KR100384936B1 - Automatic DNA Purification Apparatus - Google Patents

Abstract

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an automatic nucleic acid purification device, comprising: a solution supply unit 10 including a plurality of solution bottles, a plurality of flow passages 21, a first control valve 22, a plurality of syringes 23, and a second control valve. Dispensing apparatus 20 comprising a third control valve 25 and dispensing needle 26, plate moving means 30 comprising a rack 31 and a chucking arm 32, a vacuum block 41. ), A multi-well plate block, and an autonucleic acid purification apparatus comprising a control unit for controlling their sequential operation.

Description

Automatic DNA Purification Apparatus

The present invention relates to an automated nucleic acid purification apparatus, and more particularly, to an apparatus for separating and purifying nucleic acids from an nucleic acid-containing solution such as a cell culture solution by an automated method.

Separation and purification of nucleic acid from the nucleic acid-containing solution is an essential process required for all processes for genetic manipulation, namely gene amplification, sequencing, gene recombination, and the like. To date, various methods for separating and purifying nucleic acids from biological samples such as blood, sputum, biological tissues, animal cells, plant tissues, E. coli, viruses, electrophoretic gels, and the like are generally known.

For example, the boiling method (holmes, DS and M. Quigley, 1981, Anal. Biochem. 114: 193) and the alkaline lysis method; Birnboim, HC and J. Doly, 1979, Nucleic Acids Res 7: 1513) is known as a method for isolating and purifying plasmid DNA from transformants obtained by transforming E. coli and other bacteria.

In addition, as a method for obtaining high-purity nucleic acid, a method of separating and purifying plasmid DNA by cesium chloride density gradient centrifugation is known, but these methods are not only time-consuming but also detergent-mediated lysis process and protein. It is difficult to be adopted as a commercial method for separating and purifying nucleic acids in large quantities in a short time because it includes an azeolysis step, organic solvent purification and ethanol precipitation step.

Meanwhile, a method of separating and purifying nucleic acids in a nucleic acid-containing solution by adsorbing on a silica or glass surface using a chaotrop salt such as NaI or NaCIO ₄ is known (Marko, MA et al. 1982, A procedure for the largr). scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder.Anal. Biochem. 121: 382-387; Vogeistein.B. et al. 1979. Preparative and analytical purification of DNA from agarose.Proc.Natl.Acad. Sci. USA 76: 615-619). Such chaotrope salt induced adsorption of nucleic acids on silica or glass surfaces is a common method for separating and purifying chromosomal and plasmid DNA from cell lysates (Buffone, GJ, et al. 1991. Clin. Chem. 37., 1945).

The nucleic acid adsorption reaction is performed in a guanidine hydrochloride solution buffered at pH 4 to 5 or in a 4M sodium iodide buffered at pH 7.5 to 8 using silica particle suspension or compressed glass fibers having a surface on which the nucleic acid is adsorbed. Is performed.

Recent genome research, blood testing, crop seed testing, agricultural product quarantine, and microbial environmental testing require equipment to separate and purify DNA from multiple samples at once. Separation and purification of nucleic acids is essential for sequence recognition, gene amplification, cloning, etc. However, the separation and purification of nucleic acids by conventional manual operation requires a lot of time and labor, and the amount of nucleic acid to be purified depends on the skill of the operator. .

Accordingly, although nucleic acid separation and purification apparatuses have been developed to solve these problems, the nucleic acid purification apparatuses developed so far have limited the number of samples that can be processed at one time, and cannot continuously supply a nucleic acid-containing solution. It is impossible to purify, and since disposable dispensing tips are used for the injection of various solutions, a lot of running costs are required, and the filtration and nucleic acid adsorption processes have not been fully automated.

Accordingly, there is a need in the art for the development of an automated nucleic acid purification apparatus that can separate and purify nucleic acids from multiple samples at the same time by an automated method, and that uses a repeatable dispensing device rather than a disposable dispensing tip. In order to manufacture such an automated nucleic acid purification apparatus, a dispensing apparatus for selecting various kinds of samples in a predetermined order and quantitatively injecting them is required.

However, the rotary multi-channel valve developed so far has problems such as external leakage of the sample at the contact point of the flow path, or liquid sample remains in the form of droplets at the dispensing needle end. It could not be used in an automated nucleic acid purification device that must be precisely metered in.

Therefore, the automated nucleic acid separation and purification apparatus minimizes the external leakage of the sample, and supplies a sample in a pulse manner to prevent the formation of droplets at the dispensing needle end, thereby providing a new dispensing apparatus that enables more precise metering. in need.

In addition, in order to separate and purify nucleic acids from an automated method at the same time from various kinds of nucleic acid-containing samples, development of an apparatus for continuously supplying various kinds of nucleic acid-containing samples in a predetermined order is required.

An object of the present invention is to provide a device capable of automating all of the nucleic acid separation and purification processes and processing a large number of samples at one time to precisely separate and purify nucleic acids in large quantities in a short time.

1 is a perspective view of an essential part of a nucleic acid purification device of the present invention.

2 is a front view of the nucleic acid purification device of the present invention.

3 is a side view of the nucleic acid purification device of the present invention.

4 is a plan view of the nucleic acid purification device of the present invention.

5A is a front view of the dispensing apparatus of the nucleic acid purification apparatus of the present invention.

5B is a sectional view of a control valve of the nucleic acid purification apparatus of the present invention.

<Description of the symbols for the main parts of the drawings>

10 Solution supply part 20 Solution dispensing part

21a, 21b flow path 22 first control valve

23 syringe 24 2nd control valve

25 third control valve 26 dispense needle

30 Plate shifters 31 racks

32 Chucking arm 33 Plate storage table40 Multiwell plate block 41 Vacuum block42 Vacuum vessel 50 Vortex device

60 Cleaning block 61 Zone with sterilization solution 62 Zone with distilled water 70 Outlet

The automatic nucleic acid purification device of the present invention,

A solution supply unit 10 for supplying various nucleic acid-containing solutions and reagent solutions,

A plurality of flow paths 21 for transporting various solutions stored in the solution supply unit,

Control valves 22, 24, and 25 for controlling selection and delivery of the various solutions;

A plurality of syringes 23 for quantitatively inhaling and discharging the various solutions,

A plurality of dispensing needles 26 each connected to the plurality of syringes via a flow path;

Moving means 30 for moving the plurality of dispensing needles or multi-well plate up and down and left and right,

Vacuum block 41 for filtering the solution of the multi-well plate under reduced pressure,

A multi well plate rack 31 for mounting a plurality of the multi well plates and transferring them in a vertical direction;

A plurality of multi well plate blocks 40 on which the multi well plates transferred through the multi well plate racks are mounted;

Operation of the control valve for selection and transfer of the various solutions, operation of a plurality of syringes for metering and aspiration of the solution, movement of a plurality of dispensing needles connected to these syringes, and multiple multi mounted on a multi well plate rack And a control unit for controlling the sequential transfer of the well plate, the operation of the vortex device block, and the sequential operation of the vacuum block according to an input program.

In addition, the automated nucleic acid purification apparatus of the present invention may optionally further include a washing block 60 for washing with the dispensing needle and / or a vortex device 50 for shaking the multi well plate.

The operation of the apparatus of the present invention is controlled by the above control unit which instructs, adjusts, and controls the progress of the sequential work according to a pre-input program. By the operation of the motor during the mechanical operation of the apparatus of the present invention is controlled by the electrical signal of the control unit, the opening and closing of the valve and the movement of the solution is in the compressor and / or pump (not shown) in accordance with the electrical signal generated from the control unit It is operated by the action of the generated air pressure.

The control unit is made of a conventional computer equipped with various memory devices, arithmetic units, input / output devices, etc. already known in the general. A computer program for controlling each step of the nucleic acid separation and purification process is input to the control unit, and such a computer program uses various process control software currently on the market.

Hereinafter, the automatic nucleic acid purification device of the present invention will be described in detail with reference to the drawings. The following drawings are only intended to illustrate and explain the apparatus of the present invention, but are not intended to limit the scope of the invention.

1 is a front view of a nucleic acid purification device according to the present invention. The solution supply unit 10 is located at the bottom of the apparatus, and contains various nucleic acid-containing solutions such as cell culture solution, cell lysis buffer, nucleic acid adsorption buffer (binding buffer), nucleic acid washing buffer, elution buffer (nucleic acid desorption buffer), Containers for supplying distilled water, washing liquid, etc., and containers for waste liquid discharged from the vacuum block are arranged.

As shown in FIG. 5A, the solution dispensing part 20 is connected to the solution supply part 10 and the flow path 21, and controls the transfer of the solution through the flow path 21. ), A syringe 23 for quantitatively inhaling or discharging the solution by the action of the piston through the flow path 21, and a second control valve for controlling the solution discharged from the syringe 23 not to flow back to the solution supply part. 24) a third needle for controlling the flow of a solution between the syringe needle 26 dispensing the solution supplied from the syringe 23 into each well of the multi-well plate and the syringe 23 and the dispensing needle 26; The valve 25 is included.

When the first control valve 22 is opened, the solution is supplied from the solution supply part through the flow path 21a, but at this time, the second control valve 24 is closed so that the solution does not flow into the syringe. Subsequently, the first control valve 22 is shut off, the second control valve 24 is opened, and the solution is quantitatively sucked into the syringe 23 by the action of the piston driven by the motor. The plurality of syringes are respectively connected to the plurality of dispensing needles through a plurality of flow paths 21b and a third control valve. The solution quantitatively sucked into the plurality of syringes is quantitatively discharged by the operation of a piston driven by a motor and then passed through an open third control valve to a plurality of dispensing needles, and shuts off the second control valve. It does not flow back into the solution supply.

The solution thus transferred is injected into each well in the multi well plate through the dispensing needle 26. In this manner, by using a syringe having a piston operated by a motor, the amount of the solution injected into each well can be adjusted within the 1 µl error range. In addition, the syringe 23 can repeatedly perform the suction and discharge process of the solution by the repeated operation of the piston to pass the washing buffer or the washing solution to wash the dispensing needle 26, or to mix different solutions You can also do

FIG. 5B is a detailed view of the control valves, which is a first control valve 22 for controlling the flow of the solution supplied from the solution supply section 10 into the syringe through the flow path, and the first control valve for controlling the backflow of the solution between the syringes. A cross section of a second control valve 24 and a third control valve 25 that controls the flow of the solution between the syringe and the dispensing needle is shown.

These consist of a combination of a plurality of blocks, in which a flow path is formed. Grooves are formed on each block, and the grooves are covered with a membrane, and a pipe for applying air pressure is connected to the groove. When air pressure is applied through the pipe, the membrane covered on the groove expands to block the flow path. Will be The solution is supplied from the solution bottle through the flow path by pressurization, and the flow to the syringe and the dispensing needle is selectively controlled through the first control valve 22, the second control valve 24, and the third control valve 25. do. In the apparatus of the present invention, a conventional solenoid valve may be used as a control valve, and preferably, a multi-channel quantitative control valve device disclosed in detail in Korean Patent Application No. 10-2000-0055968 may be used.

The various multi well plates transferred to the multi well plate block 40 through the rack 31 include a cell culture solution containing plate, a first filter plate (trapping filter plate) for filtering cell lysate, and a agent for adsorbing nucleic acid. A second filter plate (binding filter plate), and a recovery filter plate for recovering nucleic acid from the second filter plate.

Standard multi-well plates currently on the market, such as 96 (12 × 8) well plates, can be used in the device of the present invention. Wells inserted into these plates may be cylindrical tubes that are typically used for nucleic acid separation and purification experiments, and the trapping and binding filters, etc., are mounted at the bottom of the inside and a vacuum suction port may be formed at the bottom thereof to be connected to a vacuum pump. It is the well of the form which can perform the vacuum filtration by vacuum.

The trapping filter inserted into the first filter plate does not permeate the cell lysate at normal pressure, but the centrifugal force is applied to the lysate or the protein, genomic DNA, and various cell debris from the cell lysates during the filtration under reduced pressure. Without it, the plasmid-containing solution performs the function of permeation. Conventional hydrophobic filters known to have this function can be used as trapping filters in the apparatus of the present invention. A trapping filter suitable for use in the apparatus of the present invention is a double layer filter in which a polyolefin resin filter layer and a hydrophobically treated glass filter layer are combined, and described in detail in Korean Patent Application No. 10-2000-0054999.

The binding filter inserted into the second filter plate is a multilayer filter consisting of a filter layer (nucleic acid adsorption layer) that selectively adsorbs nucleic acids in the presence of a chaotrope salt and a hydrophobic filter layer that prevents permeation of the nucleic acid-containing solution at atmospheric pressure. The nucleic acid adsorption layer selectively adsorbs nucleic acids in the presence of a chaotrope salt but adds an elution buffer such as TrisHCl solution to desorb the nucleic acid. The hydrophobic filter layer prevents permeation of the solution at normal pressure, but functions to permeate the solution when centrifugal force is applied to the solution or when the pressure is filtered under reduced pressure. Various kinds of conventional filters known to have this function can be used as binding filters in the apparatus of the present invention, and preferably the multilayer binding filters described in Korean Patent Application No. 10-2000-0054999 can be used.

The apparatus of the present invention includes a rack 31 and a chucking arm 32 for transporting a multi well plate. As shown in FIG. 2, the rack 31 includes a plate storage table 33 that accommodates a plurality of plates to enable vertical movement, and drive means for moving the storage table 33 up and down. . The driving means comprises a timing belt, shafts provided on both sides of the timing belt, a ball bush and a motor mounted on the lower surface, and a multi-well plate block having a multi-well plate on which the plate storage table 33 is moved by the rotation of the shaft. Transfer to 40.

Various solutions are injected into the multi well plate transferred to the multi well plate block through the rack 31. The chucking arm 32 performs the function of moving the multi-well plate thus processed up and down and left and right to the vortex device 50 or the vacuum block 41. In addition, the chucking arm 32 is equipped with a vacuum suction pad for vacuum suction of the plate cover to separate from the plate.

As shown in FIG. 4, the moving means 30 is used to move the dispensing needle 26 and the chucking arm 32 in the front-rear (Y-axis), left-right (X-axis), and up-down (Z-axis) direction axes. Means. Linear motion (LM) guides, timing belts and stepping motors are installed in each direction axis to move the dispensing needle and the chucking arm 32.

In order to move up and down (Z-axis), a pair of ball splines are mounted on both sides of the left and right (Y-axis) LM guides, and a connecting bracket is mounted on the Y-axis LM guide in the Z-axis direction. Two timing belts are mounted on both sides, and the dispensing needle and the chucking arm 32 are independently connected by rotating the ball spline by a motor by connecting the dispensing needle and the chucking arm 32.

The vacuum block 41 is composed of a vacuum container 42 having a vacuum suction tube formed on the bottom surface and a cover covering the edge of the upper end of the vacuum container. The vacuum suction pipe on the bottom surface of the vacuum container 42 is connected to the vacuum pump to reduce the pressure inside the vacuum container 42. The cover of the vacuum block is opened and closed by driving of the motor, and serves to seal the end of the multi-well plate in close contact with the upper edge of the vacuum vessel.

As shown in the top view of the apparatus of the present invention of FIG. 4, the cleaning block 60 used as the washing means of the dispensing needle is a means which is selectively employed in the apparatus of the present invention, and the zone 61 through which the sterilizing liquid flows. Distilled water flows into a zone (62).

As shown in FIG. 4, the apparatus of the present invention may further include an outlet 70 for discharging the multi-well plate and the plate cover out of the apparatus after use.

The method for separating and purifying nucleic acids from a nucleic acid-containing biological sample using the apparatus of the present invention as described above is as follows. The sample is transferred from the multi well plate filled with the nucleic acid containing biological sample solution to the first filter (trapping filter) plate using the dispensing needle 26.

The second filter plate (binding plate) is mounted on the lower portion of the vacuum container 42 of the vacuum block 41, the cover of the vacuum container is closed, and the first filter (trapping filter) plate is mounted thereon. The vacuum block was operated to filter the nucleic acid-containing biological sample solution filled in the first filter plate under reduced pressure, and cell debris, protein, genomic DNA, and the like were filtered out by the trapping filter of the first filter plate, and the plasmid-containing solution was filtered through the second filter ( Binding filter) plate.

The plasmid is adsorbed to the binding filter by injecting a binding buffer containing a chaotrop salt through the dispensing needle into the second filter plate containing the plasmid containing solution. The washing buffer was added to the plasmid adsorbed in this way, and the process of discharging the washing liquid was repeated several times by mounting the vacuum vessel and drying the adsorbed plasmid.

The recovery filter plate is mounted on the lower part of the vacuum block and the cover is closed. Then, the second filter plate on which the plasmid is adsorbed is mounted on the upper part, and an elution buffer such as TrisHCl solution is injected through the dispensing needle. After elution buffer is added to desorb the nucleic acid from the filter surface, the vacuum block is operated to collect the desorbed nucleic acid from the second filter plate into the recovery plate.

The automatic nucleic acid separation and purification apparatus of the present invention adopts a solution dispensing apparatus composed of a plurality of syringes and control valves, thereby simultaneously separating and purifying nucleic acids from various kinds of nucleic acid-containing samples, and also allowing more precise metering of various solutions. Make it possible. In addition, the device of the present invention by using a dispensing needle that can be washed, repeated use without being equipped with a disposable dispensing tip, it is possible to reduce the cost of consumables required for the separation and purification process of the conventional nucleic acid. In addition, the device of the present invention adopts a rack and a transfer device to sequentially transport a plurality of plates to continuously supply the nucleic acid-containing sample, and to automate the movement, mounting and vacuum filtration process of the plate by the plate By performing the nucleic acid separation and purification process in a continuous process, a high purity nucleic acid is separated and purified in a short time from a nucleic acid-containing sample such as a cell culture solution.

By using the automatic nucleic acid purification device of the present invention, the nucleic acid purification process, which is the most frequently used process for genetic manipulation, can be automatically performed more precisely, and a plurality of conventional manual operations can be performed using an automated device. Nucleic acid can be purified in a large amount from a sample in a short time.

Claims (5)

Solution supply (10), Flow passage 21 for transferring the solution stored in the solution supply unit, Control valves 22, 24, 25 for controlling the selection and delivery of the solution, Syringes 23 for quantitatively inhaling or discharging the various solutions, Dispensing needles 26, each connected to the syringe via a flow path, Moving means 30 for moving the dispensing needle and the multi-well plate, Vacuum block 41 for filtering the solution of the multi-well plate under reduced pressure, A multi well plate rack 31 for mounting the multi well plate and transferring it in a vertical direction; A plurality of multi well plate blocks 40 on which the multi well plates transferred through the multi well plate racks are mounted; Operation of the control valve for selection and transfer of the various solutions, operation of the syringe for quantitative suction and discharge of the solution, movement of dispensing needles connected to these syringes, and sequential transfer of the multi-well plate mounted to the multi-well plate rack And a controller for controlling the movement and mounting of the multi well plate to the vacuum block, the sequential operation of the vacuum block, and the like according to an input program. The automatic nucleic acid purification device according to claim 1, further comprising a vortex device (50) for shaking the multi well plate. The automatic nucleic acid purification apparatus according to claim 1 or 2, further comprising a washing block (60) for washing the dispensing needle. 4. The automatic nucleic acid purification apparatus according to claim 3, wherein the washing apparatus (60) is divided into a compartment (61) through which sterilizing liquid flows and a compartment (62) through which distilled water flows. The device according to claim 1, characterized in that the rack (31) comprises a plate storage table (33) for moving a plurality of plates and a driving means for moving them up and down.