KR102819404B1 - Heat generating device for pcr and method thereof - Google Patents

Abstract

A heat generating device for PCR (Polymerase Chain Reaction) is disclosed. The heat generating device of the present invention includes an FTO (Fluorine doped Tin Oxide) element for generating heat required for PCR, a guard for in situ PCR placed on top of the FTO element and forming an internal chamber of a predetermined height for containing a PCR reaction solution, and a sealing film configured with a larger area than the guard and placed on top of the guard so that the internal chamber containing the PCR reaction solution is closed.

Description

{ HEAT GENERATING DEVICE FOR PCR AND METHOD THEREOF }

The present invention relates to a heat generating device for PCR and a method therefor, and more particularly, to a heat generating device capable of generating heat at a desired temperature using an FTO element and a method therefor.

Genetic testing generally involves analyzing DNA and RNA or protein, which is a gene product, to detect changes in the genome. Among them, DNA is the aggregate and carrier of all genetic information, and molecular biology techniques that can analyze and diagnose DNA with this important information include Sequencing, DNA chip, southern blot, northern blot, dot blot, and PCR.

The most commonly used of these genetic analysis techniques is PCR (Polymerase chain reaction), developed by Mullis of Cetus Corporation (Chiron) in 1985, which is a technology that can copy and amplify a desired part of DNA.

PCR is a polymerase-based reaction that repeatedly amplifies a specific section of the base sequence of a target DNA to produce millions of identical genetic materials by cycling through different temperature ranges.

Referring to Figure 1, PCR generally consists of three steps: denaturation, a heat denaturation process that separates two strands of DNA, annealing, a process that binds a primer to the end of a base sequence, and elongation, a process that synthesizes DNA using DNA polymerase. Although there are some differences, the denaturation process is preferably performed at 94 to 96°C, the annealing process at 50 to 68°C or lower, and the elongation process at 72°C. Since different temperatures are required for each, the reaction is performed by repeatedly performing temperature cycles, and by repeating this process in a cyclic manner, a specific DNA sequence can be exponentially amplified.

Most PCR chips currently under development or under research require heat transfer materials such as platinum (Pt) or copper (Cu) to be embedded inside the channel, making manufacturing difficult. This also results in high costs for the microfabrication process. Therefore, the heat transfer materials must be separated from the PCR chip to produce individual, small heat transfer devices. The Peltier element is suitable for the development of such systems.

The Peltier element uses the Seebeck effect, which generates electromotive force by using the temperature difference generated by connecting two ends of different metal wires to form a closed circuit and inputting a potential difference between the two contact points at both ends, and the Peltier effect, which uses the electromotive force to perform cooling and heating.

The Peltier element has the advantage of providing two functions of heating and cooling using the same element, making temperature control easy and allowing precise temperature control when equipped with an appropriate control system. However, it has the disadvantage of being expensive and having low energy efficiency, consuming a lot of power.

However, a more advanced LAMP (Loop-mediated isothermal amplification) amplification method that can amplify at an isothermal temperature than the traditional temperature change amplification method has been developed. Unlike the conventional PCR in which the amplification reaction is performed in three steps of denaturation, annealing, and extension, the LAMP amplification method allows annealing and extension at a constant temperature without temperature change, so the reaction time is short, less than 1 hour, there is no DNA loss or damage due to temperature change, and since only a constant temperature needs to be maintained, experiments can be performed with only a simple thermostat without expensive equipment, so it has high field usability. In addition, since it uses four specific primers that recognize six regions of the gene sequence to be amplified, it has very high specificity for the target gene.

Therefore, a method is required to overcome the shortcomings of the Peltier element by replacing the existing Peltier element with an element that can generate heat at a desired temperature.

Meanwhile, Korean Patent Registration No. 10-2178672 discloses 'Composition for isothermal colorimetric detection including molecular beacon and use thereof', but does not describe the content of performing an isothermal amplification reaction by replacing the existing Peltier element as in the present invention.

The present invention is to solve the above-described problems, and an object of the present invention is to provide a heat generating device and method thereof that can perform PCR accurately and conveniently at low cost by replacing a Peltier element used in an existing PCR device.

According to an embodiment of the present invention for achieving the above-described purpose, a heat generating device for PCR (Polymerase Chain Reaction) includes an FTO (Fluorine doped Tin Oxide) element for generating heat required for an isothermal amplification reaction of PCR, a guard for in situ PCR placed on the upper part of the FTO element and forming an internal chamber of a predetermined height for containing a PCR reaction solution containing template DNA, and a sealing film configured to have a larger area than the guard and placed on the upper part of the guard so as to close the internal chamber containing the PCR reaction solution.

At this time, the FTO element may include a pair of electrodes attached to the lower portion of the FTO element to generate the heat.

In addition, the above pair of electrodes may each have a rectangular shape and may be arranged so as to form an 11 shape with the long sides being spaced apart from each other in a direction parallel to the lower side of the FTO element and the other side facing the lower side.

Additionally, the FTO device can be configured to have a thickness of less than 1 mm.

Meanwhile, a method for generating heat for PCR (Polymerase Chain Reaction) according to an embodiment of the present invention includes the steps of: arranging an in situ PCR guard forming an internal chamber of a predetermined height for containing a PCR reaction solution containing template DNA on the upper part of an FTO (Fluorine doped Tin Oxide) element for generating heat required for an isothermal amplification reaction of PCR; the step of injecting the PCR reaction solution into the internal chamber of the guard; the step of arranging a sealing film having a larger area than the guard so that the internal chamber containing the PCR reaction solution is closed on the upper part of the guard; the step of attaching a pair of electrodes, each of which has a rectangular shape and is provided so that its long sides are spaced apart from each other in a parallel direction to form an 11 shape, to be positioned respectively on one lower side of the FTO element and the other side facing the one side; and the step of applying a preset voltage to the pair of electrodes.

According to various embodiments of the present invention as described above, the shortcomings of the Peltier element can be complemented, and PCR can be performed accurately and conveniently at a low cost.

In order to more fully understand the drawings cited in the detailed description of the present invention, a brief description of each drawing is provided.
Figure 1 is a drawing for explaining the basic process of PCR according to one embodiment of the present invention.
FIG. 2 is a drawing showing a PCR thermocycler using a Peltier element according to one embodiment of the present invention.
Figures 3 and 4 are drawings showing the structure of a heat generating device according to one embodiment of the present invention.
FIGS. 5 and 6 are drawings for explaining problems occurring when using an FTO device as a heat generating medium according to one embodiment of the present invention and the structure of an electrode for solving the problems.
FIG. 7 is a drawing for explaining the temperature change of an FTO element according to the voltage applied to the electrode according to one embodiment of the present invention.
Figure 8 is a drawing showing the results of FTO-based isothermal PCR according to one embodiment of the present invention.
FIG. 9 is a flowchart briefly explaining a heat generation method for PCR according to one embodiment of the present invention.

First, the terms used in this specification and claims are general terms selected in consideration of functions in various embodiments of the present invention. However, these terms may vary depending on the intention of a person skilled in the art, legal or technical interpretation, and the emergence of new technologies. In addition, some terms may be terms arbitrarily selected by the applicant. These terms may be interpreted as defined in this specification, and if there is no specific definition of a term, it may be interpreted based on the overall content of this specification and common technical knowledge in the relevant technical field.

In addition, the same reference numbers or symbols described in each drawing attached to this specification represent parts or components that perform substantially the same function. For convenience of description and understanding, the same reference numbers or symbols are used in different embodiments to describe them. That is, even if components having the same reference numbers are all depicted in multiple drawings, the multiple drawings do not mean one embodiment.

In addition, terms including ordinal numbers such as "first", "second", etc. may be used in this specification and claims to distinguish between components. These ordinals are used to distinguish between identical or similar components, and the meaning of the terms should not be interpreted in a limited manner due to the use of these ordinals. For example, the order of use or arrangement of components combined with these ordinals should not be interpreted in a limited manner by their numbers. If necessary, each ordinal number may be used interchangeably.

In this specification, singular expressions include plural expressions unless the context clearly indicates otherwise. In this application, it should be understood that terms such as "comprise" or "comprises" are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

In addition, in the embodiment of the present invention, when it is said that a part is connected to another part, this includes not only a direct connection but also an indirect connection through another medium. In addition, when it is said that a part includes a certain component, it means that it may further include other components, rather than excluding other components, unless there is a specific description to the contrary.

Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

FIG. 2 is a drawing showing a PCR thermocycler using a Peltier element according to one embodiment of the present invention.

The conventional PCR heat generating device is implemented with the structure shown in Fig. 2, and cooling and heating are possible by utilizing the phenomenon that heat is generated on one side and heat is removed on the other side at the junction of two metals (TE). In the cooling state where the temperature of the sample block into which the PCR test tube is inserted is lowered, the upper surface of the Peltier becomes a heat-absorbing surface, and the lower surface becomes a heat-generating surface.

In order to lower the temperature of the heat-absorbing surface to a low temperature, the heat from the heating surface must be dissipated. If the heat from the heating surface is not sufficiently dissipated, the heat from the heating surface will be conducted to the heat-absorbing surface. To solve this problem, a heat sink used in PCs can be used to dissipate the heat from the Peltier.

According to one embodiment of the present invention, instead of the PCR heat generating device illustrated in FIG. 2, a heat generating device can be configured using an FTO element as a heat generating medium instead of a Peltier element, a copper coil, or a silver foil coil.

Referring to FIG. 3, the heat generating device (100) of the present invention may include a Fluorine doped Tin Oxide (FTO) element (110), a guard (120) for in situ PCR, and a sealing film (130). FIG. 3 (a) illustrates a plan view of the heat generating device (100), and (b) illustrates a front view. In FIG. 3 (a), the illustration of the sealing film (130) is omitted.

The FTO element (110) is composed of tin oxide doped with fluorine (F), a type of transparent conductive oxide film (F-doped SnO2: FTO), deposited on a glass substrate. FTO is characterized by high stability at high temperatures and low resistance and high transmittance due to the inclusion of fluorine.

Since the FTO element (110) has a surface resistance of 7Ω to 13Ω, heat required for the isothermal amplification reaction of PCR can be generated by applying voltage through the electrode.

According to one embodiment, the thickness of the FTO element (110) may be configured to be 1 mm.

As shown in (a) of Fig. 3, the in-situ PCR guard (120) placed on top of the FTO element (110) is a structure that forms an internal chamber of a certain height for containing a PCR reaction solution (1) containing template DNA. Simply put, the in-situ PCR guard (120) is a thin donut-shaped structure with an open interior, has a square shape, and can be configured to have a thickness of 1 mm.

The PCR reaction solution (1) injected into the inner chamber of the guard (120) for in-situ PCR comes into contact with the upper part of the FTO element (110) and the inner chamber wall of the guard (120) for in-situ PCR, and, for example, a volume of 100 ul can be injected.

As shown in (b) of Fig. 3, the sealing film (130) may be configured to have a larger area than the guard (120) so that the internal chamber containing the PCR reaction solution (1) is closed and may be placed on top of the guard (120).

An actual image of a heat generating device (100) according to one embodiment of the present invention is as shown in FIG. 4.

FIGS. 5 and 6 are drawings for explaining problems that occur when using an FTO device as a heat generating medium according to one embodiment of the present invention and the structure of an electrode for solving the problems.

As in the experimental results of Fig. 5, when terminals for applying positive (+) and negative (-) electrodes are contacted or bitten at each end of the FTO element (110), high heat is generated in a certain range around the contact ends of the electrodes, and there is a problem in that heat is generated unevenly because the heat is not well transferred to other distant surfaces of the FTO element (110).

According to one embodiment of the present invention to solve this problem, in order to uniformly generate heat on the surface of the FTO element (110), a long electrode surface can be attached to the FTO element (110) so that the surface can be uniformly heated.

Specifically, as illustrated in (a) of FIG. 6, the FTO element (110) may include a pair of electrodes (200-1, 200-2), and each of the pair of electrodes (200-1, 200-2) may have a rectangular shape and may be arranged so as to form an 11 shape with a long side parallel to a lower side of the FTO element (110) and the other side facing the lower side, while being spaced apart from each other.

Figure 6 (b) illustrates the experimental results in which heat is uniformly transferred to the entire surface of the FTO element (110).

FIG. 7 is a drawing for explaining the temperature change of an FTO element according to the voltage applied to an electrode according to one embodiment of the present invention.

As illustrated in FIG. 7, when a voltage of 15 V is applied to a pair of electrodes (200-1, 200-2), the temperature rapidly increases to around 70°C at the beginning and then gradually decreases to maintain an isotherm at around 60°C, forming a saturation curve graph (71), and when a voltage of 9.98 V is applied, the temperature gradually increases to around 60°C and maintains an isotherm at around 60°C, forming a saturation curve graph (72).

That is, when a voltage of a certain range is applied according to the surface resistance of the FTO element (110), heat (60°C) required for the isothermal amplification reaction of PCR can be uniformly generated on the surface of the FTO element (110).

Figure 8 is a diagram showing the results of an FTO-based isothermal PCR according to one embodiment of the present invention.

Figure 8 shows the PCR results for measuring the performance of an FTO element (110) using LAMP amplification technology, confirmed through electrophoresis, and shows a band pattern similar to that of the LAMP results using a conventional Peltier element.

In this experiment, the results of amplifying the DNA of Vibrio bacteria were expressed as a specific reaction (P), and the results of amplifying the DNA of E. coli were expressed as a non-specific reaction (N), together with a DNA size marker (M).

FIG. 9 is a flowchart briefly explaining a heat generation method for PCR according to one embodiment of the present invention.

First, a guard for in-situ PCR is placed on the upper part of the FTO element to generate heat required for the isothermal amplification reaction of PCR, forming an internal chamber of a certain height for containing a PCR reaction solution containing template DNA (S910).

Afterwards, the PCR reaction solution is injected into the inner chamber of the guard (S920).

Thereafter, a sealing film having a larger area than the guard is placed on top of the guard so that the inner chamber containing the PCR reaction solution is closed (S930).

Thereafter, a pair of electrodes, each having a rectangular shape and spaced apart from each other in a direction in which the long sides are parallel to form an 11-shape, are attached to one lower side of the FTO element and the other side facing the one side, respectively (S940).

Afterwards, a preset voltage is applied to a pair of electrodes to amplify the template DNA (S950).

According to various embodiments of the present invention as described above, the shortcomings of the Peltier element can be complemented, and PCR can be performed accurately and conveniently at a low cost.

Meanwhile, the heat generation method for PCR according to the various embodiments described above may be implemented as a program and stored in various recording media. That is, a computer program that can be processed by various processors and execute the heat generation method described above may be used in a state of being stored in a recording medium.

For example, a non-transitory computer readable medium storing a program that performs the steps of: i) arranging an in-situ PCR guard forming an internal chamber of a predetermined height for containing a PCR reaction solution containing template DNA on the upper part of an FTO element for generating heat required for an isothermal amplification reaction of PCR, ii) injecting the PCR reaction solution into the internal chamber of the guard, iii) arranging a sealing film having a larger area than the guard on the upper part of the guard so that the internal chamber containing the PCR reaction solution is closed, iv) attaching a pair of electrodes, each having a rectangular shape and each arranged so that their long sides are spaced apart from each other in a parallel direction to form a shape of the number 11, to be positioned respectively on one lower side of the FTO element and the other side facing each other, and v) applying a preset voltage to the pair of electrodes can be provided.

A non-transitory readable medium refers to a medium that semi-permanently stores data and can be read by a device, rather than a medium that stores data for a short period of time, such as a register, cache, or memory. Specifically, the various applications or programs described above can be stored and provided on a non-transitory readable medium, such as a CD, DVD, hard disk, Blu-ray disk, USB, memory card, or ROM.

Although the preferred embodiments of the present invention have been illustrated and described above, the present invention is not limited to the specific embodiments described above, and various modifications may be made by those skilled in the art without departing from the gist of the present invention as claimed in the claims. Furthermore, such modifications should not be individually understood from the technical idea or prospect of the present invention.

100: Heat generating device 110: FTO device
120: Guard for PCR 130: Sealing film
200-1, 200-2: A pair of electrodes

Claims (5)

In a heat generating device for PCR (Polymerase Chain Reaction),
FTO (Fluorine doped Tin Oxide) element for generating heat required for isothermal amplification reaction of PCR;
A guard for in situ PCR, which is placed on top of the FTO element and forms an internal chamber of a certain height for containing a PCR reaction solution containing template DNA; and
A sealing film is provided on the upper part of the guard so as to be in direct contact with the guard and is configured to have a larger area than the guard so that the inner chamber containing the PCR reaction solution is closed;
The above FTO device,
A pair of electrodes attached to the lower part of the FTO element to uniformly generate heat on the surface of the FTO element;
The above pair of electrodes,
Each has a rectangular shape, and each is arranged so that the long sides of the lower side of the FTO element and the other side facing the lower side are spaced apart from each other in a direction parallel to each other to form an 11-shaped shape.
The above FTO device has a surface resistance of 7Ω to 13Ω,
A heat generating device characterized in that the temperature of the FTO element is maintained at 60°C by applying a voltage of 9.98 V to 15 V to the pair of electrodes. delete delete In the first paragraph,
The above FTO device,
A heat generating device characterized by having a thickness of less than 1 mm. In a heat generation method for PCR (Polymerase Chain Reaction),
A step of placing an in situ PCR guard, which forms an internal chamber of a certain height for containing a PCR reaction solution containing template DNA, on top of an FTO (Fluorine doped Tin Oxide) element for generating heat required for an isothermal amplification reaction of PCR;
A step of injecting the PCR reaction solution into the inner chamber of the guard;
A step of placing a sealing film having a larger area than the guard on the upper part of the guard so as to be in direct contact with the guard so that the inner chamber containing the PCR reaction solution is closed;
A step of attaching a pair of electrodes, each having a rectangular shape and arranged so that the long sides are spaced apart from each other in a parallel direction to form an 11 shape, to the lower side of the FTO element and the other side facing the one side, respectively, in order to uniformly generate the heat on the surface of the FTO element; and
A step of applying a preset voltage to the above pair of electrodes;
The above FTO device has a surface resistance of 7Ω to 13Ω,
A heat generation method characterized in that a voltage of 9.98 V to 15 V is applied to the pair of electrodes to maintain the temperature of the FTO element at 60° C.