KR20140067552A

KR20140067552A - A gene amplifying tube with flexible cap and gene transferring and amplifying method using this tube

Info

Publication number: KR20140067552A
Application number: KR1020120134940A
Authority: KR
Inventors: 이명호; 이태재; 이석재
Original assignee: 티엔에스(주)
Priority date: 2012-11-27
Filing date: 2012-11-27
Publication date: 2014-06-05

Abstract

The present invention relates to a gene amplification tube in which a cap of a gene amplification tube is made of a material through which a needle can pass. Conventionally, a process of injecting or extracting a gene solution in a cap- (PCR), and then extracting the gene solution with an automated machine. The present invention also relates to an amplification method for amplifying the gene solution.

Description

[0001] The present invention relates to a gene amplification tube having a flexible cap, a gene transfer method and an amplification method using the same,

The present invention relates to a gene amplification tube that is simple and easy to operate, and in a conventional laboratory, a process which was possible only by opening a cap of a gene amplification tube for injecting or extracting a gene, The present invention relates to a gene amplification tube that can be easily injected or extracted so that gene amplification can be performed conveniently, a method for transferring a gene using the same, and a gene amplification method.

Polymerase chain reaction (PCR) is a method in which only a desired portion of a DNA chain is amplified. Since the amount of DNA in a cell is very small, it is very small to be used in experiments. Therefore, only the corresponding DNA of a gene of interest is amplified and used in the experiment. Genes are amplified and widely amplified in the field of molecular biology such as sequencing, gene analysis, diagnosis of genetic diseases, and fingerprint confirmation.

In the polymerase chain reaction (PCR), the target DNA and the gene amplification reagent are put together in a thin tube-shaped plastic container with a lid, and the gene amplification tube is inserted into the heater block of the gene amplifier. The genetic amplification tube is amplified by sequentially converting the heater block surrounding the amplification tube to three temperatures: denature (95 degrees), annealing (50 degrees), and extension (72 degrees).

Therefore, to amplify the gene, it is necessary to include the gene in a gene amplification tube, and the step of extracting the gene from the gene amplification tube after gene amplification, and the extracted gene is used for the gene reading process.

However, conventionally, in the process of amplifying a gene using PCR, the gene amplification tube is taken out of the gene amplifier and the process of opening the upper cap of the gene amplification vessel is performed manually There was.

Korean Patent Registration No. 10-2010-0945556 relates to a PCR-based analyzer, which comprises a sample processor for performing a biochemical reaction using an injected sample; A thin film type thermoelectric element including a terminal whose primary surface is placed in thermal contact with the sample processing section and receives a variable power supply; An optical system detection unit disposed on the sample processing unit or the thin film type thermoelement and movable up and down with respect to the sample processing unit or the thin film type thermoelement; And a controller for supplying a variable power source to the thin film type thermoelectric element through the terminals to precisely control the temperature of the primary side to provide a temperature required for the biochemical reaction to the sample processing unit. However, the configuration of the apparatus is too large, the diffusion of heat is not performed at a sufficiently high speed, and the amplification process takes a long time. Therefore, it is still difficult for the general consumer to use it easily and quickly.

Polymerase chain reaction (PCR) is a widely used method in molecular biology and requires accuracy in gene reading processes. However, the process of injecting or extracting a gene into a gene amplification tube has been inconvenienced by the human hand performing the opening and closing of the cap of the tube, and the gene solution is contaminated in the process, .

In order to solve this problem, the present invention aims to simplify and simplify the experiment by manually or automatically performing a process of injecting or extracting a gene into a gene amplification tube without opening the cap in the gene amplification tube, So that the gene reading process can be accurately performed.

In order to achieve the above object,

The present invention relates to a method for producing a gene amplifying tube by preparing a cap or a cap-weighted portion of a gene amplification tube using a flexible material, and by using a fluid pump connected to a syringe with a needle or a needle through a flexible material portion, The needle was inserted into the gene amplification tube so that the gene could be injected or extracted. In addition, the process of injecting or extracting genes can be done manually, but the gene can be injected or extracted automatically from the gene amplifier.

In addition, the side surface of the cap was tightly attached to the outer wall of the amplification container to have a curved frame structure so as to have a hermeticity so that there was no leakage of the sample solution in the amplification tube during the polymerase chain reaction (PCR).

We also used a material that maintains physical or chemical stability even at high temperatures in the genetic amplification vessel and cap.

In the present invention, gene transfer to an external device for further processing or analysis after gene amplification is carried out by using a gene amplification container having a flexible cap, in a state in which a cap on the upper part of the container is not opened and a syringe or needle Using a connected fluid pump, it can be manually or automatically transferred from the genetic amplification vessel to other external devices.

In addition, gene transfer can be done either manually or automatically with the cap unopened, making the process simpler and more convenient for the worker.

Finally, it is possible to prevent the inexperienced operator from making a mistake and to prevent contamination of the gene solution from the outside, which may be caused by the influence of the operator or the surrounding environment in the gene transfer process.

1 is a configuration diagram of a gene amplification tube of the present invention.
FIG. 2 is a schematic view of a cap of a genetic amplification tube and a genetic amplification tube made of a material through which a needle can penetrate at the center of a cap stopper.
Fig. 3 is a configuration diagram of a cap of a gene amplification tube having a ring-shaped protrusion structure at the sealing portion of the cap.
Fig. 4 is a flowchart showing steps of amplifying a gene.
FIG. 5 is a flowchart showing steps of transferring amplified genes from an amplification tube to an automated machine.
6 is a block diagram showing the principle of transferring amplified genes from an amplification tube to an automated machine.

One embodiment of the gene amplification tube of the present invention, a gene amplification method using the same, and a gene transfer method after completion of gene amplification will be described with reference to the accompanying drawings.

FIG. 1 is a block diagram of a gene amplification tube according to the present invention. The gene amplification tube 100 includes a gene amplification vessel 110 and a cap 120. The gene amplification vessel 110 is tubular so as to contain the gene solution. The cap 120 has a hollow cylindrical shape with no upper and lower surfaces. The cap 120 has a sealing part (130) and a stopper (140) having an area larger than the entrance of the gene amplification vessel and capable of penetrating the injection needle.

The material of the gene amplification vessel 110 may be selected from the group consisting of polypropylene (PP), chlorinated polyethylene, ethylene propylene dimethyl, silicone rubber, acrylic resin, amide resin, epoxy resin, phenol resin, polyester resin, Rubber, a polyvinyl butyral resin, a polyurethane resin, and a nitrile-butadiene rubber. However, the present invention is not limited thereto.

The cap 120 is made of a material having a low hardness such that the injection needle can pass through the cap. The material of the cap 120 includes at least one of silicone, urethane, PVC and PDMS, but is not limited thereto Of course not.

2 is a perspective view showing a state in which the entire cap 120 of the gene amplification tube 100 is not made of a material through which the injection needle can penetrate, An amplification tube 100 and a cap 120 according to an embodiment of the present invention. The cap 120 is composed of a cap 140 and a sealing part 130. The cap 140 is composed of a center part 160 and a peripheral part 150. The cap part 120 has a center part 160 of the cap part 140, It is possible to inject or extract the gene solution through the central portion 160 of the stopper portion 140 without opening the cap 120. At this time, The peripheral portion 150 of the cap 140 and the sealing portion 130 of the cap 120 are made of a material having such a high hardness that the injection needle can not penetrate, but the central portion 160 and the peripheral portion 150 are different from each other The cap 120 made of a material can be manufactured by heterogeneous injection molding. The material of the peripheral portion 150 may be at least one selected from the group consisting of polypropylene (PP), chlorinated polyethylene, ethylene propylene dimethyl, silicone rubber, acrylic resin, At least one of an amide resin, an epoxy resin, a phenol resin, a polyester resin, a polyethylene resin, an ethylene-propylene rubber, a polyvinyl butyral resin, a polyurethane resin and a nitrile-butadiene rubber. Of course not.

3 is a schematic view of a cap 120 having an outer surface of a sealing part 130 having a plurality of ring-shaped protrusion structures 131. The protrusion structure 131 closely contacts the wall surface of the inlet of the amplification container You can have it. The cap 120 and the gene amplification vessel 110 may be separately manufactured by an injection molding method, or may be manufactured at one time by a heterogeneous injection molding method.

FIG. 4 is a flow chart showing a step-by-step method of amplifying a gene, which includes preparing a gene solution containing a gene to be amplified, injecting a needle through a cap 120, Injecting the gene solution into the gene amplification tube 100 through the cap 120, inserting the gene amplification tube 100 into which the gene solution is injected into the gene amplifier 200, The gene can be amplified by repeating heating and cooling according to polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) is a step of heating to cause a polymerization reaction to synthesize a complementary base of DNA, a step of heating for thermal denaturation to break hydrogen bonding of a complementary base of DNA, The gene may be amplified by repeating the step of cooling the gene. Specifically, 1) heating the heater module to 95 ° C at normal temperature, 2) holding the temperature of the heater module at 94 ° C for 1 minute, 3) keeping the temperature of the heater module at 94 ° C for 1 second, 4) 5) Keep the temperature of the heater module at 50 degrees for 1 second, 6) Heat the heater module at 72 ℃, 7) Keep the temperature of the heater module at 72 degrees for 5 seconds, 8) 94), 9) repeating the process from steps 2) to 8) 30 times, 10) maintaining the temperature of the heater module at 72 ° C for 1 minute, 11) cooling the temperature of the heater module to 4 ° C, It is of course possible to perform, but not limited to, a chain reaction (PCR).

5 is a flowchart showing a stepwise process of transferring the amplified gene from the gene amplification tube 100 to an automated machine. The needle probe 310 connected to the fluid pump 300 and the tube 320 is connected to a gene amplifier 200 through the needle assembly 310. The needle assembly 310 passes through the cap 120 of the gene amplification tube 100 located inside the gene amplifier 200, 100, a step of extracting the gene solution in the gene amplification tube 100 with the needle probe 310 with the fluid pump 300, the step of separating the needle probe 310 from the gene amplifier And the amplified gene is transferred from the gene amplification tube to the automated machine.

6, the gene amplifier 200 includes an upper cover 210, a heating block 230, and a lower thermal insulator 240. The needle amplifier 310 passes through the gene amplifier 200 A hole 220 is formed in the upper cover 210 so that the amplified gene can be transferred from the gene amplification tube 100 to an automated machine.

The fluid pump 300 may transfer the amplified gene from the gene amplification tube 100 to an automated machine using a syringe pump, but is not limited thereto.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it should be understood that various changes and modifications will be apparent to those skilled in the art. Obviously, the invention is not limited to the embodiments described above. Accordingly, the scope of protection of the present invention should be construed according to the following claims, and all technical ideas which fall within the scope of equivalence by alteration, substitution, substitution, Range. In addition, it should be clarified that some configurations of the drawings are intended to explain the configuration more clearly and are provided in an exaggerated or reduced size than the actual configuration.

100: gene amplification tube 110: gene amplification vessel
120: cap 130: sealing portion of the cap
131: ring-shaped projection structure 140:
150: peripheral portion of the plug portion 160: central portion of the plug portion
200: Gene amplifier 210: Upper cover
220: hole of upper cover 230: heater block
240: bottom insulating material 300: fluid pump
310: Needle Assay 320: Tube

Claims

In the gene amplification tube,
Wherein the gene amplification tube comprises a gene amplification vessel and a cap,
The gene amplification vessel is tubular so as to contain the gene solution,
Wherein the cap comprises a sealing portion and a cap portion,
Wherein the sealing part is in the shape of a hollow cylinder having no upper and lower surfaces and is in sealing contact with the inner wall of the gene amplification vessel,
Wherein the plug is larger in area than the inlet of the gene amplification and the needle can penetrate.

The method according to claim 1,
Wherein the cap is made of a material having a low hardness such that the entire needle of the cap can pass through the cap.

The method according to claim 1,
The cap portion of the cap is constituted by a central portion of the cap portion and a peripheral portion of the cap portion,
The central portion of the stopper is made of a material having a low hardness enough for the injection needle to penetrate,
Wherein the peripheral portion of the stopper portion and the sealing portion are made of a material having a high hardness such that the needle can not penetrate therethrough.

The method according to claim 1,
The material of the gene amplification vessel is selected from the group consisting of polypropylene (PP), chlorinated polyethylene, ethylene propylene dimethyl, silicone rubber, acrylic resin, amide resin, epoxy resin, phenol resin, polyester resin, polyethylene resin, A polyvinyl butyral resin, a polyurethane resin, and a nitrile-butadiene rubber.

3. The method of claim 2,
Wherein the material of the cap comprises at least one of silicon, urethane, PVC, and PDMS.

The method of claim 3,
The material of the central portion of the cap includes at least one of silicone, urethane, PVC, and PDMS,
The material of the peripheral portion of the cap may be selected from the group consisting of polypropylene (PP), chlorinated polyethylene, ethylene propylene dimethyl, silicone rubber, acrylic resin, amide resin, epoxy resin, phenol resin, polyester resin, polyethylene resin, A polyvinyl butyral resin, a polyurethane resin, and a nitrile-butadiene rubber.

The method according to claim 1,
Wherein the outer surface of the sealing portion of the cap has a plurality of ring-shaped protrusion structures.

In a method for amplifying a gene,
(i) preparing a gene solution containing a gene to be amplified;
(ii) injecting the gene solution into a gene amplification tube according to any one of claims 1 to 7, wherein the needles penetrate the cap or an injection needle mounted on the robot using an automated system passes through the cap ;
(iii) inserting the gene amplification tube into which the gene solution is injected into the gene amplifier;
(iv) heating the gene amplification tube for gene amplification;
(v) cooling the gene amplification tube in a gene amplifier;
(vi) repeating steps (iv) and (v) according to a polymerase chain reaction (PCR) to polymerize the gene to a desired level;
And amplifying the amplified gene.

10. The method of claim 8, wherein step (iv)
(a) heating to cause a polymerization reaction to synthesize a complementary base of DNA;
(b) heating for thermal denaturation breaking the hydrogen bond of a complementary base of DNA;
And a gene amplification method.

A method for transferring an amplified gene from a gene amplification tube to an automated machine,
(i) passing a needle assay connected to the fluid pump through a gene amplifier;
(ii) passing the needle assay through a cap of the gene amplification tube according to any one of claims 1 to 7 located inside the gene amplifier;
(iii) contacting the needle assay with a gene solution inside the gene amplification tube;
(iv) extracting the gene solution inside the gene amplification tube with the fluid pump with a needle assay;
(v) separating and transferring the needle assay from the gene amplifier;
A method for transferring an amplified gene from an amplification tube to an automated machine.

The method according to claim 10, wherein in step (i)
Wherein the gene amplifier comprises an upper cover, a heating block, and a lower thermal insulation material. The method for transferring an amplified gene from an amplification tube to an automated machine.

12. The method of claim 11,
Wherein the upper cover has a hole so that the needle assay can be moved into the gene amplifier. The method for transferring an amplified gene from an amplification tube to an automated machine.

The method according to claim 10, wherein in step (i)
Wherein the fluid pump is a syringe pump. 6. A method for transferring an amplified gene from an amplification tube to an automated machine.