The present invention relates to a nucleic acid analyzer.
A nucleic acid analyzer is known as a device for analyzing base sequences of a deoxyribonucleic acid (DNA). The nucleic acid analyzer is a device for analyzing the base sequences of DNA by denaturing DNA fragments into a single strand, using the single strand as a model, extending a nucleic acid attached with a fluorescent label by one base each time, and sequentially capturing a fluorescent image. When analysis is performed, a substrate in which a flow path is provided in a plate made of a partially or entirely transparent material is prepared, and colonies containing a plurality of cloned DNA fragments denatured into a single strand are fixed in a reaction field provided in the flow path of the substrate. In order to enable identification of four types of nucleotides (adenine, cytosine, guanine, thymine) forming DNA for the colonies containing the plurality of DNA fragments, a reagent that fluorescently labels each base of DNA, a reagent that cleans the flow path, and the like are alternately sent. The base sequences of DNA can be sequentially analyzed by capturing, as a fluorescent image, a process in which the colonies are restored to contain the double-stranded DNA fragments.
In analysis of base sequences of DNA in the nucleic acid analyzer, first, for the colonies containing the plurality of DNA fragments fixed in the reaction field provided in the flow path of the substrate, a reagent required for each reaction process is selected from a plurality of types of reagents and is sent to the flow path of the substrate, and thus each base of the colonies containing the DNA fragments in the flow path is fluorescently modified. Further, the colonies containing the fluorescently modified DNA fragments are observed. The base sequences are analyzed as described above.
In such a nucleic acid analyzer, it is considered to increase an area of the reaction field by providing a plurality of flow paths on the substrate in order to improve throughput. A nucleic acid analyzer using a substrate having a plurality of flow paths is reported in PTL 1.
PTL 1 describes a configuration of a nucleic acid analyzer that sends a reagent to a substrate having a plurality of flow paths. However, the nucleic acid analyzer in PTL 1 requires a branched flow path structure for connecting reagents with a plurality of substrate flow paths in order to introduce the reagents into the plurality of flow paths, and the substrate flow paths and the branched flow path are both required to be replaced with the reagents, and thus a reagent consumption amount increases.
In addition, when it is considered to use substrates of different sizes in order to cope with a plurality of types of throughput (for example, a substrate having a half number of flow paths is used in order to implement nucleic acid analysis at half throughput), a branched flow path that is not connected to the substrate is generated in the branched flow path structure. A branched flow path portion that is not connected to the substrate becomes a dead volume, and a remaining drug solution or bubbles cannot be replaced with the reagents, thereby causing contamination. Therefore, it is not desirable to use substrates of different sizes.
In view of the above problems, an object of the invention is to provide a nucleic acid analyzer capable of mounting a plurality of types of substrates having different numbers of flow paths while preventing an increase in reagent consumption amount due to a branched flow path structure.
In the nucleic acid analyzer according to the invention, a first substrate includes an inlet portion connected to an introduction path, a first outlet portion connected to a first discharge path, a second outlet portion connected to a second discharge path, a first flow path configured to guide a reagent from the inlet portion to the first outlet portion, a second flow path configured to guide the reagent from the inlet portion to the second outlet portion, and a branch portion configured to branch the reagent from the inlet portion to the first flow path and the second flow path, in which the first flow path and the second flow path are connected to each other only at the branch portion.
According to the nucleic acid analyzer of the invention, by providing a branch point of the flow paths in the substrate, it is possible to minimize the number of introduction flow paths and eliminate an increase in reagent consumption amount due to an increase in the number of flow paths of the substrate. Further, by stabilizing the number of inlet portions of the substrate regardless of the number of flow paths on the substrate, it is possible to mount a plurality of types of substrates having different numbers of flow paths without generating a dead volume in the same device configuration.
The substrate 107 includes at least two flow paths 101 and 102, at least one inlet portion 103, at least two outlet portions 104 and 105, and at least one flow path branch point 106. The flow paths 101 and 102 are used to fix colonies containing DNA fragments and analyze base sequences of DNA. Each reagent is introduced into the substrate 107 from the inlet portion 103. The reagent is discharged from the outlet portions 104 and 105. The flow path branch point 106 is a location at which a flow path is branched into flow paths (a merging point of the flow paths). The flow path 101 connects between the inlet portion 103 and the outlet portion 104, and the flow path 102 connects between the inlet portion 103 and the outlet portion 105.
The introduction flow path 108 is connected to the inlet portion 103. The reagent is introduced into the substrate 107 via the introduction flow path 108 and the inlet portion 103. The discharge flow path 109 is connected to the outlet portion 104, and the discharge flow path 110 is connected to the outlet portion 105. The reagent aspiration mechanism 111 aspirates the reagent flowing through the discharge flow path 109, and the reagent aspiration mechanism 112 aspirates the reagent flowing through the discharge flow path 110. The control unit 113 controls the reagent aspiration mechanism 111, and the control unit 114 controls the reagent aspiration mechanism 112. The imaging mechanism 115 captures a fluorescent image of the colonies containing the DNA fragments. Reagents are contained in the reagent containers 116. The reagent selection mechanism 117 selects a reagent to be introduced into the substrate 107 by selectively connecting to any one of the reagent containers 116.
In
As described above, in the substrate having a plurality of flow paths, the number of the introduction flow paths 108 can be minimized by providing the flow path branch point 106 on the substrate. Therefore, even when a branched flow path structure is provided, it is not necessary to replace a branched flow path on a device side with a reagent as in the related art, and thus it is possible to prevent excessive reagents from being consumed.
As shown in
In the nucleic acid analyzer 100 according to the first embodiment, the substrate 107 includes the flow path branch point 106, and a reagent is branched from the flow path branch point 106 to each flow path on the substrate 107. In other words, the flow path branch point 106 is disposed on a substrate 107 side. Accordingly, it is sufficient to provide the minimum number of introduction flow paths 108 on the device side (if one inlet portion 103 is provided, one introduction flow path 108 is also provided). Therefore, it is not necessary to replace the branched flow path on the device side with the reagent as in the related art, and thus it is possible to prevent a reagent consumption amount.
In the nucleic acid analyzer 100 according to the first embodiment, even when the substrate 107 is replaced with the substrate 301, no branched flow path that is not connected to the substrate 301 is generated, and thus unnecessary dead volume does not occur between the reagent containers 116 and the inlet portion 303. Therefore, it is possible to prevent contamination of the reagent or the bubbles remaining in the branched flow path portion that is not connected (not used) to the substrate 301 as in the related art.
In the second embodiment, the control unit 404 (a) opens the first solenoid valve 401 when a desired amount of reagent is to be introduced into the flow path 101, and controls the reagent aspiration mechanism 403 via the control unit 404 of the reagent aspiration mechanism in a state where the second solenoid valve 402 is closed, and (b) opens the second solenoid valve 402 when a desired amount of reagent is to be introduced into the flow path 102, and controls the reagent aspiration mechanism 403 in a state where the first solenoid valve 401 is closed.
Specifically, when the substrate 107 is used, the first solenoid valve 401 is opened to introduce the reagent, and then the second solenoid valve 402 is opened to introduce the reagent; when the substrate 301 is used, only the first solenoid valve 401 is opened to introduce the reagent.
The nucleic acid analyzer 100 according to the second embodiment can reduce the number of reagent aspiration mechanisms and the number of control units as compared with the first embodiment. Accordingly, it is possible to simplify a structure particularly after the discharge flow paths.
In
The block 501 includes a plurality of branched flow paths connected to the inlet portion 103. When the first reagent container 505 is connected to the substrate 107, the reagent is aspirated by the reagent aspiration mechanisms 111 and 112 in a state where the first reagent selection solenoid valve 502 is opened and the second reagent selection solenoid valve 503 and the third reagent selection solenoid valve 504 are closed. Similarly, when the second reagent container 506 and the third reagent container 507 are connected to the substrate 107, a desired reagent is selectively introduced into the substrate 107 by opening the corresponding second reagent selection solenoid valve 503 or the corresponding third reagent selection solenoid valve 504. The same applies to a case of using the substrate 301.
In the block 501, the branched flow paths from reagent inflow ports merge at a merging point 606 and reach the outflow port 602 through which the reagents flow out to the substrate. Although three reagent inflow ports are provided in
When placed on the stage 705, the substrate 107 has a size and a shape to cover both the grooves 701 and 702. Similarly, when the substrate 107 is used, the substrate 107 is placed on the grooves 701 and 702, and the pumps 703 and 704 aspirate the substrate 107 via the grooves 701 and 702, respectively. Accordingly, the substrate 107 can be fixed on the stage.
As in the fourth embodiment, a mechanism that fixes the substrate is divided into a plurality of (two grooves 701 and 702 in
Although two pumps 703 and 704 are shown in
In
The casing 901 has a planar size slightly larger than the substrate 301. Accordingly, when the substrate 301 is accommodated in the casing 901 and placed on the stage 705, the discharge flow path 110 on a not-used side can be closed. If the discharge flow path 110 is opened for a long time (for example, from about several hours to about several days) without being used, dust or the like may clog the inside of the discharge flow path 110. By attaching the casing 901, such clogging can be prevented even when the substrate 107 is replaced with the substrate 301.
The invention is not limited to the embodiments described above, and includes various modifications. For example, the above-described embodiments are described in detail for easy understanding of the invention, and the invention is not necessarily limited to those including all the configurations described above. Further, a part of a configuration according to one embodiment can be replaced with a configuration according to another embodiment, and the configuration according to another embodiment can be added to the configuration according to one embodiment. A part of a configuration according to each embodiment can be added, deleted, or replaced with another configuration.
In the above embodiments, an imaging range of the imaging mechanism 115 when the substrate 107 is used is larger than that when the substrate 301 is used. Therefore, when the substrate is moved within the imaging range of the imaging mechanism 115, a moving range is larger when the substrate 107 is used. For example, when the substrate is placed on the stage 705 and moved with the stage 705, a moving range of the stage 705 is larger when the substrate 107 is used. Alternatively, if the imaging mechanism 115 can scan an imaging range or an imaging location, the imaging range is larger when the substrate 107 is used.
In the embodiments described above, the control units 113, 114, and 404 can be implemented by hardware such as a circuit device in which functions of the control units are implemented, and can be implemented by an arithmetic device executing software in which the functions of the control units are implemented.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/JP2020/022025 | 6/3/2020 | WO |