JP2005073578A - Nucleic acid extraction apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid extraction apparatus enabling the objective nucleic acid to be extracted in high efficiency through simplifying the extraction step and low in running cost. <P>SOLUTION: The nucleic acid extraction apparatus includes a flat plate-like stationary table 1, a control unit 2 fixed at the center of the stationary table 1, a waste liquid port 3 for retrieving waste liquid provided on one side of the control unit 2, a Y-axis table 4 provided on the other side, a Y-axis driving unit 5 mounted on the Y-axis table 4, and a sample retrieving section 6 placed on the Y-aix driving unit 5. In this apparatus, there is provided a collecting section 8 on a turntable 7, the collecting section 8 being conically trapezoidal, i.e. being of such shape as to be removed perpendicularly by a given height from the apex of a conical form so that the top face is horizontal, and the inside of the collecting section 8 is embedded with a circularly columnar conductor 9. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a nucleic acid extraction apparatus that extracts nucleic acids such as DNA and RNA from biological samples such as whole blood, plants, cells, hair, and yeast.

Conventional nucleic acid extraction apparatuses extract nucleic acids with high efficiency using magnets and minute magnetic particles (see, for example, Patent Document 1).
FIG. 6 shows a conventional nucleic acid extraction apparatus.
In FIG. 6, the conventional nucleic acid extraction apparatus includes a reaction tank portion 41 for collecting nucleic acids such as DNA and RNA contained in a biological sample such as whole blood on the surface of fine silica particles, and an unnecessary other than nucleic acid. A drain cup portion 42 for recovering a solution containing components, a recovery tube 43 for recovering an extract containing a target nucleic acid, a reaction tank portion 41, a support plate 44 for supporting the drain cup portion 42 and the recovery tube 43, A discharge pipe 45 for discharging a solution containing unnecessary components or an extract containing nucleic acid from the reaction tank portion 41 to the drain cup portion 42 or the recovery tube 43, and connection of the discharge pipe 45 and the drain cup portion 42 or the recovery tube 43. Rotating member 46 for switching between, and the rotating member 46 are integrally formed, and pressure is applied to the reaction tank portion 41 from the chemical solution injection passage or from the outside. And a obtain passage 47.

FIG. 7 shows a schematic diagram of use of a container for nucleic acid extraction.
In the chemical solution injection process of FIG. 7A, a syringe or a micropipette is inserted into the entrance of the through pipe 47, and the chemical solution is injected. Next, in the mixing step (b), mixing of the magnetic silica particles, the biological sample containing the nucleic acid, and the nucleic acid extraction solution in the reaction tank portion 41 is performed by the vertical movement of the ring electrode 49. In the separation step (c), the magnetic carrier 51 (magnetic silica particles) to which the nucleic acid is bound is separated from the liquid by a magnetic field, and is applied to the bottom of the reaction vessel portion containing the biological sample, the solution for nucleic acid extraction, and the liquid comprising the magnetic carrier 51. By bringing the magnet 50 closer, only the magnetic carrier 51 is collected at the bottom of the reaction tank portion 41 and separated from an unnecessary component liquid such as a nucleic acid extraction solution. In the unnecessary component liquid discharging step (d), a dispensing nozzle 48 connected to a pressure pump (not shown) is set in the through pipe 47, and when pressure is applied from the pressure pump in this state, the reaction tank portion The liquid in 41 is pushed out along the flow path of the discharge pipe 45 and discharged to the drain cup portion 42. In the recovery step (e), when the rotating member 46 is rotated 180 degrees, the discharge pipe 45 is connected to the recovery tube 43 from the drain cup portion 42. Next, when the inside of the reaction tank part 41 is pressurized from the dispensing nozzle set in the through pipe 47, the liquid containing the nucleic acid in the reaction tank part 41 is pushed out to the collection tube and collected in the tube. Yes. On the other hand, the gas filled in the drain cup portion 42 is discharged out of the system from the exhaust nozzle 52.
As described above, the conventional nucleic acid extraction apparatus performs extraction with high efficiency by forming a processing unit in which the inside of the apparatus is a closed system and contamination of nucleic acids such as DNA and RNA contained in a biological sample is prevented.

Japanese Patent Laid-Open No. 11-187862 (page 3-4, FIGS. 1 and 6)

However, in the above-described conventional nucleic acid extraction apparatus, a very small amount of nucleic acid is collected from the biological sample on the surface of the magnetic silica particles, and then TE (1.0 mM Tris) with the magnet 50 brought close to the reaction vessel portion 41 is obtained. -Hydrochloric acid salt, 1.0 mM EDTA; pH 8.0) A buffer solution or sterilized water is injected to elute nucleic acids such as DNA and RNA from the surface of the magnetic silica particles to separate them from unnecessary components other than nucleic acids. However, since it is necessary to discard the container containing the magnetic silica particles that are no longer necessary after the nucleic acid is separated, the operation process becomes complicated and the extraction process takes time. In addition, when dealing with a large number of biological samples, it is necessary to sequentially inject new magnetic particles, which causes problems such as an increase in running cost.
Furthermore, there has been a problem that the nucleic acid that has entered the precipitate such as denatured protein in the separation step is not properly recovered and is discarded as it is. In addition, in order to improve the recovery rate of nucleic acid, even when silica particles are excessively injected, the elution reagent does not reach the nucleic acid between the magnetic silica particles and remains on the surface of the silica particles. was there.
Accordingly, the present invention has been made in view of such problems, and it is intended to provide a nucleic acid extraction apparatus with a low running cost that can simplify the extraction process and extract a target nucleic acid with high efficiency. Objective.

The first configuration of the present invention collects a waste liquid provided in a flat plate-shaped fixing base, a control unit fixed to a central portion of the fixing base, and one side portion of the control unit on the fixing base. Placed on the Y-axis drive unit, the Y-axis table provided on the other side of the control unit on the fixed base, the Y-axis drive unit provided on the Y-axis table, and the Y-axis drive unit A nucleic acid extraction apparatus including a sample collection unit, wherein a cylindrical turntable is joined to an upper part of the control unit, and a truncated cone is formed on the turntable so that an upper surface thereof is horizontal. Provided with a collecting part shaped like a vertical height removed from the top of the truncated cone, and a cylindrical conductor embedded in the collecting part. It is.
According to a second configuration of the present invention, in the first configuration, the collection unit is provided with only one minute channel formed in a concave shape along the side surface from the center of the upper surface of the frustoconical shape, and the waste liquid A cleaning port is provided at a position rotated by 90 degrees from the port, and the sample recovery section is composed of a plurality of sample tubes.

The third configuration of the present invention includes a fixed base, a frame fixed to both sides of the fixed base, an X-axis table supported by the frames on both sides, and an X-axis attached to the X-axis table. Nucleic acid extraction apparatus comprising: a drive unit; a Z-axis drive unit attached to the X-axis drive unit; a Y-axis table fixed on the fixed base; and an extraction container held on the Y-axis table The arm is attached to the Z-axis drive unit, an extraction head for extracting nucleic acid is fixed to the tip of the arm, and a current-carrying unit is fixed on the fixing base, A circular container is placed on the upper surface of the energization section, and a rectangular collection base having an inclined upper surface is fixed in the container.
According to a fourth configuration of the present invention, in the third configuration, a conductor that is smaller than the collection table and has the same shape is embedded in the collection table, and a positive direct current is provided in the energization unit. A power supply for applying the voltage is connected.
According to a fifth configuration of the present invention, in the third or fourth configuration, the collection base is a polymer resin selected from polytetrafluoroethylene, polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, and polymethyl methacrylate. It is manufactured by at least one kind of polymer resin, or has a metal material surface coated with an insulating polymer resin.
According to a sixth configuration of the present invention, in any one of the third to fifth configurations, the energizing unit includes an ultrasonic transducer that minutely vibrates the metal plate at a constant cycle, and any one of the side surfaces of the metal plate via the insulating unit. It is characterized by being joined to the crab.

According to the present invention, the following effects can be obtained.
According to the first configuration of the present invention, nucleic acid extraction is performed on a biological sample such as whole blood, a plant, or a cell by applying a positive DC voltage to the conductor to positively charge the surface of the collection unit. From the suspension produced by adding the solution, only the target nucleic acid is retained on the surface of the collection part, while unnecessary denatured protein precipitates and nucleic acid extraction solutions are recovered from the waste liquid outlet. can do.
According to the second configuration of the present invention, the target nucleic acid separation / purification time can be shortened and a continuous nucleic acid extraction process can be performed.
According to the third configuration of the present invention, precipitates such as denatured proteins present in the extraction solution can be easily washed away by separating and collecting nucleic acids using gravity and electrostatic action. Only the nucleic acid collected on the collection table can be collected in the extraction container.
According to the 4th structure of this invention, only the target nucleic acid can be collected on a collection stand, without replacing | exchanging an extraction container by charging only on the surface of a collection stand plus. it can.
According to the fifth configuration of the present invention, by suppressing specific adsorption / residue of coexisting components such as denatured protein, the extraction process is simplified, and the target nucleic acid extraction accuracy is stably maintained. can do.
According to the sixth configuration of the present invention, precipitates such as denatured proteins can be removed from the collection table in a short time by applying minute vibrations to the collection table.

  Hereinafter, specific embodiments of the present invention will be described with reference to FIGS.

FIG. 1 is a configuration diagram illustrating a nucleic acid extraction apparatus according to Embodiment 1 of the present invention.
In FIG. 1, a control unit 2 is fixed at the center of a fixed base 1, a waste liquid port 3 for collecting waste liquid is provided around the control unit 2, and a Y-axis table 4 is fixed. A Y-axis drive unit 5 is attached on the Y-axis table 4, and a sample recovery unit 6 is also placed on the Y-axis drive unit 5. Further, a metal turntable 7 is joined to the upper part of the control unit 2, and a polymer resin collection part 8 is attached to the upper surface of the turntable 7.

Next, the operation will be described.
First, biological samples such as whole blood, plants, cells, hair, and yeast are placed in polypropylene (PP) microtubes, and then chaotropic substances such as guanidine, potassium iodide, sodium perchlorate, and urea. Buffers such as Tris-hydrochloride, sodium tetraboron-hydrochloric acid, potassium dihydrogen phosphate-sodium tetraboron, anionic surfactants and nonionic surfactants such as Triton X, Tween 20, and N-lauroyl sarcosine sodium Then, a mixed solution composed of a thiol reducing agent such as 2-mercaptoethanol or dithiothreitol is added to prepare a nucleic acid extraction solution. Next, a positive (+) potential is applied to the rotary table 7 from the control unit 2 fixed to the fixed table 1, and the surface of the collecting unit 8 is charged positively via a conductor 9 having good electrical conductivity. be able to.
Therefore, when a nucleic acid extraction solution is dropped little by little on the surface of the collection unit 8 using a micropipette (not shown), a precipitate such as a denatured protein or a reagent solution contained in the nucleic acid extraction solution is collected in the collection unit. 8 flows down along the slope of 8 and is finally collected in the waste liquid port 3 installed on the fixed base 1. On the other hand, the target nucleic acid is a higher-order structure that is negatively charged under physiological conditions due to the presence of a phosphate group (—PO ₃ H) and a sugar hydroxyl group (—OH), which are basic components. It is generally known to form Therefore, only the nucleic acid can be held on the surface of the collection unit 8 by electrostatically bonding the positive charge formed on the surface of the collection unit 8 and the phosphate group or hydroxyl group of the nucleic acid.

FIG. 2 is a plan view of FIG. A concave micro flow channel 10 is formed along the side surface slope from the center of the upper portion of the collecting portion 8, and is rotated 90 degrees to the right from the waste liquid port 3 in a region corresponding to the lower portion of the side surface of the collecting portion 8. A cleaning port 11 is provided. The sample collection unit 6 has a plurality of 1.5 mL to 3 mL polypropylene (PP) sample containers set in the Y-axis direction.
As described above, the control unit 2 rotates the collection unit 90 to the right by 90 degrees, moves the microchannel 10 to the cleaning port 11, and dissolves the reagent components and the like using a micropipettor (not shown). Is dropped on the surface of the collection part 8. Then, residues such as unnecessary reagent components and residual denatured protein can be collected in the cleaning port 11. Next, the microchannel 10 is rotated 90 degrees to the right and moved to the sample tube 12, and a nucleic acid solution (for example, TE buffer) is placed on the upper surface of the collection unit 8 using a micropipette (not shown). When dripping and stopping the supply of electricity from the control unit 2, the nucleic acid can be collected in the sample tube 12 together with the nucleic acid solution. Further, the nucleic acid extraction process from the biological sample can be continuously performed by driving the Y-axis drive unit 5 to move the sample tube 12 and repeating the above-described operation.
Note that it is preferable to use a polymer resin such as polytetrafluoroethylene (PTFE) or polydimethylsiloxane (PDMS) as the material of the collection part 8 because it is necessary to prevent specific adsorption of nucleic acids and the like. .

FIG. 3 is a configuration diagram showing the configuration of the nucleic acid extraction apparatus. In the figure, a frame 22 is fixed to the left and right of a fixed base 21, and an X-axis table 23 is supported by the frame 22, and an X-axis drive unit 24 that moves a Z-axis drive unit 25 in the X direction on the X-axis table 23. Is provided. The Z-axis drive unit 25 is equipped with an arm 28 to which an extraction head 29 for extracting nucleic acid is attached at the tip. On the other hand, a Y-axis table 26 and an energization unit 30 are fixed on the fixed base 21, an extraction container 27 is set on the Y-axis table 26, and a circular container 31 is placed on the upper surface of the energization unit 30. The collection base 32 having an inclined upper surface is fixed inside the container 31.
In FIG. 4, a conductor 33 that is smaller than the collector 32 and has the same shape is inserted into the collector 32, and the energization unit 30 is connected to the positive (+) side of the power supply 34. ing.
In this way, when direct current electricity is supplied from the power supply 34 to the energization unit 30, the surface of the collection base 32 is positively charged via the conductor 33. Next, a pump (not shown) mounted on the Z-axis drive unit 25 is operated to extract a nucleic acid extraction solution in which a predetermined amount of an extraction reagent is added to a biological sample such as whole blood, plant, or cell. After being collected in 29, it is dropped on the upper surface of the collection table 32.

Then, since the upper surface of the collection table 32 is inclined, precipitates such as denatured proteins and extraction reagents will flow down along the inclined surfaces due to gravity. On the other hand, nucleic acids such as DNA and RNA are composed of phosphate, sugar and base. As the action of a phosphate group (-PO ₄ H) or sugar a hydroxyl group (-OH), a is in solution anions (-) of becoming a conformation are generally known. Therefore, since direct current electricity is continuously supplied from the power source 34 to the conductor 33 during the nucleic acid extraction process, the water (H ₂ O) in the solution for nucleic acid extraction and the cleaning solution. Even if anionic components such as sodium thiocyanate and SDS (sodium dodecyl sulfate) are present, the surface charge on the collection base 32 is not neutralized, so the collected nucleic acid is collected from the collection base 32. It will not flow down. Therefore, only the nucleic acid can be stably held on the collection table 32.
Further, the metal arm 28 is moved from a control unit (not shown) built in the Z-axis drive unit 25 while operating the Z-axis drive unit 25 to lower the extraction head 29 and bring it close to the surface of the collection base 32. When a positive direct current is applied to the surface, the surface of the extraction head 29 is positively charged. Therefore, when the supply of direct current electricity from the power supply 34 is stopped, the nucleic acid collected on the collection table 32 is attracted to the surface of the extraction head 29 and collected. Next, the X-axis drive unit 24 is driven to move the extraction head 29 to the upper part of the extraction container 27, and then the Z-axis drive unit 25 is driven to lower the extraction head 29 to extract the extraction container 27. The target nucleic acid can be recovered in the nucleic acid dissolving solution by immersing in the nucleic acid dissolving solution (for example, TE buffer) and stopping the supply of electricity to the extraction head 29.

  Furthermore, the collection base 32 is made of a polymer resin such as polytetrafluoroethylene (PTFE), polypropylene (PP), polyethylene (PE), polycarbonate (PC), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), or poly Since the surface of a metal material such as copper or aluminum is coated with an inert polymer resin such as tetrafluoroethylene (PTFE) or polydimethylsiloxane (PDMS), the surface of the collection table 32 is coated with. Coexisting components such as denatured proteins and extraction reagents (components such as guanidine salts, sodium iodide, potassium iodide, sodium thiocyanate, urea, EDTA, Triton X-100, Tris-HCl buffer) do not adsorb and remain. Therefore, simplification of the extraction process can be realized, and the target nucleic acid extraction accuracy can be stably maintained.

FIG. 5 is a diagram illustrating the configuration of the fourth embodiment. In the energization unit 30, an ultrasonic transducer 36 that applies minute vibration is fixed to a metal plate 35 such as copper, aluminum, or SUS with an insulating unit 37 interposed therebetween.
In this way, since minute vibrations generated from the ultrasonic transducer 36 are applied to the collection table 32, precipitates such as denatured proteins contained in the extraction solution are briefly collected from the collection table 32. Can be removed in time. Further, since an insulating portion 37 having a low electrical conductivity formed of a Teflon (PTFE) sheet or nitrile rubber is disposed between the metal plate 35 and the ultrasonic vibrator 36, the metal plate The direct current electricity supplied to 35 is not transmitted to the ultrasonic transducer 36. Therefore, it is possible to prevent the ultrasonic transducer 36 from being electrically deteriorated.

  The present invention simplifies the extraction process, and as a low-cost nucleic acid extraction apparatus capable of extracting a target nucleic acid with high efficiency, from a biological sample such as whole blood, plant, cell, hair, yeast, The present invention can be usefully applied to the field of extracting nucleic acids such as RNA.

It is a block diagram of the nucleic acid extraction apparatus which shows Example 1 of this invention. It is a top view of the nucleic acid extraction apparatus which shows Example 2 of this invention. It is a sectional side view of the nucleic acid extraction apparatus which shows Example 3 of this invention. It is sectional drawing which shows the collection method of the nucleic acid extraction apparatus in Example 3 of this invention. It is a block diagram of the nucleic acid extraction apparatus which shows Example 4 of this invention. It is a sectional side view of the conventional nucleic acid extraction apparatus. It is a schematic diagram of a nucleic acid extraction method using a conventional container for nucleic acid extraction, (a) is an injection step, (b) is a mixing step, (c) is a separation step, (d) is an unnecessary component removal step, (e ) Is a nucleic acid recovery step.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fixing base 2 Control part 3 Waste liquid port 4 Y-axis table 5 Y-axis drive part 6 Sample collection part 7 Rotating stand 8 Collection part 9 Conductor 10 Microchannel 11 Cleaning port 12 Sample tube 21 Fixing base 22 Frame 23 X axis Table 24 X-axis drive unit 25 Z-axis drive unit 26 Y-axis table 27 Extraction container 28 Arm 29 Extraction head 30 Energization unit 31 Container 32 Collection base 33 Conductive unit 34 Power supply 35 Metal plate 36 Ultrasonic vibrator 37 Insulation unit

Claims (6)

A flat plate-shaped fixing table, a control unit fixed to a central part of the fixing table, a waste liquid port provided on one side of the control unit on the fixing table, and a waste liquid port for collecting the waste liquid, on the fixing table A Y-axis table provided on the other side of the control unit, a Y-axis drive unit provided on the Y-axis table, and a sample recovery unit placed on the Y-axis drive unit A nucleic acid extraction apparatus comprising:
A cylindrical turntable is joined to the upper part of the control unit, a truncated cone shape is formed on the turntable, and a certain height is removed from the top of the truncated cone shape in a vertical direction so that the upper surface is horizontal. A nucleic acid extraction apparatus comprising: a collecting portion shaped like a cylinder, and a cylindrical conductor embedded in the collecting portion.   The collection part is provided with only one minute channel formed in a concave shape along the side surface from the center of the upper surface of the frustoconical shape, and provided with a cleaning port at a position rotated 90 degrees from the waste liquid port, The nucleic acid extraction apparatus according to claim 1, wherein the sample recovery unit includes a plurality of sample tubes. A fixed base, a frame fixed to both sides of the fixed base, an X-axis table supported by the frames on both sides, an X-axis drive unit attached to the X-axis table, and the X-axis drive unit A nucleic acid extraction apparatus comprising: a Z-axis drive unit attached to the Y-axis table; a Y-axis table fixed on the fixed table; and an extraction container held on the Y-axis table,
An arm is attached to the Z-axis drive unit, an extraction head for extracting a nucleic acid is fixed to the tip of the arm, an energization unit is fixed on the fixing base, A nucleic acid extraction apparatus, wherein a circular container is placed on an upper surface, and a rectangular collection base having an inclined upper surface is fixed in the container.   A conductor that is smaller than the collection table and has the same shape is embedded in the collection table, and a power supply for applying a positive DC voltage is connected to the energization unit. The nucleic acid extraction apparatus according to claim 3, wherein   The collector is made of at least one polymer resin selected from polytetrafluoroethylene, polypropylene, polyethylene, polycarbonate, polyethylene terephthalate, polymethyl methacrylate, or on the surface of a metal material. 5. The nucleic acid extraction apparatus according to claim 3, wherein the nucleic acid extraction apparatus is coated with an insulating polymer resin.   6. The current-carrying portion is characterized in that an ultrasonic transducer that minutely vibrates the metal plate at a constant cycle is joined to one of the side surfaces of the metal plate via an insulating portion. The nucleic acid extraction apparatus according to any one of the items.

Images