JP2004337137A - Automatic nucleic acid extraction and automatic nucleic acid extractor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an automatic nucleic acid extraction and an automatic nucleic acid extractor that can automatically carry out the process in a short time with simplified steps and simplified devices through the whole process from the nucleic acid extraction to the purity determination of the recovered nucleic acids. <P>SOLUTION: A pestle crashes the sample and removes the nucleic acids by adsorbing the nucleic acid with magnetic supports and a plurality of tank type of extraction vessels each of which includes nucleic acids-extracting solvent which includes the magnetic supports, the washing solvent and eluent solvent are used to extract nucleic acids from the sample, isolate the nucleic acids and recovers them by washing and eluting and simultaneously determining the concentration and purity of the extracted nucleic acids instantaneously. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a nucleic acid extraction method and a nucleic acid extraction device for extracting a nucleic acid from various large amounts of a nucleic acid-containing specimen, for example, a biological sample using a magnetic carrier, and measuring the concentration and purity of the nucleic acid.
[0002]
[Prior art]
In recent years, in the field of genetic testing, in addition to biochemical testing, molecular biology techniques such as PCR (Polymerase Chain Reaction) have been increasingly used, and genomic DNA extraction is an essential step prior to testing. It is becoming. Conventional methods for extracting genomic DNA include techniques such as the phenol / chloroform method and the alkali method. However, the use of a harmful organic solvent, its operability, and the time spent spend it on researchers. is the current situation.
[0003]
Conventionally, various devices for extracting nucleic acids from various specimens containing nucleic acids have been proposed. For example, Japanese Patent Application Laid-Open No. Hei 3-125972 discloses an apparatus which uses an industrial articulated robot to prevent virus infection and increase the efficiency of the extraction process. Further, for example, Japanese Patent Application Laid-Open No. 4-131076 describes an apparatus for improving nucleic acid recovery efficiency from a small amount of blood or the like. Further, for example, Japanese Patent Application Laid-Open No. 9-47278 describes an apparatus employing a filter system using a vacuum pump. Furthermore, Japanese Patent Application Laid-Open No. 11-215978 discloses an apparatus which aims to automate nucleic acid extraction requiring complicated operations based on the structure of an extraction container suitable for a nucleic acid extraction method using a magnetic carrier without fear of contamination. .
[0004]
On the other hand, in the market, mini-prep DNA purification kits for the purpose of plasmid purification, whose simplicity and performance have been proven, are marketed by various companies.
[0005]
By the way, usually, the nucleic acid extraction operation includes erythrocyte hemolysis, leukocyte enrichment, cell membrane decomposition, and nucleic acid purification, and the operations common to the extraction by the above-described apparatus are injection operation of a sample or a solution, mixing operation for reaction, and solidification operation. It is performed by a liquid separation operation, an unnecessary component liquid removal operation or a nucleic acid recovery operation. The purity and concentration of the nucleic acid after the extraction are separately measured by a spectrophotometer.
[0006]
[Problems to be solved by the invention]
However, in the solid-liquid separation operation during the extraction operation process using the above-described apparatus, there are devices having a centrifugal separation or filter separation configuration, but it is necessary to use a device such as a centrifuge or a vacuum pump in the automation device. However, there have been problems such as an increase in size, complexity, and cost of the apparatus.
[0007]
Further, according to the above-described apparatus, the operation of dispensing the magnetic carrier that binds the nucleic acid to the sample (eg, blood) and the nucleic acid in the extraction container is performed by the operator in advance as a preparation stage for operating the apparatus. In consideration of its safety, it has to be carried out in clean pliers or the like, which is extremely inefficient and inferior in the simple and quick required for the entire process from nucleic acid extraction to recovery and purity test.
[0008]
Furthermore, according to the miniprep DNA purification kit described above, it is pointed out that the extraction operation is complicated when a small amount of sample is used. Further, the measurement of the purity and the concentration of the nucleic acid after the extraction had to be separately measured by a spectrophotometer, which was inconvenient. In this respect, the efficiency was low and the simple and quick operation was inferior.
[0009]
An object of the present invention is that the entire process from nucleic acid extraction from a sample to measurement of the purity of the recovered nucleic acid can be automatically processed in a short time by a simplified extraction method and a simplified extraction device. An object of the present invention is to provide an automatic nucleic acid extraction method and an automatic nucleic acid extraction device.
[0010]
[Means for Solving the Problems]
In order to achieve the above object, in the automatic nucleic acid extraction method according to claim 1, in the automatic nucleic acid extraction method for extracting nucleic acid from a sample containing nucleic acid using a magnetic carrier, different reagent solutions enter and are adjacent to each other. Among the extraction vessels composed of a plurality of divided tanks, the processing member coated with the sample is inserted into the tank in which the reagent liquid and the magnetic carrier are mixed, and the sample is crushed and crushed by the processing member. The nucleic acid isolated from the sample is bound to the magnetic carrier, then the processed member is magnetized, the magnetic carrier is adsorbed to the processed member, and then the nucleic acid bound to the magnetic carrier on the processed member is Wash in a tank containing a different reagent solution, further elute the nucleic acid of the magnetic carrier in a tank containing a different reagent solution, then release the processed member from magnetization, remove the high-purity nucleic acid, and remove the nucleic acid collection tube. Equipped tank Recovered, is characterized by measuring simultaneously, the nucleic acid concentration and nucleic acid purity in the tube that is recovered.
[0011]
(Action)
Since the entire process from nucleic acid extraction to recovery and nucleic acid purity measurement is automatically processed by a simplified extraction method, nucleic acid can be separated from the sample in a short time, and nucleic acid can be extracted and recovered in a short time. The concentration and purity can be measured instantaneously.
[0012]
According to a second aspect, in the automatic nucleic acid extraction method according to the first aspect, the washing and elution of the nucleic acid performed in the tanks containing different reagent solutions are further performed a plurality of times in the adjacent tank. I have.
[0013]
(Action)
By performing further washing and elution of the nucleic acid a plurality of times in a tank adjacent to the tank in which the nucleic acid has been washed and eluted, a nucleic acid having a higher concentration and purity can be extracted from the magnetic carrier on the processed member.
[0014]
According to a third aspect of the present invention, in the automatic nucleic acid extraction method according to the first aspect, the magnetization or demagnetization of the processing member is performed by a magnetized medium inserted into and removed from the processing member.
[0015]
(Action)
The processing member is magnetized by inserting the magnetized medium into the processing member, and the processing member adsorbs the magnetic carrier to which the nucleic acid is bound and moves to each of the compartments of the extraction container, washing and eluting the nucleic acid, In addition, the magnetization of the processed member is released by detaching the magnetized medium from the processed member, and high-purity nucleic acids are separated and recovered in a tank having a collection tube of an extraction container, thereby performing conventional nucleic acid extraction. The intermediate step can be omitted without using a harmful organic solvent, and the nucleic acid extraction operation can be performed safely and the extraction time can be reduced. Further, since the magnetic carrier is used as a carrier for nucleic acid extraction in the nucleic acid extraction process, the entire process from nucleic acid extraction to nucleic acid purity measurement can be automated, and the processing speed of the entire process can be improved.
[0016]
According to a fourth aspect of the present invention, in the automatic nucleic acid extraction method according to the first aspect, the crushing and pulverization of the sample are performed by ultrasonic vibration of the processing member in the tank and crushing and dispersing processing of vertical movement.
[0017]
(Action)
The sample adhered to the processing means is efficiently crushed and pulverized by the crushing dispersion processing by the ultrasonic vibration and the vertical movement of the processing means, and is uniformly dispersed in the tank, and furthermore, the precipitation of the magnetic carrier is prevented. Promotes the binding of the nucleic acid isolated from the nucleic acid to the magnetic carrier.
[0018]
An automatic nucleic acid extraction device according to claim 5, wherein in the automatic nucleic acid extraction device for extracting nucleic acid from a sample containing nucleic acid using a magnetic carrier, a tank containing a reagent solution and a magnetic carrier mixed with a reagent solution different from each other. Providing an extraction container comprising a plurality of divided tanks in which adjacent tanks are divided adjacently, wherein the extraction container has processing means for crushing and crushing the attached sample, and magnetizing means for adsorbing and removing the magnetic carrier. A nucleic acid collection tube provided in one of the plurality of tanks for washing nucleic acids bound to the magnetic carrier, eluting the nucleic acids in the magnetic carrier, releasing the magnetizing means, and providing high-purity nucleic acids in one of the plurality of tanks A processing member having means for collecting is provided, and a measuring device for measuring nucleic acid concentration and nucleic acid purity in the nucleic acid collection tube is provided, and the processing member can be moved up and down, and the processing member and Serial are characterized by being intermittently movable either the workpiece or the extraction vessel such that the dividing tank of the plurality tank is matched positions.
[0019]
(Action)
The extraction container is composed of a plurality of divided tanks in which a tank in which a reagent solution and a magnetic carrier are mixed and a tank in which different reagent solutions are mixed are arranged adjacent to each other, and the processing member is a processing means for crushing and pulverizing the sample. And a magnetizing means for adsorbing and removing the magnetic carrier, a collecting means for collecting the high-purity nucleic acid in the nucleic acid collecting tube, and a measuring means for measuring the nucleic acid concentration and purity of the nucleic acid collecting tube, so that the processing member can be vertically moved. In addition, since either the processing member or the extraction container is configured to be intermittently movable, the entire apparatus is simplified, and the automation of the apparatus is facilitated. Therefore, the operability is easy so that the entire process can be automatically operated by a simple button operation without the complicated and inconvenient operability of a commercially available miniprep DNA extraction kit and without the need for complicated nucleic acid extraction. The nucleic acid can be extracted within a short time.
[0020]
According to a sixth aspect of the present invention, in the automatic nucleic acid extraction apparatus according to the fifth aspect, in the extraction container, the plurality of tanks radially divided from an axis are formed in a circular shape adjacent to each other in a circumferential direction. It is characterized by being done.
[0021]
(Action)
Since the extraction container or the processing member is intermittently rotated about the axis of the circular extraction container to perform nucleic acid extraction, the amount of movement of the extraction container or the processing member is small, and the nucleic acid extraction can be performed quickly. Can be reduced in volume and size, so that the entire apparatus can be made compact.
[0022]
According to a seventh aspect of the present invention, in the automatic nucleic acid extraction apparatus according to the fifth aspect, the magnetizing means in the processing member is made of a magnetization medium that can be inserted into and removed from the processing member.
[0023]
(Action)
The magnetized medium is inserted into and removed from the processing member to magnetize or demagnetize the processing member. Therefore, the magnetic carrier to which the nucleic acid isolated from the sample is bound by the processing means can be easily absorbed and removed, and then moved to the next sorting tank. Can be collected. Therefore, nucleic acid extraction can be performed quickly, easily, and safely as compared with the organic solvent means used in conventional nucleic acid extraction.
[0024]
According to an eighth aspect of the present invention, in the automatic nucleic acid extraction apparatus according to the fifth aspect, the processing means in the processing member comprises an ultrasonic vibration driving unit, a vertical driving unit, and a sample attachment unit. I have.
[0025]
(Action)
In the tank containing the reagent solution and the magnetic carrier, the sample adhered to the processing member is crushed and dispersed by the ultrasonic vibration and the vertical movement of the processing member, and can be efficiently crushed and crushed to be uniformly dispersed.
[0026]
According to a ninth aspect, in the automatic nucleic acid extraction apparatus according to the fifth or sixth aspect, a plurality of the extraction containers and the processing members are installed in combination.
[0027]
(Action)
Since the extraction container has a circular shape and the amount of movement of the extraction container or the processing member is small, even if a set of the extraction container and the processing member are installed in the apparatus in a plurality, the installation area does not take up much space. By means of the member, nucleic acid can be rapidly and simultaneously extracted from many samples.
[0028]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described. FIG. 1 is a schematic view showing an automatic nucleic acid extraction apparatus according to an embodiment of the present invention, wherein (a) is an external view thereof, and (b) is a perspective view showing an arrangement of an extraction container with a lid opened. It is. FIG. 2 is a perspective view showing an extraction container and a processing member of the automatic nucleic acid extraction device of FIG. 1, (a) is a perspective view showing a processing member coated with a sample above the extraction container, b) is a perspective view showing the processing member inserted above the extraction container and into which the magnetized medium is inserted, (c) is a perspective view showing the processing member inserted into the extraction container equipped with the collection tube, and (d) is a recovery. FIG. 4 is an explanatory perspective view showing an embodiment in which a tube is irradiated with ultraviolet and visible light to measure a nucleic acid concentration and purity.
FIG. 3 is a plan view of an extraction container in the automatic nucleic acid extraction device of FIG. FIG. 4 is a flowchart of the automatic nucleic acid extraction method.
[0029]
As shown in FIGS. 1 (a) and 1 (b), the automatic nucleic acid extraction device includes an apparatus main body 1 and a lid 2, and the apparatus main body 1 includes an operation panel section 3 on which operation buttons are arranged, An ultraviolet-visible spectrophotometer 17 as a measuring instrument for measuring the concentration and purity of nucleic acids, particularly nucleic acids, is provided inside the extraction container section 4 and the operation panel section 3.
[0030]
As illustrated in FIG. 2, the extraction container unit 4 includes a bottomed extraction container 5 and a processing member 6 (hereinafter, referred to as a pestle) such as a pestle provided above the extraction container 5.
[0031]
As shown in FIG. 3, the extraction container 5 is composed of tanks divided radially around the rotation axis core 7 in the radial direction, for example, into eight equal parts. It is composed of a division tank 8, a second division tank 9, a third division tank 10, 4a to 4n division tanks 11, 12, 13, 14, and a fifth division tank 15.
[0032]
The extraction container 5 has a circular shape having an appropriate thickness as its shape, and is made of a material conventionally used, for example, glass, polypropylene, polyvinyl chloride, or silicon coating on the surface. Various things such as a general resin can be adopted. Among them, polypropylene is particularly preferred because it does not adsorb nucleic acids as target components.
[0033]
The rotation shaft core 7 is connected to an intermittent rotation drive unit (not shown), and rotates the extraction container 5 intermittently in the direction shown by the arrow B so that each of the division tanks 8 to 15 of the extraction container 5 matches the position of the pestle 6. Making it possible.
[0034]
The pestle 6 is located above the extraction container 5 and has an ultrasonic vibration drive unit and a vertical motion drive unit, which will be described later, in the upper part, and a bar-shaped projection 6a in the lower part. 9, the third sectioning tank 10, the 4a to 4n sectioning tanks 11, 12, 13, 14 and the lower section are formed so as to be inserted into the fifth sectioning tank 15. The five-section tanks 8, 9, 10, 11, 12, 13, 14, and 15 can be inserted and removed.
[0035]
The rod-shaped protrusion 6a of the pestle 6 is preferably made of a polypropylene resin to which a sample or a nucleic acid component does not penetrate, and a long hole is formed in the center of the axis of the rod-shaped protrusion 6a in the axial direction of the material. The lower part of the rod-shaped projection 6a is finished to have a surface with a higher friction coefficient so that the sample A can be applied or adhered easily.
[0036]
Further, the rod-shaped protrusion 6 a is ultrasonically vibrated and vertically moved to break and pulverize the sample A at the bottom of the liquid reservoir of the first division tank 8.
[0037]
That is, the ultrasonic vibration of the rod-shaped projection 6a is oscillated by the electromagnet rod 20 connected to an ultrasonic transducer (not shown), and the vertical movement of the rod-shaped projection 6a is applied to a vertical movement drive source (not shown). The connected vertical bar 21 vertically moves up and down with the pestle 6.
[0038]
The electromagnet rod 20 is formed so as to be able to be inserted and removed through an elongated hole of the rod-shaped projection 6a so as to provide ultrasonic vibration and exhibit a function as a magnetization medium in the rod-shaped projection 6a.
[0039]
The upper and lower operating rods 21 are removably fitted to the rotary shaft core 7 of the extraction container 5, the lower end is connected to a vertical driving source, and the upper end is attached to and detached from the pestle 6 by a suction means such as a magnet. It is joined as possible.
[0040]
In the extraction vessel 5, the nucleic acid extraction solution and the magnetic carrier are mixed in the first sectioning tank 8, the washing liquid is put in the second sectioning tank 9 adjacent to the first sectioning tank 8, and further the second sectioning tank is put in the second sectioning tank. The eluate is placed in the adjacent third compartment 10.
[0041]
The nucleic acid extraction solution preferably contains a reducing agent such as 2-mercaptoethanol or dithiothreitol for the purpose of denaturing and inactivating proteins contained in the sample, particularly ribonuclease.
[0042]
The 4a to 4n division tanks 11 to 14 adjacent to the third division tank 10 include a tank 11 containing a cleaning liquid as in the second division tank 9 and a tank 12 containing an eluate as in the third division tank 10. A tank 13 containing a washing solution and a tank 14 containing an eluate are adjacent to each other.
[0043]
Further, a fifth sectioning tank 15 adjacent to the tank 14 among the 4a to 4n sectioning tanks and adjacent to the first sectioning tank 8 contains an eluate, and has a high level capable of being desorbed into the liquid reservoir. A nucleic acid collection tube 16 having a purity is mounted.
[0044]
The nucleic acid extraction solution is a solution having a function of destroying cells containing nucleic acids in a sample, exposing the nucleic acids, and binding the nucleic acids to a magnetic carrier.
[0045]
The washing solution may be any solution that does not promote the elution of nucleic acids from the magnetic carrier and that prevents binding of proteins, saccharides, and lipids to the solid phase. It is preferable to wash with 100% ethanol.
[0046]
The eluate may be any one that promotes elution of the nucleic acid from the magnetic carrier to which the nucleic acid is bound. For example, water or a TE buffer (10 mM Tris-hydrochloride, 1.0 mM EDTA; pH 8.0) is preferable.
[0047]
The nucleic acid collection tube 16 is made of quartz or glass in the visible region as a material, and is preferably rectangular or conical with a downward tapering to form a treatment liquid collection part.
[0048]
The ultraviolet-visible spectrophotometer 17 performs a spectrophotometric analysis using light having a wavelength in the ultraviolet to visible region (200 nm to 780 nm) that causes electron energy transition of a molecule, for example, and measures the absorbance of the nucleic acid in the collection tube 16. The numerical value obtained by measuring the concentration and purity of the nucleic acid is incorporated in the apparatus main body 1 so as to be displayed on the display window 3a of the operation panel section 3.
[0049]
Next, a method for performing a nucleic acid extraction method using the above-described automatic nucleic acid extraction apparatus will be described with reference to the flowchart shown in FIG.
[0050]
Before starting the automatic nucleic acid extraction apparatus, nucleic acids in which a magnetic carrier is mixed as a reagent solution in each of the first to fifth compartments 8, 9, 10, 11, 12, 13, 14, 15 of the extraction container 5 The extraction container 5 containing the extraction solution, the washing solution and the eluate is set in the apparatus main body 1.
[0051]
First, as shown in FIG. 4, the sample A is applied to the vicinity of the tip of the bar-shaped projection 6 a of the pestle 6, and the pestle 6 is installed on the apparatus main body 1 so as to be located above the extraction container 5.
[0052]
This sample is a sample containing nucleic acids, and includes biological materials derived from animals such as whole blood, serum, plasma, urine, saliva, body fluid, and other non-animal biological materials such as plants and microorganisms. It also includes cells and cultured cells separated from these biological materials.
[0053]
Further, the nucleic acid in the present invention means DNA or RNA, and the DNA includes double-stranded DNA, single-stranded DNA, plasmid DNA, genomic DNA, cDNA and the like. In addition, the RNA includes endogenous RNAs derived from organisms that produce these biological materials, in addition to RNAs derived from foreign parasites such as viruses, bacteria, and fungi, and t-RNA, m-RNA, r-RNA and the like.
[0054]
Then, the operation button of the operation panel unit 3 is pressed, and the automatic operation of nucleic acid extraction is started.
[0055]
When the operation button is pressed and started, the vertical driving means such as a motor (not shown) is driven to move down the pestle 6 and insert the rod-shaped projection 6 a into the first sectioning tank 8 of the extraction container 5.
[0056]
When the rod-shaped projection 6a of the pestle 6 inserted into the first division tank 8 approaches the liquid reservoir bottom of the first division tank 8, the electromagnet rod 20 is inserted. Then, the ultrasonic vibrator is activated to vibrate the electromagnet bar 20 and resonate the bar-shaped projection 6a.
[0057]
Further, the vertical movement drive source is activated, and the vertical operating rod 21 for attracting and joining the pestle 6 by the magnet starts vertical movement to move the rod-shaped projection 6a up and down.
[0058]
As a result, the rod-shaped projection 6a is further subjected to ultrasonic vibration in the up-and-down motion, and the crushing and dispersing action of the sample A using the nucleic acid extraction solution and the magnetic carrier as a medium starts at the bottom of the liquid reservoir of the first division tank 8.
[0059]
The nucleic acid is isolated from the sample crushed and pulverized by the crushing and dispersing action, and the nucleic acid is bound to the surface of the magnetic carrier mixed in the nucleic acid extraction solution in the first compartment 8.
[0060]
At this time, the sample in the first sorting tank 8 is efficiently crushed and crushed by the ultrasonic vibration wave and the crushing and dispersing action of the vertical motion of the rod-shaped projection 6a of the pestle 6, and is uniformly dispersed, and at the same time, The magnetic carrier is also uniformly dispersed and does not settle at the bottom of the liquid reservoir of the first sectioning tank 8. Therefore, the nucleic acid isolated from the sample A is bound to the magnetic carrier with a high probability.
[0061]
When the crushing and crushing of the sample A is completed while the rod-shaped projection 6a is inserted into the first sectioning tank 8, the electromagnet rod 20 is magnetized, and the surface of the rod-shaped projection 6a is also magnetized as a magnetic medium. . Due to the magnetization of the rod-shaped protrusion 6a, the magnetic carrier to which the nucleic acid is bound is adsorbed on the surface of the rod-shaped protrusion 6a.
[0062]
The magnetic carrier is, for example, a magnetic carrier for nucleic acid binding which is made of magnetic silica particles containing a superparamagnetic metal oxide, binds nucleic acids, and enables solid-liquid separation by a magnetic field.
[0063]
Next, the vertical operating rod 21 is pushed upward so as to disengage the rod-shaped projection 6a of the pestle 6 inserted into the first sectioning tank 8 from the first sectioning tank 8, and the extraction container 5 is rotated intermittently to form the second section. The sorting tank 9 is moved to a position below the rod-shaped projection 6a.
[0064]
The rod-shaped projection 6a adsorbing the magnetic carrier to which the nucleic acid is bound is lowered and inserted into the second division tank 9. The rod-shaped projection 6a inserted into the second division tank 9 is suspended in the washing liquid in the second division tank 9 and washed without eluting the nucleic acid of the magnetic carrier adsorbed to the rod-shaped projection 6a, that is, unnecessary. Remove proteins and contaminants.
[0065]
When the removal of the unnecessary components is completed, the rod-shaped projection 6a is detached upward from the second division tank 9, the extraction container 5 is intermittently rotated in one direction, and the third division tank 10 is moved to the rod-shaped projection 6a of the pestle 6. Move to lower position.
[0066]
The rod-shaped projection 6a of the pestle 6 on which the magnetic carrier that binds the nucleic acid from which the unnecessary components have been removed is adsorbed is lowered and inserted into the third sectioning tank 10. The rod-shaped projections 6a inserted into the third sectioning tank 10 are suspended in the eluate in the third sectioning tank 10, and promote the elution of nucleic acids from the nucleic acid of the magnetic carrier adsorbed on the rod-shaped projections 6a, that is, the elution. .
[0067]
When the elution of the nucleic acid is completed, the pestle 6 is detached upward from the third sectioning tank 10, the extraction vessel 5 is intermittently rotated in one direction, and the sectioning tank 11 of the 4a to 4n sectioning tanks is rod-shaped. It moves to a position below the protrusion 6a.
[0068]
Then, the rod-shaped protrusion 6a of the pestle 6 adsorbing the magnetic carrier that binds the eluted nucleic acid is lowered and inserted into the division tank 11 of the 4a to 4n division tanks. The rod-shaped protrusion 6a inserted into the separation tank 11 of the 4th to 4n separation tanks is suspended in the cleaning liquid in the separation tank 11, and cleans the nucleic acid of the magnetic carrier adsorbed on the rod-shaped protrusion 6a.
[0069]
Further, the rod-shaped projection 6a of the pestle 6 is separated from the division tank 11 of the 4a to 4n division tanks, inserted into the division tank 12 adjacent to the division tank 11, and suspended in the eluate in the division tank 12. To elute the nucleic acids on the magnetic carrier.
[0070]
Further, in the division tanks 13 and 14 of the 4th to 4n division tanks as well, similarly to the division tanks 11 and 12, the rod-shaped projections 6a of the pestle 6 are put into the tank 13 containing the washing liquid and then the eluate. The suspension is inserted and suspended in the order of the tank 14, and the nucleic acid on the magnetic carrier is washed and eluted.
[0071]
Then, the rod-shaped projection 6a of the pestle 6 detached from the division tank 14 of the 4a to 4n division tanks is inserted into the fifth division tank 15 provided with the nucleic acid recovery tube 16, and the eluate in the fifth division tank 15 is inserted. During the suspension, the electromagnet bar 20 is pulled out. At this time, the rod-shaped protrusion 6a is released from the magnetization of the electromagnet rod 20, separates high-purity nucleic acid from the surface of the pestle 6 together with the magnetic carrier, and pushes the separated high-purity nucleic acid into the collection tube 16 to collect.
[0072]
Next, the upper and lower operating rods 21 to be suction-bonded to the pestle 6 are pulled down strongly, and the upper and lower operating rods 21 are detached from the pestle 6 to put the pestle 6 on the extraction container 5.
[0073]
Then, the pestle 6 which has become free is picked up, and after removing the rod-shaped protrusion 6a of the pestle 6 from the extraction container 5, the measuring instrument removes light having a wavelength of from ultraviolet to visible part (200 nm to 780 nm) in the collection tube 16. And instantaneously measure the concentration and purity of the nucleic acid. That is, the absorbance of the nucleic acid obtained by the light irradiation is converted into an electric quantity, the output signal is further processed, and the measured value of the concentration and purity of the nucleic acid is instantaneously displayed on the display window 3a of the operation panel unit 3. Digitally displayed.
[0074]
As described above, the invention made by the present inventor has been specifically described based on the embodiment. However, the present invention is not limited to the above-described embodiment, and can be variously modified without departing from the gist thereof. Needless to say.
For example, the extraction vessel 5 is intermittently rotated to move the first to fifth compartments 8 to 15 so as to match the position of the pestle 6. The pestle 6 may be intermittently rotated by intermittently rotating the pedestal 21 and the pestle 6 may be moved to each of the first to fifth divisional tanks 8 to 15.
[0075]
【The invention's effect】
The effects obtained by typical aspects of the invention disclosed in the present application will be briefly described as follows.
[0076]
In other words, the entire process from nucleic acid extraction of a sample to recovery of high-purity nucleic acid and measurement of the purity of the recovered nucleic acid is automatically processed by a simplified extraction method and a simplified extraction device, so that The nucleic acid can be extracted from the sample in a short time, and the concentration and the purity can be instantaneously measured from the nucleic acid recovered in a short time. Therefore, the entire process is automatically operated by simple button operation without the need for complicated parameter input and troublesome operation as in the commercially available mini-prep DNA extraction kit, and without the need for complicated extraction time. Nucleic acid can be extracted and measured in a short time.
Further, since the magnetic carrier is used as the nucleic acid extraction carrier in the nucleic acid extraction process, the nucleic acid can be efficiently separated in a short time. Therefore, no harmful organic solvent which has been conventionally used is used, so that the intermediate step can be omitted and the extraction and recovery of nucleic acid can be performed safely.
Furthermore, since the sample is crushed and crushed by the crushing and dispersing action of the processing member in the extraction container to uniformly separate nucleic acids and the like, a conventionally used centrifugal separator and a vacuum pump are not required, and the entire apparatus is simplified. Can be compact.
Furthermore, since the structure of a set of extraction devices composed of an extraction container and a processing member has been simplified, even if a plurality of sets of the extraction devices are installed in the device main body, the installation space is not increased, and a compact device is used. Nevertheless, high-purity nucleic acid extraction can be performed quickly and efficiently from a plurality of different samples by using the plural sets of extraction devices.
[Brief description of the drawings]
FIG. 1 is a schematic view showing an automatic nucleic acid extraction apparatus according to an embodiment of the present invention, wherein (a) is an external view thereof, and (b) is a perspective view showing an arrangement of an extraction container with a lid opened. It is.
FIG. 2 is a perspective view showing an extraction container and a processing member of the automatic nucleic acid extraction device of FIG. 1, and FIG. 2 (a) is a perspective view showing a processing member above a extraction container and coated with a sample. , (B) is a perspective view showing the processing member inserted above the extraction container and having the magnetized medium inserted therein, (c) is a perspective view showing the processing member inserted into the extraction container equipped with the collection tube, (d) FIG. 3 is an explanatory perspective view showing an embodiment in which a collection tube is irradiated with light having a wavelength in the range from ultraviolet to visible to measure nucleic acid concentration and nucleic acid purity.
FIG. 3 is a plan view of an extraction container in the automatic nucleic acid extraction device of FIG.
FIG. 4 is a flowchart of an automatic nucleic acid extraction method.
[Explanation of symbols]
A sample
1 Device body
2 Lid
3 Operation panel
3a Display window
4 Extraction container
5 Extraction container
6 pestle (processed member)
6a rod-shaped protrusion
7 Rotating shaft core
8 First sorting tank
9 Second division tank
10 Third division tank
11-14 4th to 4n division tanks
15 fifth division tank
16 Nucleic acid collection tube
17 UV-visible spectrophotometer (measuring instrument)
20 Electromagnet rod (magnetic medium)
21 Vertical operating rod

Claims (9)

In an automatic nucleic acid extraction method for extracting a nucleic acid from a sample containing a nucleic acid using a magnetic carrier, the reagent solution and the magnetic carrier are contained in an extraction container comprising a plurality of division tanks containing different reagent solutions and divided adjacent to each other. The processing member coated with the sample is inserted into the mixed tank, the sample is crushed and pulverized by the processing member, and at the same time, the nucleic acid isolated from the sample is bound to the magnetic carrier. The member is magnetized, the magnetic carrier is adsorbed to the processing member, and then the nucleic acid bound to the magnetic carrier on the processing member is washed in a tank containing a different reagent solution, and further a tank containing a different reagent solution The nucleic acid of the magnetic carrier is eluted in the step, then the magnetization of the processing member is released, and high-purity nucleic acid is recovered in a tank provided with a nucleic acid recovery tube, and simultaneously the nucleic acid concentration and the nucleic acid purity in the recovered tube To Automated nucleic acid extraction method, characterized by a constant. 2. The automatic nucleic acid extraction method according to claim 1, wherein the washing and elution of the nucleic acid performed in the plurality of compartments containing different reagent solutions are further performed a plurality of times in a plurality of adjacent compartments. Automatic nucleic acid extraction method characterized by the following 2. The automatic nucleic acid extraction method according to claim 1, wherein the magnetic carrier is adsorbed and removed from the processing member by a magnetic medium inserted into and removed from the processing member. 2. The automatic nucleic acid extraction method according to claim 1, wherein the crushing and crushing of the sample are performed by crushing and dispersing the ultrasonic vibration and vertical movement of the processing member in the plurality of compartments. . In an automatic nucleic acid extraction apparatus for extracting nucleic acid from a sample containing nucleic acid using a magnetic carrier, a plurality of separation tanks in which a tank containing a reagent solution and a magnetic carrier and a tank containing a different reagent solution are adjacently partitioned. Provided with an extraction container comprising processing means for crushing and pulverizing the adhered sample, and magnetizing means for adsorbing and removing the magnetic carrier, for washing the nucleic acid bound to the magnetic carrier. And a processing member having means for eluting the nucleic acid of the magnetic carrier, releasing the magnetizing means and collecting a high-purity nucleic acid into a nucleic acid collection tube provided in one of the plurality of tanks, A measuring device for measuring the nucleic acid concentration and the nucleic acid purity in the collection tube is provided, the processing member can be moved up and down, and the processing member and each of the plurality of tanks coincide with each other. Automated nucleic acid extraction apparatus characterized by comprising in the intermittent movable either the workpiece or the extraction vessel such that. 6. The automatic nucleic acid extraction device according to claim 5, wherein the extraction vessel is formed by forming the plurality of tanks radially divided from an axis in a circular shape adjacent to each other in a circumferential direction. Automatic nucleic acid extraction device. 6. The automatic nucleic acid extraction device according to claim 5, wherein the magnetizing means in the processing member comprises a magnetization medium that can be inserted into and removed from the processing member. 6. The automatic nucleic acid extraction device according to claim 5, wherein the processing means in the processing member comprises an ultrasonic vibration drive unit, a vertical drive unit, and a sample attachment portion. . The automatic nucleic acid extraction device according to claim 5 or 6, wherein a plurality of the extraction containers and the processing members are installed in combination.

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