KR20170029171A

KR20170029171A - Gene Amplification Apparatus and Method

Info

Publication number: KR20170029171A
Application number: KR1020150126157A
Authority: KR
Inventors: 조원창
Original assignee: 조원창
Priority date: 2015-09-07
Filing date: 2015-09-07
Publication date: 2017-03-15
Also published as: KR101780334B1

Abstract

According to the present invention, there is provided an air conditioner comprising: a lower chamber and an upper chamber communicable with each other; at least one lower fan heater installed at one side of the lower chamber and blowing air heated to an inner space of the lower chamber; A disk rotatably installed in the upper chamber and containing a plurality of tubes; And an air discharge fan installed at one side of the upper chamber to discharge air in the upper chamber to the outside; A gene amplifying apparatus is provided.

Description

Gene amplification apparatus and gene amplification method therefor

The present invention relates to a gene amplification apparatus and a gene amplification method using the same, and more particularly, to a gene amplification apparatus and method for amplifying a gene by amplifying the temperature of a sample tube and a sample by using a polymerase chain reaction (PCR) And a reagent mounting system, and a gene amplification method therefor. 2. Description of the Related Art Particularly, the present invention relates to a thermal cycler used in a polymerase chain reaction (PCR). In the thermal cycling method, a tube or a chip containing a sample and an amplification reagent using air or gas is used (Gene) through maintaining, rising and falling at the desired temperature.

Polymerase Chain Reaction (PCR) technology is a technique to amplify a specific region of DNA or RNA in a large volume in a reaction vessel. In addition to the field of pure molecular biology, the field of medicine, science, agriculture, veterinary science, food science, environmental science, archeology , And anthropology. Especially, these gene amplification technologies are widely used for research, development, and diagnosis of life sciences genetic engineering and medical fields. In the polymerase chain reaction, DNA polymerase is used to amplify a specific DNA sequence in the genome as necessary. Polymerase chain reaction (PCR) can amplify a large number of genes using gene-replicating enzymes using only a small amount of genes to carry out researches using genes, experiments, and detection of microorganisms. In order to amplify these genes, a temperature regulator that regulates the temperature of the gene sample is essential.

Polymerase chain reaction generally involves three processes. That is, it includes denaturation, annealing, and extension, and DNA can be exponentially amplified by repeating this process repeatedly. More specifically, in the denaturation step, double stranded DNA is treated at 90 ° C or higher and separated into single stranded DNA. In annealing, two kinds of primers are bound to complementary single stranded DNA, respectively, and they are maintained at 55 to 60 ° C for 30 seconds to several minutes. In the extension, a DNA polymerase is activated to extend the primer. The time required for the elongation reaction depends on the concentration of the template DNA, the size of the amplified fragment, and the reaction temperature. When using Thermusaquaticus (Taq) polymerase, which is commonly used, it takes about 30 seconds to several minutes at 72 ℃.

The thermal denaturation process is usually carried out at 95 ° C, and the binding reaction and the polymerization reaction are carried out at a temperature of about 55 ° C to 75 ° C, which is lower than the above temperature. Therefore, it is necessary to repeatedly raise and lower the sample temperature have.

On the other hand, until now, the method of polymerase chain reaction has mainly been to isolate and confirm the amount of the product after the reaction, but it is becoming increasingly necessary to perform accurate quantification more rapidly. In fact, the target amount in the sample in Gene Expression (RNA) Analysis, Gene Copy Assay (human HER2 gene in breast cancer or HIV virus burden amount measurement), genetyping (knockout mouse analysis) Accurate measurement of the temperature is very important. However, the conventional method only shows the qualitative result of DNA amplified by using gel-end-point at end-point, and quantitative detection of DNA is inaccurate.

Therefore, in order to minimize the error of the existing end-point measurement method, a quantitative competitive PCR method is used. In this method, a competitor who already knows the quantity is put into the reaction solution and at the same time, the PCR is performed under the same conditions, and then the amount of the original target is deduced by comparing the amount of the product made in the target with the product made in the competitor. However, this method should be used to create the most appropriate competitor per PCR, and to match the appropriate ratio of target and competitor (at least 1/10 to 1: 1, but most accurate at 1: 1) It is very complicated to perform experiments at various concentrations and it is known that the probability of actual success is also small.

In view of the above problems, a real time PCR technique for monitoring the progress of each cycle in order to measure the PCR step within the exponential phase has recently been introduced.

At the same time, in order to quickly measure the progress of each cycle, the fluorescence detection method is also used as an in-the-tube measuring means instead of the gel separation method. Developed. In 1992, Higuchi et al. Developed a method of measuring the amount of fluorescence accumulated per cycle using a CCD camera after UV irradiation with ethidium bromide at every amplification. PCR We were able to capture the amplification plots of the whole process.

However, in a conventional DNA polymerase chain reaction real time monitoring device, a metal block such as a Peltier device is used in most cases. Therefore, when the PCR temperature condition is the same for each well or chip, Many samples can be repeated at the same time under the same conditions, but there are many limitations in performing PCR reaction on different samples at different temperature conditions. In addition, since the Peltier device involves the use of a metal block, the temperature transition rate is in the range of 1 to 3 ° C./second, so that a long time is required for the temperature transition. Therefore, the reaction time is generally 1 to 2 hours or more. There is a problem that it takes much time to inspect by the method. Also, the accuracy of temperature is limited to about ± 0.5 ℃. Therefore, there is a limit in controlling the temperature quickly and precisely, and there is a limit to the sensitivity and the specificity of the reaction due to the insufficient temperature uniformity.

On the other hand, a Peltier element, a silicon element, a ceramic element, or the like is used as a heating element (heater) for performing a PCR reaction. However, these devices are solid-state heating elements and must closely contact the heating element and the heating period in order to transfer the temperature to the container containing the sample. If not closely adhered, a temperature difference occurs between the space away from the heating element and the space close to the heating element. In order to compensate for this, it is necessary to experimentally calculate the temperature distribution for each position to compensate for the temperature uniformity, but this is not the best way to maintain a uniform temperature distribution.

As one example of the prior art, semiconductor silicon having a high temperature rising rate can be used to increase the temperature transfer rate. The rate of temperature rise of the semiconductor silicon is higher than that of the Peltier device. However, since this method is also a heat conduction method through contact between solids, heat is not uniformly transferred to the container. In order to overcome such disadvantages, a separate liquid such as oil may be additionally installed near the vessel to uniformly transfer the heat to the vicinity of the heating element. However, this may cause problems such as additional maintenance and control of the liquid and cost increase have.

In another example, a peltier device is a technically stable device, which is advantageous in developing a gene amplification device. However, due to the characteristics of a solid thermoelectric device, a phenomenon occurs in which the device is over- The amplitude is more than uniformity, then gradually decreases, and the stabilization phase is entered. In addition, it is under-shooting below the desired temperature during the temperature cooling, and it is also difficult to make an accurate and constant temperature gradient by the process step.

Furthermore, the Peltier device can not be directly contacted with the sample, and the temperature of the sample is raised or lowered through a heating block interposed between the sample and the tube containing the amplifying reagent. Sample and heating block, since both surfaces of the rigid contacts (hard contact) to a temperature transfer by contact between the solid, which may cause differences in temperature transferred from the contact surface in accordance with the manufacturing conditions, in general, the temperature uniformity (unoformaity) up to + Respectively.

1 is a plan view and a front view of a heater using a Peltier element in a gene amplification apparatus of the prior art.

Referring to the drawings, a plurality of tubes T are disposed in a heating block B attached to a Peltier element P. At this time, as can be seen from the drawing, the temperature distribution is uneven depending on the position of the tube TB. That is, a temperature difference occurs between the edge and the center, which may adversely affect the gene amplification in which the cycle is repeated dozens of times.

2 is a perspective view showing another example of a heater using a Peltier element in a gene amplification apparatus of the prior art.

Referring to the drawings, a temperature sensor S is disposed on the bottom surface of a heating block B in which a tube TB is disposed, so that heat from the Peltier element P can be sensed. As shown in FIG. 2, even if temperature correction is performed using a plurality of temperature sensors and a correction program, a temperature uniformity of more than ± 0.50 ºC to 1.00 ºC is usually exhibited for each tube (TB).

On the other hand, in the polymerase chain reaction, there is a cycle of raising and lowering the temperature of the sample. In fact, the time during which the sample is maintained at a constant temperature is generally less than one minute, but it takes a long time to raise and lower the temperature. As a result, a typical PCR chain reaction requires more than an hour as a whole. This is because heat can not be transferred directly from the Peltier element or other solid-state device to the sample tube, and the heating block is used as the intermediate medium. That is, the time required for heating and cooling the metal body constituting the heating block becomes longer.

3 shows the polymerase chain reaction of a sample performed in an amplification apparatus using a Peltier element as a change in temperature over time.

Referring to the drawing, since the temperature rise rate per second is about 2-3 degrees Celsius per second, it takes about 15 to 20 seconds to lower the temperature of the coupling step to 55 degrees Celsius at 95 degrees Celsius during the denaturation step, It takes 15 ~ 20 seconds to rise to a degree. Therefore, a temperature transfer time of 30 to 40 seconds occurs in each PCR cycle, which is a dead time in which no gene amplification occurs. Therefore, there is a need to increase the temperature rise and fall speed and shorten the transition time to enable rapid inspection.

It is an object of the present invention to provide an improved gene amplification apparatus and a gene amplification method therefor.

It is another object of the present invention to provide a gene amplification apparatus including temperature control means capable of uniformly maintaining a temperature distribution of a sample and a gene amplification method therefor.

It is another object of the present invention to provide a gene amplification apparatus using high temperature air and a gene amplification method using the same so that the heat transfer to the sample tube can be made uniform irrespective of position, .

According to an aspect of the present invention, there is provided an air conditioner comprising: a lower chamber and an upper chamber communicable with each other; at least one lower fan heater installed at one side of the lower chamber and blowing air heated to an inner space of the lower chamber; A disk rotatably installed in the upper chamber and containing a plurality of tubes; And an opening / closing device installed on one side of the upper chamber and operated by a fan or a valve for discharging the air in the upper chamber to the outside; A gene amplifying apparatus is provided.

According to an aspect of the present invention, there is further provided an air circulation fan provided on the upper portion of the disk in the upper chamber.

According to another aspect of the present invention, the at least one fan heater includes a heat line and a fan, and controls the temperature and the flow rate of the heated air by controlling power supplied to the heat line and the fan.

According to another aspect of the present invention, the disc is installed on a rotary shaft extending through an upper chamber and a lower chamber, and a fan positioned below the disc is installed on the rotary shaft. By rotating the rotating shaft, the temperature of the container mounted on the disk can be made uniform.

According to another aspect of the present invention, there is further provided an optical system provided adjacent to the tube disposed on the disc at one side of the upper chamber, and the optical system detects the degree and state of gene amplification of the sample contained in the tube.

According to the present invention, there is also provided a method comprising: receiving the plurality of containers containing a sample on a disc; Blowing air heated to the lower chamber and the upper chamber using the fan heater to the containers housed in the disk unit; Discharging air using the air discharge fan or opening / closing apparatus according to the temperature of the upper chamber or the temperature of the upper chamber; Raising or lowering the temperature of the chamber by the fan heater after a predetermined period of time of the container temperature in the upper chamber; And repeating the step of blowing the air heated to the lower chamber and the step of discharging the air.

According to the present invention, when there are a plurality of fan heaters, each of the fan heaters can be used at the same time, or can be applied separately for temperature rising and temperature lowering depending on their respective roles. In this case, it is possible to further shorten the transition time by increasing the temperature rising and falling speed.

In the gene amplification apparatus and the gene amplification method according to the present invention, not only hot air heated by hot wire but also air circulation fan attached to the upper chamber for uniform delivery of hot air is used The problem of irregularities in the temperature distribution of the container can be solved by rotating the air in the upper chamber or using the motor to rotate the disk. Also, in the present invention, the temperature rise and the air temperature can be rapidly increased by using the hot wire and the fan, so that the polymerase chain reaction can be rapidly performed.

1 is a plan view and a front view of a heater using a Peltier element in a gene amplification apparatus of the prior art.
2 is a perspective view showing another example of a heater using a Peltier element in a gene amplification apparatus of the prior art.
3 shows the polymerase chain reaction of a sample performed in an amplification apparatus using a Peltier element as a change in temperature over time.
FIG. 4 is a schematic diagram of a gene amplification apparatus according to an embodiment of the present invention.
5 is a front view of the gene amplification apparatus shown in FIG.
FIG. 6 is a perspective view illustrating a state where a disk provided in the upper chamber is exposed.
7 is a cross-sectional view of the disk shown in Fig.
FIG. 8 schematically shows circulation of air in the gene amplification apparatus according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in more detail with reference to an embodiment shown in the accompanying drawings.

FIG. 4 is a schematic diagram of a gene amplification apparatus according to an embodiment of the present invention. FIG. 5 is a front view of the gene amplifying apparatus shown in FIG. 4, and FIG. 6 is a perspective view showing a disk provided in the upper chamber exposed.

The gene amplifying apparatus according to the present invention includes a lower chamber 10 and an upper chamber 20 capable of interacting with each other and a lower chamber 10 provided at one side of the lower chamber 10, A disk 21 which is rotatably installed in the upper chamber 10 and supports a plurality of tubes 23 and a lower chamber 21 which is provided at one side of the upper chamber 20, And a temperature discharging device using an installed air discharging fan 15 or an opening / closing device (not shown). Although not shown in the drawing, the opening and closing device may be formed by forming an opening at one side of the upper chamber 20, for example, and providing a door to the opening. The door (not shown) is controllably opened and closed to raise and lower the temperature. In the upper chamber 20, a fan 19 for air circulation may be further provided on the disc 21.

In the embodiment shown in the drawings, the lower chamber 10 is formed in a truncated cone shape as a whole, and the large-diameter cross section is arranged to face upward. The upper chamber 20 is formed in a generally cylindrical shape. The upper chamber 20 is supported by a second support S2 fixed to the table T and the table T is supported by a first support S1 supported on the base B.

The lower chamber 10 is fixed to the lower portion of the upper chamber 20. [ A space communicably connected to the upper chamber 20 and the lower chamber 10 is formed in the upper chamber 20 and the lower chamber 10, respectively. At least one fan heater 13 is installed on the outer peripheral surface of the lower chamber 20. The fan heater 13 is provided with a heating line (not shown) and a fan (not shown). The fan heater 13 heats air with heat generated from the heating line by electric power, It can blow. In the example shown in the figure, three fan heaters 13 are installed around the lower chamber 10, but in other embodiments, one or more fan heaters 13 may be installed. The electric power supplied to the hot wire and the fan can be controlled so that the temperature and the flow rate of the hot air blown to the lower chamber 10 can be controlled. The plurality of installed fan heaters may simultaneously raise or lower the temperature in the chamber, or each fan heater may be used to separate the roles for temperature rise and temperature decrease.

The disk 21 provided inside the upper chamber 20 is rotatably installed by the rotary shaft 11. [ The rotary shaft 11 extends through the upper chamber 20 and the lower chamber 10 and the lower end of the rotary shaft 11 extends outside the lower chamber 10. The lower end of the rotary shaft 11 is connected to a motor M provided on the table T and thus can be controllably rotated by the motor M. [

In the interior of the lower chamber 10, a fan 12 is installed on the rotary shaft 11. The fan 12 is for air circulation and rotates together when the rotary shaft 11 rotates. The hot air introduced into the lower chamber 10 by the fan heater 19 can be uniformly dispersed in the lower chamber 10 by the rotation of the fan 12. [

A fan 15 for discharging air is provided at one side of the upper chamber 20. In the embodiment shown in the drawing, the air discharge fan 15 is provided with two air discharge fans 15 at 180 degrees on the side of the upper chamber 20. The air discharge fan 15 may be operated to discharge the air inside the upper chamber 20 to the outside. That is, when it is necessary to lower the temperature inside the upper chamber 20, the high-temperature air in the upper chamber 20 can be discharged using the air discharge fan 15.

In the upper chamber 20, a fan 19 may be installed above the disc 21. The fan 19 is for uniformly maintaining the internal temperature of the upper chamber 20 as a whole by dispersing the air in the upper chamber 20. The fan 19 installed on the upper part of the disk 21 may be optionally provided with a hot wire. The fan 19 can blow air into the upper chamber 20 while heating the air by the hot wire.

Referring to FIG. 6, the upper chamber 20 can separate the upper portion to expose the disc 21. A disc 21 is provided in the upper chamber 20 and a plurality of sample tubes 23 are arranged along the circumference of the disc 21. The sample tubes 23 are arranged in the circumferential direction. At least one position recognition hole 21a is formed in the disk 21. The rotational position of the disk 21 can be detected through the position recognition hole 21a and the optical sensor (not shown). It is possible to determine the rotational position of the disc 21 by detecting whether or not light incident from an optical sensor (not shown) provided on the upper portion or the lower portion of the disc 21 passes through the position recognition hole 21a have. In the example shown in the figure, the position recognition holes 21a are formed in a plurality of circular shapes.

It is preferable that the air circulation holes 21b are formed in the disk 21 at uniform intervals around the central axis. The air from the lower chamber 10 can smoothly communicate with the upper chamber 20 through the air circulation hole 21b. A communication hole 20a wider than the plane surface area of the disk 21 is formed between the bottom surface of the upper chamber 20 and the upper surface of the lower chamber 10 so that air can smoothly circulate between the upper chamber and the lower chamber have.

On one side of the upper chamber 20, an optical system 17 is provided at a position adjacent to the sample tube 23 disposed on the disk. The optical system 17 is, for example, a fluorescence optical system for confirming in real time the amplification state of a sample contained in the sample tube 23. The degree and state of gene amplification of the sample can be diagnosed through the optical system 17.

One or more temperature sensors (not shown) may be provided inside the upper chamber 20 and the lower chamber 10. [ In particular, by providing a temperature sensor at a position adjacent to the sample tubes 23, it is possible to detect the temperature of each sample contained in the sample tube 23 and judge whether or not the temperature is properly maintained.

Fig. 7 shows a cross-sectional view of the disk shown in Fig.

Referring to the drawings, the edge of the plate member forming the disc 21 is formed to be inclined, and an insertion port 21c into which the sample tube 23 can be inserted is formed at the edge. By forming the edge of the disk 21 to be inclined as described above, the sample tube 23 is disposed obliquely on the disk 21. By arranging the sample tube 23 obliquely, it is possible to align the longitudinal direction axis of the sample tube with the direction of the composite force of centrifugal force and gravitational force generated when the disk 21 rotates. The insertion ports 21 may be formed at equal intervals on the edge of the disk 21, for example, 48 pieces may be formed on the edge of the disk 21. [

FIG. 8 schematically shows circulation of air in the gene amplification apparatus according to the present invention.

Referring to the drawings, the hot air generated by the heater fan 13 flows into the lower chamber 10, which is indicated by an upward sloping arrow. The hot air of the lower chamber 10 moves to the upper chamber 20 along an arrow indicated in the vertical upward direction. The hot air also flows vertically upward through the hole 21b formed in the disk 21. [ The air circulating fan 19 disposed on the upper side of the disk 21 blows the hot air downward as indicated by an inclined downward direction so that air flows in the upper chamber 20 only at the upper and lower portions of the disk But the sample tubes can be uniformly dispersed and circulated toward the edge region of the disposed disc. As a result, the internal temperature of the upper chamber 20 can be kept uniform, and thus a uniform temperature can be maintained in all the sample tubes 23 as well. When it is necessary to lower the temperature of the upper chamber 20, the fan 15 discharges air as indicated by a horizontal arrow.

Hereinafter, the operation of the gene amplification apparatus of the present invention will be schematically described.

In the gene amplification apparatus configured as described above, preparation for gene amplification is performed by inserting a sample tube 23 containing a sample into the insertion section 21c formed on the disk 21. [ Next, the fan heater 13 and the motor M are driven to perform gene amplification. That is, the hot air is injected into the inner space of the lower chamber 10 using the fan heater 13, and the sample tube 23 disposed on the disk 21 by rotating the rotary shaft 11 with the motor M Can be rotated.

The hot air injected into the lower chamber 10 flows into the upper chamber 20 through the communication hole 20a formed between the lower chamber 10 and the upper chamber 20 and the hole 21b formed in the disk 21. [ It can flow. At this time, the fan 12 installed on the rotary shaft 11 serves to raise the air upward. The air introduced into the upper chamber 20 can be uniformly dispersed in the upper chamber 20 by the air circulation fan 19 installed in the upper portion of the disk 21. [

When it is necessary to lower the air temperature inside the upper chamber 20, the air discharge fan 15 is operated. The operation of the hot wire provided in the fan heater 13 is stopped and the fan 15 for air discharge is operated to discharge the air inside the upper chamber 20 to the outside so that the air temperature inside the upper chamber 20 .

10. Lower chamber 12. Fan
13. Fan heater 20. Upper chamber
21. Disc 23. Tube

Claims

A lower chamber and an upper chamber capable of interacting with each other,
One or more lower fan heaters installed at one side of the lower chamber to blow air heated to the inner space of the lower chamber;
A disk receiving a plurality of containers in the upper chamber; And
And an air discharge device installed at one side of the upper chamber to discharge air in the upper chamber to the outside.

The method according to claim 1,
Further comprising an air circulation fan installed on the upper portion of the disk in the upper chamber.

The method according to claim 1,
Wherein the one or more fan heaters include a heat wire and a fan, and the temperature and flow rate of the heated air are controlled by controlling power supplied to the hot wire and the fan, and when a plurality of fan heaters are installed, Wherein the first and second amplifying units are driven in the same or different manner.

The method according to claim 1,
Wherein the disk is installed on a rotary shaft extending through the upper chamber and the lower chamber, and the rotary shaft is rotated by a motor.

The method according to claim 1,
And a fan is further provided under the disk.

The method according to claim 1,
Further comprising an optical system provided adjacent to a container disposed on the disc at one side of the upper chamber, wherein the optical system detects the amplification degree and state of the gene in the sample contained in the container.

The method according to claim 1,
Wherein the air discharging device is a rotating fan or an opening / closing device for discharging or shutting off the air in the upper chamber.

A gene amplification method using the gene amplification apparatus according to claim 1,
Receiving the plurality of containers containing the sample on the disc;
Blowing air heated by the fan heater to a container mounted on the disk;
Discharging air using the air discharge device when the upper chamber reaches a predetermined temperature;
Controlling the fan heater to raise or lower the temperature of the upper chamber; And
Repeating the step of blowing air heated to the lower chamber and discharging the air.