KR20060116984A - Heating block apparatus for amplifying dna - Google Patents

Abstract

A heating block apparatus for amplifying DNA is provided to minimize errors according to temperature change by uniformizing DNA chip temperature, reduce the reaching time to desired temperature and DNA expression time by separating heating block into the preheating block and expression heating block, and analyze numerous DNA chips simultaneously. The heating block apparatus(2) for amplifying DNA comprises at least three pairs of preheating block(4) and expression heating block(6), a chip-introducing portion(10) in one side of a first preheating block connected with one expression heating block, a chip-releasing portion(12) in one side of a final expression heating block connected with one preheating block, and a partition portion(14) between the preheating block and expression heating block, wherein the preheating block contains at least one heater and temperature sensor; and the expression heating block contains at least one temperature sensor, heater and optical channel(8).

Description

Heating Block Apparatus for Amplifying DNA {Heating Block Apparatus for Amplifying DNA}

1 is a perspective view of a heating block according to the present invention,

2 is a cross-sectional view of the heating block according to the present invention;

3 is a configuration diagram of a chip input unit and a chip discharge unit according to the present invention;

4 is a cross-sectional view of a preheating heating block equipped with a chip input unit and an expression heating block equipped with a chip discharge unit according to the present invention;

5 is another cross-sectional view of a preheating heating block equipped with a chip input unit and an expression heating block equipped with a chip discharge unit according to the present invention;

6 is a block diagram of a temperature control device of the preheating heating block and the expression heating block according to the present invention;

7 is a view showing a spatial temperature measurement of the preheating heating block and expression heating block according to the present invention.

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

2: heating block 4: preheating heating block

6: expression heating block 8: optical channel

10: chip input unit 12: chip discharge unit

14 bulkhead portion 16 shaft

20: LM Guide 22: Rack Gear

24: lift support 26: power unit

28: control unit 30: temperature sensor

32: heater 34: chip recognition sensor

36: chip transport part 42: chip feed tray

44: spring 46: partition wall groove

The present invention relates to a PCR (Polymerase Chain Reaction) apparatus for amplifying a DNA chip by heating a DNA chip, and more specifically, by separating a heating block for each temperature unit and observing an amplification process of a DNA chip according to temperature change. The present invention relates to a PCR device that enables accurate measurement according to temperature compensation, and forms a structure capable of moving a DNA chip and amplifying and observing a DNA chip within the block.

DNA amplification is widely used for research and development and diagnostic purposes in the life sciences, genetic engineering and medical fields. In particular, DNA amplification by polymerase chain reaction (hereinafter referred to as PCR DNA amplification) is widely used. It is utilized.

In the field of PCR DNA amplification, various devices and methods have been developed and used to automate the configuration of PCR devices to amplify various DNA samples more efficiently and quickly. The basic principles of operation are as follows.

Commercially available PCR DNA amplification techniques include template DNA to be amplified, oligonucleotide primer pairs having sequences complementary to specific sequences of each single strand of template DNA, and high temperature stable DNA polymerases. Samples containing polymerase and deoxyribonucleotide triphosphates (dNTPs) are prepared, and the sequence of specific regions of the template DNA is amplified by repeating a temperature cycle that sequentially changes the temperature of the samples.

Specifically, the amplification technique uses a three- or two-step temperature cycling cycle. The process of achieving DNA amplification by temperature change is as follows.

The first step is the denaturation step, in which the double-stranded DNA is separated into single-stranded DNA by heating the sample to a high temperature. The second step is an annealing step, in which the sample subjected to the denaturation step is cooled to an appropriate temperature, where the single-stranded DNA and the primer are double-stranded and partially double-stranded DNA-primer complexes (DNA-primer). to form a complex). The third step is a polymerization step, in which the primer of the DNA-primer complex is extended by the polymerase by polymerization to maintain the sample subjected to the annealing step at an appropriate temperature. The step is to clone a new single stranded DNA with complementary sequences.

By repeating these three steps 20 to 40 times in sequence, DNA between two primers is replicated every cycle, resulting in millions of times or more of DNA amplification.

The temperature in the denaturation step is a value in the range of 90 ℃ to 95 ℃, the temperature in the annealing step is appropriately adjusted according to the melting point (Tm), that is, Tm value of the primer used, typically 40 ℃ to 60 It is a value in the range of ° C. Temperature in the polymerization step according to the mainly sseomeoseu high temperature stability TAG DNA polymerase extracted from aqua tea kusu (Thermus aquaticus) using 72 ℃ the optimum activation temperature of (Taq DNA Polymerase), to use a three-step temperature cycle cycle the Since the active temperature range of the TAG DNA polymerase is quite wide, a two-step temperature cycle is also used in which the temperature of the annealing step and the polymerization step are equalized to circulate the temperature.

Here, if the maintenance of a constant temperature and the temperature change step by step is not made quickly, if the temperature is lowered in the annealing step, since the primer is not attached to the position to be amplified, it has a great effect on the yield.

As described above, a conventional PCR apparatus for amplifying DNA by heating a DNA chip bundles a plurality of small tubes containing DNA and amplifies the PCR in the same temperature cycle, or forms a module at the bottom of the DNA chip to raise and lower the temperature by a silicon heater. This is how you do it.

The above-mentioned device has the advantage of analyzing DNA samples of several patients at once, but it is difficult to test several types of DNA because the temperature conditions for maximizing amplification are different for each DNA type.

On the other hand, the PCR amplification method for constructing the module to raise and lower the temperature by the heater has a problem that the heat distribution is caused by the heating by contact, not only the heat distribution is uneven, but also the coating surface of the heater is damaged. Cause of failure, there is a problem that does not amplify DNA.

In addition, the air temperature in the empty space inside the module has a great influence on the temperature on the chip, and generates a difference between the control temperature and the measured temperature according to the gap between the chip and the silicon heater.

It is very important to control the temperature of each step by repeating the denaturation, annealing, and polymerization process, which is the temperature mechanism of PCR amplification, and the amplification temperature should be maintained at an error range of ± 0.05. Therefore, because there are many variations, there are many variations among modules, variations between equipments, and between specimens, which reduces the reliability of equipment.

The present invention has been made to solve the above problems, by maintaining a constant temperature through the heating block to minimize the error due to temperature changes by uniformizing the temperature of the DNA chip, the heating block preheating heating block and expression heating block There is a technical problem to provide a heating block of the PCR device that can be separated to shorten the time to reach the desired temperature, shorten the DNA expression time, and can analyze multiple DNA chips at the same time.

According to the present invention, at least three pairs are repeatedly installed by using a preheating block and an expression heating block as a pair, and a chip input unit is mounted on one side of the first preheating heating block in which only one expression heating block is connected and installed. It provides a heating block of the PCR device equipped with a chip outlet on one side of the final expression heating block is connected to the preheating heating block only.

Heating block according to the present invention, if necessary, the partition can be installed between the preheating heating block and the expression heating block.

The heating block is repeatedly installed at least three pairs of preheating block and expression heating block in a pair, and by alternately installing the preheating block and expression heating block so that the DNA chip can quickly reach the temperature required for PCR amplification It also allows multiple DNA chips to be analyzed simultaneously.

The preheating heating block is for preheating the DNA chip to a temperature required for amplification, and one or more heaters and one or more temperature sensors are installed at one side inside the preheating heating block to provide a temperature for DNA amplification.

On the other hand, the expression heating block is for expressing the DNA chip amplified in the preheating block, one or more heaters and one or more temperature sensors required for temperature control is installed on one side inside the expression heating block, the analysis of the DNA chip The optical channel required for penetrating the upper surface of the expression heating block is installed to analyze the DNA chip located inside the expression heating block.

The preheating heating block and the expression heating block constituting the heating block provides a constant temperature required for PCR amplification, and temperature compensation over a large number can significantly reduce the temperature deviation.

Here, when the DNA chip having a temperature difference is introduced into the preheating heating block or the expression heating block, heat is rapidly spread by the principle of entropy to maintain the equilibrium of the system.

The chip input unit is to be inserted into the DNA chip to be amplified into the preheating block, is formed on one side of the first preheating heating block is connected to only one expression heating block.

The chip discharge part is to allow the amplified DNA chip to be discharged to the outside of the expression heating block, and is formed on one side of the final expression heating block in which only one preheating heating block is connected.

The partition wall is provided as a partition wall that separates the preheating heating block and the expression heating block and blocks the movement of the heat, and is opened when the DNA chip is moved. As a preferred example, the partition wall body has a flat plate shape to block the movement of the heat while providing the partition. It is provided at the top of the partition body and the partition wall is lowered when opening the partition wall to provide a passage for the preheating heating block and expression heating block, provided in the bottom of the partition wall portion is composed of a spring compressed or restored when the partition wall portion.

If necessary, the heating block according to the present invention includes a chip transporting device for transporting the DNA chip, the transporting device is a preheat heating block and the DNA chip seated on the expression heating block to the preheating heating block or expression heating block It is for moving to another preheating heating block or expression heating block that is connected to, and may be used as long as it can achieve this purpose, but it is preferable to use a transportation device commonly used in the art. Most preferably, a transportation device using a piston or an air cylinder as a power source is preferable.

Hereinafter, exemplary embodiments of the present invention will be described with reference to the accompanying drawings.

1 is a perspective view of a heating block according to the present invention, Figure 2 is a cross-sectional view of the heating block according to the present invention, Figure 3 is a block diagram of the chip input unit and the chip discharge unit according to the present invention, Figure 4 according to the present invention A cross-sectional view of a preheating heating block equipped with a chip input unit and an expression heating block equipped with a chip discharge unit, and FIG. 5 is another cross-sectional view of a preheating heating block equipped with a chip input unit and an expression heating block equipped with a chip discharge unit according to the present invention. 6 is a configuration diagram of a temperature control apparatus of a preheating heating block and an expression heating block according to the present invention, and FIG. 7 is described together with a view showing a spatial temperature measurement mode of the preheating heating block and the expression heating block according to the present invention. .

In the heating block 2 according to the present invention, as shown in FIGS. 1 to 7, at least three pairs or more are repeatedly installed by using the preheating heating block 4 and the expression heating block 6 as one pair. The chip input unit 10 is mounted on one side of the first preheating heating block 4 in which only the expression heating block 6 is connected, and only one preheating heating block 4 is connected to the final expression heating block 6. It consists of a heating block (2) of the PCR device is equipped with a chip outlet 12 on one side.

In particular embodiments, the heating block 2 may include a preheating heating block 4 and at least one temperature sensor 30 equipped with at least one heater 32 and at least one temperature sensor 30, as shown in FIG. 1. Initial preheating in which at least three or more pairs are repeatedly installed with one or more expression heating blocks 6 equipped with one or more heaters 32 and optical channels 8 and only one expression heating block 6 is connected and installed. A chip input unit 10 into which a DNA chip is introduced at one side of the heating block 4 is mounted, and a chip in which the DNA chip is discharged at one side of the final expression heating block 6 in which only one preheating heating block 4 is connected and installed. The discharge part 12 is mounted, and is provided between the preheating heating block 4 and the expression heating block 6 to serve as a partition wall separating the preheating heating block 4 and the expression heating block 6 from the DNA chip. It may be composed of a heating block (2) of the PCR device including a partition portion 14 that is opened during movement.

The preheating heating block 4 preheats the DNA chip so that the DNA chip can quickly reach the amplification temperature, and forms a pair with the expression heating block 6 connected to each preheating heating block 4 to amplify the DNA chip. Provide the required temperature.

The preheating heating block 4 measures the spatial temperature with at least one or more temperature sensors 30, and the heater 32 mounted with at least one or more heats the DNA chip to a temperature required for amplification. In one preferred embodiment, the DNA chip is heated to about 95 ° C. in the denaturation step, about 55 ° C. in the annealing step, and about 75 ° C. in the polymerization step.

The expression heating block 6 constitutes a pair with the preheating heating block 4 connected to each expression heating block 6 by observing the change while maintaining the DNA chip having reached the required amplification temperature in the same temperature range. Maintain the temperature required for amplification of the chip. In a preferred embodiment, the DNA chip may be maintained at about 95 ° C in the denaturation step, about 55 ° C in the annealing step, and about 75 ° C in the polymerization step.

The space temperature is measured by at least one temperature sensor 30 mounted inside the expression heating block 6, and maintains a proper temperature by the heater 32 mounted with at least one, and the expression heating block 6 ) The optical channel 8 penetrating the outer top surface is mounted so that the change of the DNA chip can be directly observed. In a particular embodiment, the heating block 2 according to the present invention is the preheating heating block 4 and the expression heating block 6 are sequentially and repeatedly installed in three pairs of the preheating heating block 4 and the expression heating block 6. It consists of a structure that performs amplification to about 95 ℃ in the denaturation step, amplification to about 55 ℃ in the annealing step, amplification to about 75 ℃ in the polymerization step at a time.

As shown in FIG. 2, the partition wall 14 is installed as a partition wall connected between the preheating heating block 4 and the expression heating block 6 and separating the preheating heating block 4 and the expression heating block 6. At the same time it is opened upon movement of the DNA chip.

As a specific embodiment, the partition wall 14 according to the present invention is installed in a flat plate-shaped connection between the preheating heating block 4 and the expression heating block 6, the size of the DNA chip is movable on the top of the partition wall (14) It has a groove having. When the partition 14 serves as a partition wall, the partition wall groove 46 is seated on the partition between the preheating heating block 4 and the expression heating block 6 to be blocked. The partition surface refers to a portion in which one surface of the preheating heating block 4 and the expression heating block 6 contacting the partition wall 14 is closed when the partition wall 14 is inserted. In addition, when the DNA chip is moved from the preheating block 4 or the other expression heating block 6 to the preheating heating block 4 or the expression heating block 6, the partition 14 is the control unit 28. When the partition wall 14 is lowered, the spring 44 at the bottom of the partition wall 14 is compressed, and the partition wall groove 46 provided at the top of the partition wall 14 is lowered to express the preheating heating block 4. The spaces of the heating block 6 are connected to each other.

When the movement of the DNA chip is completed, the partition 14 is removed from the load applied to the partition 14 by the control unit 28 and is partitioned by the restoring force of the spring 44 provided at the lower end of the partition 14. Groove provided in (14) rises to separate the space that the preheating heating block 4 and the expression heating block 6 is connected.

As shown in FIG. 3, the chip input unit 10 and the chip discharge unit 12 are mounted on one side of the first preheating heating block 4 in which only one expression heating block 6 is installed. The DNA chip is inserted, and the chip discharge part 12 is mounted on one side of the final expression heating block 6 in which only one preheating heating block 4 is connected, and the DNA chip is discharged.

The chip input unit 10 and the chip discharge unit 12 are provided with at least one chip recognition sensor 34 connected to one side adjacent to the chip input unit 10 and the chip discharge unit 12, and the DNA chip is chipped. When seated on the song tray 42, the chip recognition sensor 34 recognizes the DNA chip, and the recognized signal is sent to the control unit 28 to transfer the chip transfer tray 42 to the preheating heating block 4 and the expression heating block ( 6) move inside or outside. Here, the chip input unit 10 and the chip output unit 12 includes a driving device for transporting the DNA chip.

At this time, a preferred control sequence in which the DNA chip is inserted into the preheating heating block 4 through the chip input unit 10 is a chip transfer tray 42 for inserting the DNA chip into the preheating heating block 4. 4) When moved to the outside, the DNA chip is mounted on the chip transport tray 42, the chip recognition sensor 34 detects the seated DNA chip, and if the DNA chip is detected, the chip recognition sensor 34 controls the controller 28. ) Transmits an electrical signal, and the rack gear 22 receiving the electrical signal from the controller 28 guides the chip transfer tray 42 into the preheating heating block 4. Preferably, a button is provided at the chip input unit, and more preferably, the tray is discharged when the button is pressed, such as a CD-ROM.

As shown in FIGS. 4 and 5, the driving device for transporting the DNA chip includes a preheating heating block 4 in which the chip input unit 10 is connected and an expression heating block 6 in which the chip discharge unit 12 is connected. A chip transport unit 36 for transferring DNA chips therein, a rack gear 22 provided at both lower ends of the chip transport tray 42 to move the chip transport tray 42, and the rack gear 22 and the rack. It is provided between the gears 22 and the shaft 16 for seating the DNA chip after the preheating or heating step, and is installed at the lower center of both rack gears 22 to move to the next step when the DNA chip reaches the amplification temperature. A lift supporter 24 for raising the shaft 16 and a lower center portion of the chip transfer tray 42 are provided inside or outside the preheating heating block 4 or the expression heating block 6. To the LM guide 20, the LM guide 20 or the rack gear 22, It includes a power unit 26 provided on one side inside the block for supplying power, and a control unit 28 provided on one inside of the block for controlling the above-described configuration. Here, the power unit 26 may use a conventional motor or the like.

The control unit 28 is a rack gear 22, the lift supporter 24, the power unit 26, chip recognition sensor 34, chip transfer tray 42, temperature sensor 30, heater 32, partition wall The internal circuits are respectively connected to the unit 14, and electrical signals are transmitted / received to drive the heating block 2 device.

Referring to Figure 6, the configuration for controlling the temperature of the heating block 2 according to the present invention is equipped with at least one heater 32 in the preheating heating block 4 and the expression heating block 6, the space temperature At least one temperature sensor 30 necessary for controlling is distributed and inserted into the block. As a preferable example with respect to the distributed installation of the heater 32 and the temperature sensor 30, the heater 32 is mounted one each up / down inside the block, and the temperature sensor 30 before / after / left / right A total of eight inserts, one at each corner of the top and bottom, and two at the middle of the left and right sides are controlled to control the space temperature to reduce the compensation range of the temperature as much as possible.

Here, the temperature compensation method inside the block is a method of measuring the spatial temperature according to the thermal distribution of each temperature sensor 30, as shown in FIG. After the dog calculates the average value of the temperature deviation, two to three seconds later, four temperature sensors 30 near the exit from which the DNA chip is discharged from the other side of the inlet calculate the average value of the temperature deviation, and then the left and right side temperature sensors 30 Compute the temperature by calculating the average value of the temperature deviation. The reason for obtaining the average value of the temperature deviation after 2 to 3 seconds is to cause the diffusion of heat for a predetermined time.

Initially, the temperature sensor 30 of the preheating heating block 4 and the expression heating block 6 must compensate the temperature to calculate the proper space temperature inside the block, but the temperature set through the initial temperature compensation is used semi-permanently. This is possible.

If necessary, the heating block 2 according to the present invention includes a chip transport device 36 for transporting the DNA chip, the chip transport device 36 is a preheating heating block 4 and the expression heating block 6 In order to achieve this purpose, the DNA chip seated on the c) is transferred to another preheating block 4 or the expression heating block 6 connected to the preheating block 4 or the expression heating block 6. Any thing may be used as long as it is present, but it is preferable to use a transportation device commonly used in the art. Most preferably, a transportation device using a piston or an air cylinder as a power source is preferable.

The operation of the heating block 2 according to the present invention configured as described will be described as an example of the automated heating block 2 as follows.

First, the denaturation step, the first step for DNA amplification, presses a button installed on the chip input to heat the DNA chip, the sample to be amplified. Preferably, by operating the drive by pressing a button, such as a CD-ROM, the chip transfer tray 42 is discharged to the outside. Then, when the chip transfer tray 42 is discharged to the outside of the heating block 2, the DNA chip is placed on top of the chip transfer tray 42.

Next, when the DNA chip is recognized by the chip recognition sensor 34, the recognition signal of the chip recognition sensor 34 is sent to the controller 28 to operate the driving device based on the signal sent to the controller 28. The chip feed tray 42 is drawn into the preheating heating block 4. Next, the DNA chip seated on the preheating heating block 4 is heated to about 95 ° C., which is a DNA amplification temperature.

Next, the heated DNA chip is moved to the expression heating block 6 installed in connection with the preheating heating block 4 by opening the partition 14 and measuring the temperature with a temperature sensor 30 mounted therein. The DNA chip is heated by the heater 32 to maintain an appropriate temperature, and the change of the DNA chip is directly observed through the optical channel 8 mounted on each expression heating block 6.

As such, by heating to a high temperature, the double-stranded DNA is separated into a single strand in the denaturation step.

The annealing step, the second step, is a step of lowering the denatured DNA to about 55 ° C.,

The DNA chip 40 is moved from the expression heating block 6 to the next preheating heating block 4 and kept constant at about 55 ° C. to lower the temperature of the DNA chip, and when the temperature reaches about 55 ° C., the DNA chip is expressed. Observe by moving to the heating block (6).

Here, when a DNA chip having a temperature difference is introduced into the preheating heating block 4, heat is rapidly diffused by the principle of entropy to maintain the equilibrium of the system.

The single-stranded high-temperature DNA chip is cooled to form a double-stranded DNA-primer complex by combining double-stranded DNA and primers with a double helix.

The third step, the polymerization step (Polymerization step), by moving the DNA chip subjected to the annealing step to the preheating heating block (4) and heating to about 72 ℃, DNA primer polymerase polymerase to the primers of the DNA-primer complex This is a step of replicating a new single-stranded DNA having a sequence complementary to the original DNA chip by extension and observing in the expression heating block (6).

By repeating these three steps 20 to 40 times in sequence, DNA between the two primers can be replicated every cycle to achieve DNA amplification up to millions of times or more.

As described above, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. Therefore, the embodiments described above are to be understood as illustrative and not restrictive in all aspects. The scope of the present invention is shown by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

The heating block of the PCR device according to the present invention is not configured to raise or lower the temperature by a heater by constructing a module, but to maintain a constant temperature through a heating block to uniformize the temperature of the DNA chip and thereby reduce the error according to the temperature initialization. Minimizing, dividing the block into preheating blocks and expression blocks to shorten the time to reach the temperature required for DNA amplification, shorten the DNA expression time, there is an effect that can analyze multiple DNA chips at the same time.

Claims (6)

At least three pairs are repeatedly installed by using a preheating block and an expression heating block as a pair, and a chip input unit is mounted on one side of the first preheating heating block in which only one expression heating block is connected and installed. Heating block of the PCR device is equipped with a chip outlet on one side of the final expression heating block that is installed only connected. The method of claim 1, Heating block, characterized in that the preheating block is equipped with one or more heaters and one or more temperature sensors. The method of claim 1, Expression heating block is a heating block, characterized in that equipped with one or more temperature sensors, one or more heaters and optical channels. The method of claim 1, Heating block, characterized in that the partition wall connection is installed between the preheating heating block and the expression heating block. The method of claim 1, Heating block, characterized in that the chip recognition sensor is connected to the chip input unit and the chip discharge unit. The method of claim 1, And a driving device for transporting the DNA chip in the preheating heating block connected to the chip input unit and the expression heating block connected to the chip discharge unit.

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