JP2012024000A - Apparatus for analyzing nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus for analyzing nucleic acid that has a small dead volume between a reagent bottle and a flow cell without using a valve between the reagent bottle and the flow cell.SOLUTION: The apparatus for analyzing nucleic acid includes: a flow cell 2 including an injection port injecting solution, a reaction area, a flow path for connecting the injection port and the reaction area; and a discharge port for discharging the solution; a nozzle 7 injecting the solution to the flow cell 2; a nozzle conveying unit 8 for driving the nozzle 7; and a detection unit 9 detecting the reagent in the flow cell 2.

Description

  The present invention relates to a nucleic acid analyzer.

  New techniques for determining the base sequences of DNA and RNA have been developed.

  In the currently used method using electrophoresis, a cDNA fragment sample synthesized in advance by reverse transcription reaction from a DNA fragment or RNA sample for sequencing is prepared, and a dideoxy reaction is performed by a well-known Sanger method. After that, electrophoresis is performed, and the molecular weight separation development pattern is measured and analyzed.

  On the other hand, in recent years, a method has been proposed in which a large number of DNA fragments as samples are fixed to a substrate and the sequence information of many fragments is determined in parallel.

  A technique is known in which fine particles are used as a carrier for carrying DNA fragments, and PCR is performed on the fine particles. Thereafter, fine particles carrying PCR-amplified DNA fragments are put in a plate provided with a number of holes in which the hole diameter is adjusted to the size of the fine particles, and read by the pyro sequence method.

  In addition, a technique of performing PCR on a microparticle using a microparticle as a carrier for supporting a DNA fragment is already known. Thereafter, the microparticles are dispersed and fixed on the glass substrate, an enzyme reaction (ligation) is performed on the glass substrate, a fluorescent dye is incorporated, and the sequence information of each fragment is obtained.

  Furthermore, a technique for fixing a number of DNA probes having the same sequence on a substrate is known. In addition, after cutting the DNA sample, a DNA probe sequence and an adapter sequence of a complementary strand are added to the end of each DNA sample fragment. By hybridizing these on the substrate, the sample DNA fragments are immobilized on the substrate randomly one molecule at a time. In this case, the DNA elongation reaction does not occur on the substrate, and after loading the substrate with the fluorescent dye, the unreacted substrate is washed and the fluorescence is detected to obtain the sequence information of the sample DNA.

  As described above, a method for determining the sequence information of a large number of fragments in parallel by fixing a large number of nucleic acid fragment samples on a substrate has been developed and put into practical use.

  In these methods, since a sequence reaction is performed on the substrate, it is necessary to supply many types of solutions onto the substrate and to raise and lower the temperature on the substrate. For example, a technique is known in which after a solution containing an anchor primer is first supplied, the substrate is heated and cooled, then a cleaning solution is supplied onto the substrate, and unreacted anchor primer on the substrate is cleaned. Thereafter, a solution for ligation reaction and a solution for decomposing the complex of anchor primer and fluorescent primer are supplied. It is also necessary to raise and lower the temperature and to wash with a washing liquid during the supply of these solutions.

  As apparatuses for performing these analyses, Patent Documents 1 and 3 disclose apparatuses that supply a reagent solution stored in a container to a flow cell that is a reaction field via a tube.

JP 2009-532031 A Special table 2008-506969 JP 2008-151772 A

  However, in the apparatus through the tube as described above, the capacity of the tube between the reagent container and the flow cell becomes a dead volume, and the amount of reagent necessary for one analysis increases, so there is a problem that the running cost of the analysis becomes high. .

  Patent Document 2 discloses a method of using a valve between a reagent bottle and a flow cell, but there is a problem that the apparatus configuration becomes complicated in order to realize a valve opening / closing mechanism.

  An object of the present invention is to provide a nucleic acid analyzer that has a small dead volume between a reagent bottle and a flow cell and does not use a valve between the reagent bottle and the flow cell.

  The nucleic acid analyzer of the present invention includes an injection port for injecting a solution, a reaction region, a flow channel connecting the injection port and the reaction region, a flow cell having a discharge port for discharging the solution, a nozzle for injecting the solution into the flow cell, The nozzle drive part which drives a nozzle, and the detection part which detects the sample in a flow cell are provided.

  A dead volume between the reagent bottle and the flow cell is small, and a nucleic acid analyzer that does not use a valve between the reagent bottle and the flow cell can be provided.

It is a figure for demonstrating an example of a structure of the nucleic acid analyzer of this invention. It is a figure for demonstrating an example of a structure of the flow cell of this invention. It is a figure for demonstrating an example of a structure of the holder unit of this invention. It is a figure for demonstrating an example of a structure of the holder unit of this invention.

The above and other novel features and effects of the present invention will be described below with reference to the drawings.
Here, specific embodiments will be described in detail in order to understand the present invention, but the present invention is not limited to the contents described here. Moreover, each structure can be combined suitably and this specification is also disclosing about the said combination form.

  FIG. 1 shows an example of a nucleic acid analyzer according to the present invention. The nucleic acid analyzer 1 fixes a flow cell 2 and a flow cell 2 in which flow paths are formed, a holder unit 3 for adjusting the temperature of the flow cell 2, a stage unit 4 for moving the flow cell 2 and the holder unit 3, a plurality of reagents and washing A reagent container 5 containing water, a liquid feeding unit 6 as a driving source for sucking and injecting the reagent contained in the reagent container 5 and injecting the reagent into the flow cell 2, the reagent container 5 and the flow cell 2 are actually used when the reagent is aspirated / discharged. A nozzle 7 for accessing the nozzle 7, a nozzle transport unit 8 for transporting the nozzle 7, a detection unit 9 for observing a sample fixed to the flow cell 2, and a waste liquid container 10 for storing waste liquid from the flow cell 2.

  The operation of the nucleic acid analyzer 1 will be described with reference to FIG. First, the flow cell 2 holding the DNA to be measured is installed in the holder unit 3. Next, the nozzle 7 accesses the reagent container 5 and sucks the reagent by the liquid feeding unit 6. The nozzle 7 is transported to the upper surface of the flow cell 2 by the nozzle transport unit 8 and injects a reagent from the injection port 11 into the flow cell 2. Then, the holder unit 3 reacts by adjusting the temperature of the DNA fragment and the reagent contained in the flow cell 2.

  In order to observe the reacted DNA fragment, the flow cell 2 is moved so that the detection region to be observed by the stage unit 4 can be observed by the detection unit 9, and the fluorescence of a plurality of DNA fragments in the detection region is applied by applying excitation light. Is detected. After detection, the operation of moving the flow cell 2 minutely and detecting in the same manner is repeated a plurality of times. When the observation is completed in all the detection regions, the washing water accommodated in the reagent container 5 is sucked by the liquid feeding unit 6 and injected into the flow cell 2 to wash the flow path of the flow cell 2. Further, the DNA sequence can be read by repeating the operation of injecting and detecting another reagent a plurality of times. In nucleic acid analysis, it is necessary to identify four types of bases. Methods for detecting dyes labeled in four or more types are widely known.

  In the present embodiment, since the dispensing is performed through the nozzle 7, the dead volume from the reagent container 5 to the injection port (described later) 11 can be reduced.

  The flow cell 2 of the present invention will be described with reference to FIG. On the upper surface of the flow cell, there are provided an injection port 11 and a discharge port 12 which are holes for injecting and discharging the solution. The flow cell 2 has a hollow structure for passing a solution, and the hollow structure includes a flow path part 13 and a detection part (reaction region) 14 for preventing heating. As shown in FIG. 2, the flow path portion 13 is narrower than the detection portion 14 when viewed from above.

  By reducing the volume of the flow path unit 13, the dead volume of the reagent solution is reduced. By increasing the area of the detection unit 14, the number of beads immobilized with the DNA to be measured can be increased, and the number of DNA fragments that can be analyzed at a time can be increased.

  The material of the upper surface portion of the flow cell 2 is preferably a material having high light transmittance for fluorescence detection. Examples of such materials include inorganic materials such as glass and sapphire, and resin materials such as polymethyl methacrylate resin, polycarbonate resin, and cycloolefin resin. The material of the lower surface portion of the flow cell 2 is preferably a material having high thermal conductivity. Examples of such a material include an inorganic material such as silicon and glass, a metal material such as SUS, and a highly thermally conductive resin material blended with a carbon fiber and an inorganic feeler. In FIG. 2, two sets of the injection port 11, the flow path unit 13, the detection unit 14, and the discharge port 12 are arranged, but the present invention is not limited to this, and may be one or three or more. However, by providing a plurality of sets, the injection port 11, the flow path unit 13, the detection unit 14, and the discharge port 12, it is possible to inject liquid into other detection units simultaneously when one detection unit is detected. . In addition, detection and a reaction for detection can be performed simultaneously, and throughput can be increased.

  The holder unit 3 of the present invention will be described with reference to FIG. The holder unit 3 includes a temperature adjustment element 16 that enables temperature adjustment, a heat plate 15 that contacts the temperature adjustment element 16, a heat absorption plate 17, and a clamp 18. As shown in FIG. 3, a temperature adjustment element 16 is provided below the heat plate 15. With such a configuration, it is possible to realize a sequence reaction on a substrate that requires a plurality of temperature increases and decreases.

  Specifically, a part of the lower side of the detection part 14 of the flow cell 2 is in contact with the heat plate 15, and a part of the lower side of the flow path part 13 of the flow cell 2 is in contact with the heat absorbing plate 17. At the time of heating for the sequence reaction, the heat in the flow path portion 13 is radiated to the outside through the heat absorbing plate 17, so that the temperature rise in the flow path portion 13 can be suppressed and evaporation from the injection port 11 is performed. Can be prevented.

  The clamp 18 is provided with a clamp 18 having a U-shaped cross section in order to enhance the adhesion between the heat absorbing plate 17 and the lower part of the flow cell 2. The amount of heat transfer from the lower part of the flow cell 2 to the heat absorption plate 17 can be increased by the clamp 18. The material of the heat absorbing plate 17 is preferably a metal material having high thermal conductivity, small dimensional change during clamping, and high durability. Examples of such a material include aluminum and copper. In order to improve the adhesion between the flow cell 2 and the endothermic plate 17, a silicon sheet having high thermal conductivity may be used between the flow cell 2 and the endothermic plate 17. By providing such a clamp 18, it is possible to prevent liquid leakage from the adhesion interface.

  Moreover, the holder unit of this invention is demonstrated using FIG. 4 as a different form from FIG. The difference from FIG. 3 is that the flow cell 2 is stored in the holder 19 and incorporated in the cell holder unit. In this embodiment, the liquid is injected from the nozzle 7 through the injection port 20 in the holder 19. In the form of FIG. 3, when the thickness of the upper surface portion of the flow cell is thin, the contact area between the nozzle and the upper surface portion of the flow cell is small, so that liquid leakage easily occurs.In the form of FIG. 4, the contact area between the nozzle and the injection port is Since it does not depend on the thickness of the upper surface portion of the flow cell, the thickness of the upper surface portion of the flow cell can be reduced. 3 and 4, two sets of the injection port 11, the flow path unit 13, the detection unit 14, and the discharge port 12 are arranged. However, the present invention is not limited to this, and one or three or more sets may be used. Good.

A nucleic acid analysis method using the nucleic acid analyzer of the present invention will be described. After fragmenting and amplifying the DNA to be measured, beads having the DNA fragment to be measured immobilized thereon are prepared using an emulsion PCR method. Subsequently, beads are immobilized in the flow cell of the present invention using an acrylic gel. Next, the reaction device on which the beads are immobilized is installed in the nucleic acid analyzer shown in FIG. Finally,
(A) Hybridization of anchor primer (b) Ligation of fluorescent primer (c) Fluorescence detection (d) Removal of anchor primer and fluorescent primer (e) Repeat steps (a) to (d) to determine the base sequence .

DESCRIPTION OF SYMBOLS 1 Nucleic acid analyzer 2 Flow cell 3 Holder unit 4 Stage unit 5 Reagent container 6 Liquid feeding unit 7 Nozzle 8 Nozzle conveyance unit 9 Detection unit 10 Waste liquid container 11 Injection port 12 Discharge port 13 Flow path part 14 Detection part 15 Heat plate 16 Temperature control Element 17 Endothermic plate 18 Clamp 19 Holder 20 Injection port

Claims (14)

A flow cell having an inlet for injecting a solution, a reaction region, a flow path connecting the inlet and the reaction region, and an outlet for discharging the solution;
A nozzle for injecting the solution into the flow cell;
A nozzle drive unit for driving the nozzle;
A nucleic acid analyzer comprising: a detection unit that detects a sample in the flow cell.   The nucleic acid analyzer according to claim 1, wherein the flow path is narrower than the reaction region.   The nucleic acid analyzer according to claim 1, wherein the flow path and the reaction region are adjusted separately.   The nucleic acid analyzer according to claim 1, wherein an endothermic plate that absorbs heat from the flow path is disposed.   The nucleic acid analyzer according to claim 4, wherein the endothermic plate and the flow cell are fixed to each other.   The nucleic acid analyzer according to claim 1, further comprising a temperature adjusting unit that adjusts the temperature of the reaction region.   The nucleic acid analyzer according to claim 6, wherein the temperature adjustment unit includes a heat plate and a temperature adjustment element.   The nucleic acid analyzer according to claim 6, wherein the temperature adjusting unit and the flow cell are fixed to each other.   The nucleic acid analyzer according to claim 1, wherein the flow cell is fixed to a flow cell holder.   The nucleic acid analyzer according to claim 9, wherein the solution is injected from an opening provided in the flow cell holder and enters the flow cell through the injection port.   The nucleic acid analyzer according to claim 1, wherein the flow cell has a plurality of flow paths, and each of the flow paths has an inlet, a reaction region, and an outlet.   The nucleic acid analyzer according to claim 1, wherein the detection performed by the detection unit is fluorescence detection.   The nucleic acid analyzer according to claim 1, further comprising a drive unit that drives the flow cell, and switching a detection region in a reaction region detected by the detection unit by driving the flow cell.   The nucleic acid analyzer according to claim 1, wherein the flow cell is configured by stacking a member made of a light transmissive material and a member having high thermal conductivity.

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