetic particles are cleaned with the cleaning liquid. The magnetic particles are again fixed to the bottom surfaces of the amplification wells 34 by the magnetic force generating unit (not shown), and the used cleaning liquid is removed and disposed of.
At this stage, it is desirable to replace the pipette chips in order to prevent mixing of impurities, such as nucleic acids other than the target nucleic acid, into the purified target nucleic acid.
By using the dispenser unit with new pipette chips attached thereto, the elution solution stored in amplification wells in the line D is poured into the line B. Due to the elution solution, the target nucleic acid that has been adsorbed to the magnetic particles is liberated into the elution solution. The magnetic particles are fixed to the bottom surfaces of the amplification wells 34 by the magnetic force generating unit (not shown), and the liquid containing the liberated target nucleic acid is sucked by the dispenser unit 2, whereby the purification step is completed.
After the completion of the purification step, the sucked target nucleic acid is mixed with the 2nd PCR reagent contained in the amplification wells 34 in the line E, and a temperature cycle is applied thereto by the thermal cycler (not shown). This causes the 2nd PCR to proceed. In the 2nd PCR, exclusively the target nucleic acid is amplified. After the temperature cycle has been applied, the nucleic acid amplification step is all completed.
After the completion of the nucleic acid amplification step, the procedure advances to the hybridization step. The hybridization reaction unit 4 includes the stage fixing member 41 and the hybridization stage 42 mounted thereon. The hybridization stage 42 is movable in the Y-direction with respect to the stage fixing member 41. The hybridization plate 43 and the reaction cassette 45 can be arranged on the hybridization stage 42. As the hybridization plate 43, it is possible, for example, to employ one cut out from a commercially available 96-well plate of polypropylene as shown in
The nucleic acid solution having undergone the 2nd PCR is moved by the dispenser unit 2 to the hybridization wells 44 containing the hybridization reagent. When the nucleic acid solution is mixed with the hybridization reagent, the resultant mixture solution is transferred to the reaction cassettes 45. The reaction cassettes 45 are reaction cassettes which have reaction chambers where DNA microarrays are fixed, with hybridization reaction being effected within the reaction chambers. The reaction cassettes 45 are designed so that the target nucleic acid can be detected by utilizing the hybridization reaction. As the reaction cassettes 45 of this embodiment, it is possible to use well-known biochemical reaction cassettes, so a detailed description of the inner structure thereof will be omitted here.
As already stated above, when the reaction cassettes 45 are not set at predetermined positions in the cassette holders 50, futile standby time is generated when the processing in each step of the nucleic acid is stopped. To solve this problem, the detector first detects whether the reaction cassettes 45 are set in the cassette holder 50, and the dispenser unit and the nucleic acid amplification unit are controlled based on a detection signal output from the detector.
Actually, when the detector does not detect the reaction cassettes 45 set at predetermined positions in the cassette holder 50, the amplification unit and the dispenser unit operate up to the nucleic acid amplification step but do not advance to the hybridization step. The dispenser unit stops and an alarm indicator issues an alarm. The alarm indicator may be a lamp that is lighted, or one using voice. There are no particular limitations regarding the specific construction of the alarm indicator. With this construction, the user of the nucleic acid automatic examining device of the present invention can quickly ascertain stopping of the device. By accurately setting the reaction cassettes at predetermined positions, stopping of the dispenser unit 2 is detected by the detector, and control is effected so as to re-start the operation of the dispenser unit based on the detection signal therefrom. The control of the dispenser unit is conducted based on the detection signal issued upon detection of the reaction cassette 45 immediately before transition to the hybridization step.
In the following, a method of detecting whether the reaction cassettes 45 are set at the accurate positions in the nucleic acid automatic examining device of this embodiment will be described.
To effect hybridization reaction, it is necessary for the reaction cassettes 45 to be held in intimate contact with a temperature adjuster. For that purpose, it is desirable for the reaction cassettes 45 to be individually separated from each other so that they allow equalization in conformity with the surface of the temperature adjuster with a certain degree of freedom within the cassette holder 50. Further, when optically detecting the hybrid of the target nucleic acid by the detection unit, it is necessary to perform focusing and tilt adjustment.
As the light emitting element 72, it is possible to use, for example, a light emitting diode. As the light receiving element 73, it is possible to use, for example, a photo transistor. There are no particular limitations regarding the reflection plate 70 as long as it is formed of a material capable of reflecting light emitted from the light emitting element.
If even a single cassette is left unattached, the voltage level of the circuit as the detection result is H. To actually install the detection circuit of
While in the example of
The method of detecting the reaction cassettes 45 is not restricted to the optical methods as described above. For example, it would be also possible to detect the attachment of the reaction cassettes 45 to the cassette holder 50 by an electrical detection sensor through conduction when electric contacts provided on both of them come into contact with each other, or by a mechanical switch.
In the nucleic acid automatic examining device of this embodiment, the hybridization stage 42 is mounted on the stage fixing member 41 and is movable in the Y-direction. In the case of cassette detection utilizing this construction, a set of light emitting element 72 and light receiving element 73 are arranged on the main body of the examining device. In this case, after moving the hybridization stage 42 to the front side to set the cassette holder 50, it is possible to detect the reaction cassettes 45 one by one while moving to the detecting position, thus detecting whether all the reaction cassettes 45 have been attached.
In the nucleic acid automatic examining device according to the present invention, it is possible to automate the examination through computer control of each operation according to a pre-set program. The specific setting of each operation may be effected as appropriate through programming according to the kind of target nucleic acid to be examined, the construction of the detection system, etc.
While in the above embodiment the present invention is applied, by way of example, to a nucleic acid automatic examining device which has reaction steps such as nucleic acid amplification through PCR and hybridization reaction, and which uses a DNA microarray, this should not be construed restrictively.
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.
This application claims the benefit of Japanese Patent Application No. 2006-130297, filed May 9, 2006, which is hereby incorporated by reference herein in its entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-130297 | May 2006 | JP | national |
