KR101444208B1 - A Thin-film Type Gene Amplifying Chamber and A Gene Amplifying Method Using This Chamber. - Google Patents

Abstract

The present invention relates to a gene amplifier which is easy to operate and easy to carry, and a process of amplifying a gene extracted from a cell tissue, which has been possible only through experimentation for a certain period of time by researchers treating various equipments and reagents in a conventional laboratory, The present invention relates to a thin film type amplification chamber and a gene amplification method using the thin film type amplification chamber.

Description

[0001] The present invention relates to a thin film type gene amplification chamber and a gene amplification method using the thin film type amplification chamber.

The present invention relates to a gene amplifier which is easy to operate and easy to carry, and a process of amplifying a gene extracted from a cell tissue, which was possible only by experimentation for a certain period of time by researchers treating various equipments and reagents in a conventional laboratory, The present invention relates to a thin film type amplification chamber and a gene amplification method using the thin film type amplification chamber.

In recent years, imports of environmentally polluted and irresponsible foreign agricultural and fishery products have increased the number of fish, vegetables and vegetables contaminated by mad cow disease and radiation. In this regard, the government has provided measures through the introduction of the country of origin indication system, but until now, it has taken a long time to conduct the inspection of origin or species discrimination, and there is a problem that it can be performed only in a laboratory using professional equipment.

However, consumers are becoming more and more interested in safe foods, such as being illegally converted to domestic products during distribution. Therefore, consumers and retailers are demanding a technology that can easily distinguish the origin or species of food ingredients during the purchase process.

In general, genetic information on biomaterials, including species and origin of plants and animals, and even diseases, can be found by analyzing the genes of the biomaterials. The genetic information of the biomaterial is contained in the nucleic acid of the cell. By separating the chromosomes in the nucleic acid to obtain information about DNA (deoxyribonucleic acid), it is possible to identify various kinds of biomaterials. Thus, biomaterials that cause unknown biological phenomena can be identified.

In order to obtain information on the presence or absence of a specific nucleic acid and the abundance thereof, nucleic acids are extracted from the chromosomes in the cells of the biomaterial including the nucleic acid, and finally, the extracted DNA is re- The amplification process proceeds. The nucleic acid extraction process is known to use a bead. The process of amplifying the extracted nucleic acid and confirming the presence or absence of a specific nucleic acid and the presence thereof is known as a process using PCR (Polymerase Chain Reaction).

However, since these procedures were carried out using professional equipment, they have all been carried out only by experts with expert knowledge and skills in the laboratory. Therefore, it takes a long time to obtain the analysis result, and there have been problems that it is difficult for the general user to use the device easily because of using a relatively large stationary device.

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.

Gene amplification technology using PCR method is widely used in the field of molecular biology. In general, the gene amplification process is as follows. The target DNA and the gene amplification reagent are put in a plastic container with a lid. Then, 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). Until now, gene amplifiers have been used in fixed locations in hospitals and laboratories. Therefore, the gene amplification time which takes about 2-3 hours was not a big problem.

However, as the era of U-healthcare comes, there is a growing demand for a portable gene amplifier that can be used on the spot without limitation of time or space while being carried by an individual. In particular, in a situation where promptness and accuracy are required, such as diagnosis of a new influenza virus or a virus infection, the necessity of performing the gene amplification process more quickly and efficiently in a shorter time is emphasized.

In order to achieve the above-mentioned object, the present invention provides a method of controlling the temperature of a sample, such that heat generated in a heater block can be transmitted to a sample solution passing through a wall surface of a gene amplification tube, So that the heat transfer can be performed quickly. That is, the thickness of the wall of the chamber is made thinner, and the distance between the walls of the chamber is made close to allow the heat transfer between the heater block and the sample solution to be made very quickly, thereby shortening the time required for gene amplification.

In addition, the microfluidic channel structure is applied to the gene amplification chamber, and the sample solution inlet and outlet are provided at both ends of the chamber, thereby enabling the gene transfer to an external device for further processing or analysis after gene amplification and gene amplification Respectively.

The present invention relates to a method and an apparatus for analyzing a sample, which is capable of rapidly transferring heat generated from a heater block to a sample solution passing through a wall surface of a gene amplification tube, Respectively. That is, the thickness of the chamber wall is made thinner, and the distance between the chamber wall surfaces is made close to allow the heat transfer between the heater block and the sample solution to be made very quickly, so that the time required for the amplification of the gene is shortened.

In addition, the microfluidic channel structure is applied to the gene amplification chamber, and the sample solution inlet and outlet are provided at both ends of the chamber, so that the gene can be transferred to an external device for further processing or analysis after gene amplification and gene amplification Respectively.

Finally, by applying the above-described structure, the gene amplification can be performed quickly, so that a user who wants to know species, species, origin or disease status of plants and animals can quickly perform gene-based diagnosis in the field.

1 is a configuration diagram of a gene amplification chamber of the present invention.
Fig. 2 is a block diagram of an injection plug and an exhaust plug which are improved to facilitate gene injection.
3 is a block diagram showing the principle of a gene amplifier.
4 is a configuration diagram of a gene amplifier.
5 is a flowchart showing a step-by-step method of amplifying a gene.
6 is a flowchart showing steps of injecting a gene solution into a gene amplification chamber.
7 is a configuration diagram showing a method of automatically transferring a gene solution in a gene amplification chamber to a read chip.
8 is a flowchart showing a stepwise method of automatically transferring a gene solution in a gene amplification chamber to a read chip.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram of a gene amplification chamber according to a first embodiment of the present invention; FIG.

1 is a block diagram of a gene amplification chamber 100 according to the present invention. The gene amplification chamber 100 includes an upper plate 110, a lower plate 120, a fluid flow channel 150, 110 includes an upper end portion of the injection port, an upper end portion of the microfluidic channel, and an upper end portion of the discharge port. The lower plate 120 includes a lower end portion of the injection port and a lower end portion of the fluid flow channel 150 and a lower end portion of the discharge port. The stopper includes an injection plug 130 inserted into the injection port after coupling the upper plate 110 and the lower plate 120, and an exhaust plug 140 inserted into the discharge port.

The upper plate 110 and the lower plate 120 can be joined by ultrasonic welding. The material of the upper plate 110 and the lower plate 120 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, An ethylene-propylene rubber, a polyvinyl butyral resin, a polyurethane resin, and a nitrile-butadiene rubber. Preferably, polypropylene which is physically and chemically stable and hermetically maintained during heating and cooling required for gene amplification can be used as the material of the upper plate 110 and the lower plate 120, but the present invention is not limited thereto.

The sealing cap used as the cap 130 and the cap 140 is a silicone, urethane, PVC, or PDMS material. When a medical silicone material is used, there is no leakage of the sample solution from the fluid flow channel 150, But it is also possible to use similar materials which have good stability at high temperature and good hermeticity and have no leakage due to the ordinary technician's standard will be. The inlet cap 130 and the discharge cap 140 are formed to have an inner vapor pressure in order to prevent the injection cap 130 and the discharge cap 140 from being pulled out by the inner vapor pressure on the inner wall of the injection port and the discharge port. And a wrinkle structure is formed at a portion contacting the injection port or the discharge port. In order to efficiently inject the gene solution into the channel, the injection plug 131 having the injection needle guide hole and the discharge plug 140 are provided with the air discharge pin 141 as illustrated in FIG. 2 .

The width of the fluid flow channel 150 is 1 to 10 mm and the height of the fluid flow channel 150 is 1 to 5 mm and the height of the fluid flow channel 150 is 1 to 5 mm. And the thickness from the fluid flow channel 150 to the surface of the gene amplification chamber 100 can be used in a range of 0.01 mm to 0.5 mm. Preferably, the width of the fluid flow channel 150 is 2 mm, the height is 0.3 mm, the distance between the fluid flow channels 150 is 2 mm, the thickness from the fluid flow channel 150 to the surface of the gene amplification chamber 100 is 0.1 mm. However, it is needless to say that the width, height, and thickness of the fluid flow channel 150 may be modified by the user and are not limited to the present embodiment.

3 is a diagram showing the operation principle of the gene amplifier 200. The thermoelectric transducer 240, the heat sink 230 and the cooling fan 220 are symmetrical on the upper and lower sides of the gene amplification chamber 100, 210 and the lower layer portion 250 of the upper and lower layers. FIG. 4 is a block diagram of the gene amplifier 200, which includes a gene amplification chamber 100 and a heater block 210. The heater block 210 is positioned above and below the gene amplification chamber 100 and is in surface contact with the upper plate 110 and the lower plate 120 of the gene amplification chamber 100 to perform heating and cooling. The heater block 210 is composed of an upper layer portion and a lower layer portion 250. The heater block 210 has a structure capable of simultaneously heating and cooling the lower and upper portions of the gene amplification chamber 100. The heater block 210 includes a thermoelectric element 240, a heat sink 230, a cooling fan 220, and a mount for positioning the gene amplification chamber 100 to perform the heating and cooling functions. The thermoelectric module 240 may be mounted on the heat sink 230 having the cooling fan 220 inserted therein and then coupled with the upper plate 250 of the heater block to assemble the heater block 210. At this time, the thermoelectric element 240 uses a Peltier element. A thermal diffusion plate made of a metal may be used at the top of the thermoelectric element 240 in the heater block 210 to uniform the temperature distribution to be transmitted to the gene amplification chamber 100. The metal may be copper, gold, silver, or aluminum. However, it is preferable to use copper superior in economy and efficiency, but it is not limited thereto. The thickness of the thermal diffusion plate may be in the range of 0.1 to 5 mm, and it is preferable to use a copper thermal diffusion plate having a thickness of 1 mm, but the present invention is not limited thereto. The spring 260 is further provided on the upper or lower portion 250 of the heater block of the gene amplification chamber 100 so that the heater surface is brought into close contact with the surface of the gene amplification chamber 100 by the restoring force of the spring 260 So that two heating / cooling modules can be fabricated, and the two heating / cooling modules can be positioned below and above and combined to produce a gene amplifier 200. 4 shows an embodiment in which the spring 260 is further included in the lower portion 250 of the heater block 210. However, the present invention is not limited thereto.

FIG. 5 shows a flowchart of a method for amplifying a gene. (I) preparing a gene solution containing a gene to be amplified, (ii) injecting the gene solution into the gene amplification chamber 100, (iii) amplifying the gene amplification chamber into which the gene solution is injected, (Iv) heating the gene amplification chamber 100 in the heater block 210, (v) heating the gene amplification chamber 100 in the heater block 210, The gene may be amplified through a step of cooling the amplification chamber 100 and (vi) a step of repeating steps (iv) and (v) to polymerize the gene to a desired level. The PCR step comprises the steps of (a) heating for polymerization reaction to synthesize a complementary base of DNA, (b) heating in a thermal denaturation step of separating two strands of DNA, (c) The gene can be amplified by repeating the step of cooling.

A spring 260 is mounted on the upper or lower portion 250 of the heater block 210 so that the surface of the gene amplification chamber 100 and the heater block 210 are in surface contact with each other due to the restoring force of the spring 260. [ So that the efficiency of the gene amplification process can be improved.

2) maintaining the temperature of the heater module at 94 ° C for 1 minute; 3) maintaining the temperature of the heater module at 94 ° C for 1 second; and 4) 5) Keep the temperature of the heater module at 50 degrees for 1 second, 6) Heat the heater module at 72 degrees, 7) Keep the temperature of the heater module at 72 degrees for 5 seconds, 8) The temperature of the heater module is heated to 94 degrees, 9) the process from step 2) to 8) is repeated 30 times (step 8 in the last iteration is omitted), 10) 11) A method of cooling the temperature of the heater module to 4 degrees may be used, but it goes without saying that the present invention is not limited to this embodiment.

The gene solution contains the gene template, polymerase, primer (F), primer (R), DNTPs, D.I. Water is used.

6 is a flowchart showing a method of injecting a gene solution into the gene amplification chamber 100. FIG. The injection needle containing the gene solution is inserted into the injection plug 130 of the gene amplification chamber 100 and the inside air of the thin film type gene amplification chamber 100 is discharged to the discharge cap 140 of the gene amplification chamber 100 The gene solution is injected into the channel 150, and the gene solution is injected into the gene amplification chamber 100 (step < RTI ID = 0.0 > ). ≪ / RTI > The step of injecting the gene solution into the gene amplification chamber 100 can be done manually by a person or can be injected using a mechanically automated system.

The injection needle guide hole shown in FIG. 2 for injecting the gene into the gene amplification chamber 100 can be easily injected by using the injection plug 131 and the air discharge pin 141 of the discharge cap. At this time, an injection needle containing a gene solution is inserted into an injection plug 131 having an injection needle guide hole, the air release pin 141 of the discharge plug is pulled out, and a syringe containing the gene solution is pushed, Into the fluid flow channel 150, inserting the air discharge pin 141 into the discharge cap, and removing the syringe. The step of injecting the gene solution into the gene amplification chamber 100 can be done manually by a person or can be injected using a mechanically automated system.

7 and 8 illustrate a method of automatically transferring the gene solution in the gene amplification chamber 100 to the reading chip 300 after completion of gene amplification. From the injection pump 400, the needle detector 410 is moved to the right The needle aspirator 410 is inserted into the fluid flow channel 150 in the injection section of the gene amplification chamber 100 and the needle assembly 410 is inserted between the gene amplification chamber 100 and the read chip 300, And transferring the gene solution in the gene amplification chamber 100 to the read chip 300 by supplying air to the injection pump 400 to automatically read the gene solution in the gene amplification chamber 100. [ (300). At this time, the injection pump 400 may use 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 chamber 110: top plate of gene amplification chamber
120: lower plate of the gene amplification chamber 130:
131: injection plug with injection needle guide 140:
141: pin for discharging air from the discharge cap 150: fluid flow channel
200: gene amplifier 210: heater block
220: cooling fan 230: heat sink
240: thermoelectric element 250: lower layer of heater block
260: spring 300: read chip
400: Infusion pump 410: Needle assay

Claims (20)

In the thin film type gene amplification chamber,
An upper plate, a lower plate, a fluid flow channel, and a sealing cap,
Wherein the upper plate comprises an upper end of the injection port, an upper end of the fluid flow channel, and an upper end of the discharge port,
The lower plate includes a lower end of the injection port, a lower end of the fluid flow channel, and a lower end of the discharge port,
Wherein the sealing cap includes an injection cap inserted into the injection port after the upper plate and the lower plate are coupled to each other, and an exhaust cap inserted into the discharge port,
The material of the upper plate and the lower plate 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, , A polyvinyl butyral resin, a polyurethane resin, and a nitrile-butadiene rubber,
Wherein the fluid flow channel has a width of 1 to 10 mm and a height of 0.1 to 5.0 mm and an interval between channels of 1 to 5 mm and a surface of the gene amplification chamber has a planar structure capable of direct surface contact with the heater Thin Film Genetic Amplification Chamber.
delete delete The method according to claim 1,
Wherein the thickness from the fluid flow channel to the surface of the gene amplification chamber is 0.01 mm to 0.50 mm.
5. The method of claim 4,
Wherein the upper and lower plates of the gene amplification chamber are joined by ultrasonic welding.
6. The method of claim 5,
A wick structure is formed in the injection port and the inner wall of the discharge port to prevent the injection stopper and the discharge stopper from being pulled out by the internal vapor pressure,
Wherein the injection cap and the discharge cap are formed with a wrinkle structure at a portion contacting the injection port or the discharge port so as to withstand internal vapor pressure.
The method according to claim 6,
The injection cap has an injection needle guide hole,
Wherein the exhaust cap has an air exhausting pin.
In the gene amplifier,
A gene amplification chamber according to any one of claims 1 and 4 to 7;
And a heater block which is in surface contact with the upper plate and the lower plate of the gene amplification chamber to perform heating and cooling,
Wherein the heater block is composed of an upper layer portion and a lower layer portion,
Wherein the upper layer portion includes a mounting portion for positioning the thermoelectric element, the heat sink, the cooling fan, and the gene amplification chamber,
Wherein the lower layer portion includes a mounting portion for positioning the thermoelectric element, the heat sink, the cooling fan, and the gene amplification chamber.
delete 9. The method of claim 8,
Wherein the thermoelectric element is a Peltier element.
9. The method of claim 8,
Wherein the heater block further comprises a thermal diffusion plate made of a metal material at an upper end of the thermoelectric device to uniformly distribute a temperature distribution to the gene amplification chamber.
12. The method of claim 11,
Wherein the thickness of the thermal diffusion plate is 0.1 to 5.0 mm, and the metals include at least one of copper, gold, silver, and aluminum.
9. The method of claim 8,
Wherein the heater block further comprises a spring mounted inside the upper layer portion or the lower layer portion,
And the surface of the heater block is brought into close contact with the surface of the gene amplification chamber by the restoring force of the spring.
In a method for amplifying a gene,
(i) preparing a gene solution containing a gene to be amplified;
(ii) injecting the gene solution into the gene amplification chamber of any one of claims 1 and 4 to 7;
(iii) inserting the gene amplification chamber into which the gene solution is injected into a heater block of a thin film type gene amplifier;
(iv) heating the gene amplification chamber in the heater block;
(v) cooling the gene amplification chamber in the heater block;
(vi) repeating steps (iv) and (v) to polymerize the gene to a desired level;
/ RTI >
The gene solution includes a gene template, a polymerase, a primer (F), a primer (R), DNTPs, and DI Water that are desired to be amplified,
The step (iv)
(a) a first heating step for heating a polymerization reaction to synthesize a complementary base of DNA;
(b) a second heating step of heating in a thermal denaturation step of separating two strands of DNA;
And amplifying the amplified gene.
delete delete 15. The method of claim 14,
The step (ii)
(a) inserting an injection needle containing a gene solution into an injection plug of the gene amplification chamber;
(b) inserting an empty injection needle for discharging the air inside the thin film type gene amplification chamber into an exhaust plug of the gene amplification chamber;
(c) injecting the gene solution into the channel by pushing the syringe containing the gene solution;
(d) completing the injection and withdrawing the injection needle;
And amplifying the amplified gene.
15. The method of claim 14,
In the step (ii), the injection plug of the gene amplification chamber has an injection needle guide hole, and the exhaust plug of the gene amplification chamber has an exhaust pin.
(a) inserting an injection needle containing a gene solution into the injection plug;
(b) removing the air discharge pin of the discharge stopper;
(c) injecting the gene solution into the channel by pushing the syringe containing the gene solution;
(d) inserting an air outlet pin into the outlet cap and removing the inlet needle;
Wherein the gene amplification method comprises the steps of:
15. The method of claim 14,
A spring is mounted on the upper or lower portion of the heater block of the gene amplifier,
A gene amplification method in which the surface of a thin film type gene amplification chamber is brought into surface contact with a heater block due to a restoring force of a spring, whereby heat transfer is performed.
A method for automatically transferring a gene solution in a gene amplification chamber to a read chip after gene amplification is completed,
(i) inserting a needle assay connected to the injection pump into an inner channel in the injection section of the gene amplification chamber;
(ii) connecting a needle assay between the gene amplification chamber and the read chip;
(iii) feeding the gene solution inside the gene amplification chamber to the read chip by supplying air through the injection pump;
Wherein the gene solution in the gene amplification chamber is automatically transferred to the read chip.

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