KR20010038297A - Multiple- Process Thermal Cycler for Enzyme Reaction and Nucleic Acid Hybridization - Google Patents

Abstract

PURPOSE: Provided is a multi-functional temperature circulator which can conduct various experiments such as enzyme reaction, PCR, LCR, hybridization, DNA chip PCR, and in situ PCR, determine the most appropriate temperature during the reactions, and prevent substances from being evaporated. CONSTITUTION: A multi-functional temperature circulator includes: i) reaction blocks able to control temperature; ii) a tube binding plate (11) moved by up-and-down movement guide equipped with a spring and back-and-forth movement guide and able to control temperature circulation; iii) a separable heating cover (19) inserted into a stopper (15) which is connected with the tube binding plate (11) and covers the tube installed on the tube binding plate (11); and iv) a holding device (18) which selectively binds with the cover able to be separated from or inserted into the tube binding plate (11).

Description

Multi-Process Thermal Cycler for Enzyme Reaction and Nucleic Acid Hybridization

The present invention relates to a temperature circulating device used in PCR (nucleic acid polymerase chain reaction), LCR (ligase chain reaction), nucleic acid hybridization reaction.

Since PCR generally undergoes denaturation, annealing, and elongation processes, a mild temperature device is composed of devices capable of circulating three temperatures. The double stranded template is denatured at high temperature (about 95 ° C.) and the primer is annealed to the denatured single stranded DNA at low temperature (50-65 ° C.). At moderate temperatures (72–75 ° C.), polymerases synthesize DNA from primers. Most instruments allow you to program each temperature and time.

Conventionally, a PCR apparatus of a water bath type has been used. However, in recent years, the constant temperature bath method is almost disappeared, and most of the temperature circulating equipment is a block type machine, which performs temperature cycling by changing the temperature of the metal block. The temperature of the block is programmed and entered and adjusted to the program. In general, 0.5ml tubes are used a lot, but thin tubes or small tubes, which are designed for good heat transfer from the block to the tubes, are also used. Because it changes the temperature of the block, it takes time to raise and lower the temperature. The time it takes to change the temperature depends on the material of the block, how it is heated, and how it is cooled, which also affects the total time it takes to circulate the temperature. Depending on the type of machine and the programming method, the efficiency of the enzyme, nucleic acid hybridization reaction, etc. to be reacted will vary greatly.

Currently, there is a need for a large-capacity temperature circulator capable of simultaneously processing a large number of samples, for example, samples in 384 wells, and of a device capable of temperature cycling on slide glass capable of performing DNA microarray PCR or in situ PCR. Necessity is emerging.

In the temperature cycling reaction, the exact reaction temperature is important. There are many differences in the result of the enzyme reaction product due to the slight difference in reaction temperature. If this is not the optimal reaction temperature, other by-products will be produced in addition to the desired product. Thus, the temperature at which the optimum reaction temperature conditions can be found quickly, i.e., quickly retrieved to the optimum temperature required by the temperature gradient block with wells having various temperatures by continuously decreasing or increasing the temperature on one block. There is a need for a circulation device.

XY robot structure that includes two heating blocks capable of temperature control and one block whose temperature gradient is formed by Peltier elements installed at both ends of the block, and which can be moved between the blocks by a stepping motor controller. The present invention has been completed by developing an opening and closing heating cover.

Accordingly, it is an object of the present invention to provide a temperature circulating device that can set optimal temperature conditions in various temperature cycling experiments and that does not require the use of oil using an automated heating cover.

1 is a front view of a temperature circulating device of the present invention,

2 is a side view of the temperature circulator of the present invention;

3 is a plan view of the temperature circulator of the present invention;

4a to 4c is a front view, a side view, a plan view of a temperature gradient block of the temperature circulation device of the present invention;

5 is a front view showing a heating cover and a holding device of the present invention,

6A to 6C are a front view, a side view, a plan view of a temperature gradient block of another embodiment of the present invention,

Figure 7 shows a side view of the vertical movement guide of the temperature circulation device of the present invention.

<Brief description of the main parts of the drawings>

1: temperature gradient block 2, 3: heating block

5a, 5b: Peltier element 7: heat sink

8: Fan 11: Tube storage plate

18: holding device 19: heating cover

The present invention provides a temperature circulation device using a block method, comprising: reaction blocks (1, 2, 3) capable of adjusting three or more temperatures; One of the blocks (1) has a Peltier element (5a, 5b) is attached to both ends of the bottom surface of the block to form a temperature gradient through the block by cooling or heating the temperature at both ends separately, and on the reaction block An XY robot type tube storage plate 11 capable of adjusting the temperature circulation by moving up and down and back and forth by the vertical movement guide 12 and the front and rear movement guide 13; A separate heating cover 19 inserted into a stopper 15 mounted at both ends of the tube storage plate to cover the tube mounted on the tube storage plate, which is integrated with the storage plate 11; ; The cover is provided with a temperature circulating device, characterized in that it comprises a removable detachable holding device 18, the cover is inserted and detached from the tube storage plate by the XY robot method.

In addition, the present invention is characterized in that the spring 17 is installed in the vertical movement guide of the tube storage plate so that when the tube is inserted into the well in the block, the tube is adhered to the block at a constant pressure so that the heat is transferred from the block to the tube well. Provided is an apparatus.

In addition, in one embodiment of the present invention, the block provides a device characterized in that it has a well of the type in which the slide is inserted.

In another embodiment of the present invention, a hole is formed in a bottom surface of one of the blocks to insert an optical fiber, and a sensor is mounted to detect a fluorescence emitted by projecting light onto the optical fiber. It provides a device characterized in that.

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

As shown in FIG. 3, the block of the present invention includes a first reaction block 1, a second reaction block 2, and a third reaction block 3 not shown. On the upper surface of the block of the present invention, a plurality of wells 4 having a form into which a tube is inserted are formed. The block of the present invention may be inserted with a plurality of tubes or multi-well plate. The second (2) and the third block (3) of the block is a constant temperature heating block set to a constant temperature, the first block (1) is a temperature gradient block is formed a temperature gradient. 4 is a front view, a side view, and a plan view of the temperature gradient block of the present invention. The first block has Peltier 5a, 5b elements attached to both ends of the bottom surface of the block, and the Peltier element 5a, 5b. There are a plurality of temperature sensors 6 attached therebetween. A heat sink 7 is provided directly under the Peltier elements 5a, 5b, and a fan 8 is mounted at one end of the heat sink. Plate heaters and temperature sensors are attached to the bottom surfaces of the second and third blocks.

The present invention includes a tube storage plate 11 for inserting the tube 10 into the block as shown in FIG. 1, which is similar in size to the block and has a plurality of holes for receiving the tube. Up and down movement guides 12 are connected to both sides of the tube storage plate 11, and the front and rear movement guides 13 are connected to the ends of the up and down movement guides, and each guide has a stepping motor 14a, 14b) is attached. A stopper 15 is attached to both sides of the tube storage plate, and a spring 16 is provided between the stopper 15 and the tube storage plate 11. In addition, the vertical movement guide 12 on both sides is provided with a spring 17 in the vertical movement axis direction as shown in FIG. The spring 17 presses the tube at a constant pressure to improve heat transfer from the block to the tube when the tube in the tube storage plate is inserted into the block.

5 shows a holding device 18 and a heating cover 19 for fixing the heating cover of the present invention. As shown, the ball flanza 20 is disposed in the holding device 18 and a sensor 21 is attached to the ball flanza. A rod 22 is formed in the cover, and is engaged with the ball planar 20.

6 shows another embodiment of a block of the invention. When using slide glass without the use of tubes or multi-well plates, the shape of the block shall be such that the slide can be inserted. The present invention includes a block having a well 4 in which a slide is inserted. As in Fig. 4, the Peltier elements 5a and 5b are provided at both ends of the block just below the block, and the heat sink 7 is provided under the block. A fan 8 is attached to one end of the heat sink.

The temperature circulating device of the present invention forms a hole in the bottom of the well of any one block of the block to insert the optical fiber, the sensor is connected to the optical fiber to project light through the optical fiber, the detector for detecting the fluorescence emitted from the optical fiber Can be mounted.

Hereinafter, the operation of the present invention.

If the temperature of the Peltier elements 5a, 5b at both ends of the temperature gradient block 1 of the present invention is set differently, a temperature gradient is formed around the Peltier elements 5a, 5b. In a preferred embodiment of the present invention, the temperature gradient block 1 may be formed a temperature gradient over 35 ℃ to 70 ℃. The temperature gradient formed in the block differs depending on the temperature set in the Peltier elements at both ends. If the Peltier elements at both ends are set at 50 ° C and 60 ° C, the block will form a temperature gradient between 50 ° C and 60 ° C. The temperature of both Peltier elements 5a, 5b can be set differently according to various enzyme reaction purposes. The upper and lower portions of the Peltier element have a temperature difference caused by heat or cold formed by the Peltier element. The heat sink 7 serves to disperse the heat or cold air generated by the Peltier element, and the fan 8 serves to discharge the heat or cold air dispersed by the heat sink to the outside. In a preferred embodiment, the temperature gradient block 1 of the invention can be used as an annealing block. The annealing temperature is very important in the annealing step in the PCR reaction. By using the temperature gradient block of the present invention in the annealing step, it is possible to determine the correct annealing temperature. The heating block is equipped with a heater at the bottom of the block and is kept constant at the set temperature. In the present invention, the heating block may be preferably used in an elongation step and a denaturation step. Usually, the expansion step is carried out between 70 ° C and 75 ° C, and the denaturation step is carried out at 90 ° C to 95 ° C.

The tube storage plate 11 of the present invention is moved up and down and back and forth by the vertical movement guide 12, the front and rear movement guide 13, and the stepping motors 14a and 14b. The up-and-down movement guide 12 has an up-and-down motion of inserting the tube storage plate 12 into or out of the block, and the up-and-down movement guide has a back-and-down movement action of moving the tube storage plate between blocks.

The tube storage plate 11 and the heating cover 19 are integrally formed, and the heating cover 19 is inserted under the stopper 15 on both sides of the tube storage plate 11 to be integrated with the tube storage plate. When the heating cover 19 is separated from the tube storage plate 11, the stopper 15 moves horizontally on the upper surface of the heating cover, so that the heating cover is separated from the tube storage plate. The heating cover of the present invention is equipped with a heater to maintain the reaction temperature during the PCR reaction is to prevent the evaporation of the reactants. The heating cover 19 is formed with a rod 22 is inserted into the insertion portion of the holding device is engaged with the ball planar 20. The sensor 21 detects whether the ball planar and the rod are inserted. Usually, the heating cover is fixed to the holding device. When the PCR reaction is started, the tube storage plate is moved toward the heating cover fixed to the holding device by moving up and down and back and forth of the tube storage plate to insert the heating cover under the stopper 15. Thereafter, the downward movement by the downward movement guide is greater than the force engaged with the ball planner, and the heating cover 19 is separated from the holding device 18. During the PCR reaction, the heating cover is moved integrally with the tube storage plate. When the PCR reaction is completed, the heating cover is fixed back to the holding device, the cover is opened, and then the multi-well plate is recovered from the tube storage plate. Thus, the heating cover 19 of the present invention can be detachably automatically, so that after the completion of the reaction, the recovery of the multi-well reaction plate is simplified, and the finished reactant can be readily used in the next process. All of these processes are controlled by the program. The device of the present invention is capable of a PC interface and can be controlled by a PC.

In another embodiment of the present invention, the block is a well in the form of inserting the slide, the slide itself can perform a PCR reaction, nucleic acid hybridization reaction, etc. can perform a DNA chip, or in situ PCR .

According to the present invention, 96-well or 384-well plates can be processed within a short time, and the optimum reaction temperature can be determined at a time by a temperature gradient, and there is a heating cover, so there is no fear of evaporation, and thus no oil needs to be used.

As described above, according to the present invention, a large volume of samples can be circulated to a desired temperature within a short time, and various enzyme reactions, PCR, LCR (ligase chain reaction), hybridization reaction, DNA chip PCR, and in situ PCR are performed. It is possible to determine the optimum reaction temperature at a time by the temperature gradient block, and the heating cover which is automatically opened and closed simplifies the processing process and eliminates the fear of evaporation of the reactants.

Claims (4)

A temperature cycling device using a block method, comprising: reaction blocks (1, 2, 3) capable of adjusting three or more temperatures; One of the blocks is formed with Peltier elements 5a and 5b at both ends of the bottom of the block to form a temperature gradient through the block by individually cooling or heating the temperature at both ends, and the upper and lower motion guides 12 XY robot-type tube storage plate 11 that can adjust the temperature circulation by moving up and down, back and forth by the front and rear movement guide 13; A detachable heating cover (19) covering the tube mounted on the tube storage plate, which is inserted into the stopper (15) mounted at both ends of the tube storage plate and integrated with the storage plate; And the cover is detachable from the tube storage plate and is inserted and detachable holding device (18). The method according to claim 1, wherein when the tube (10) is inserted into the well (4) in the block by installing a spring (17) in the vertical movement guide of the tube storage plate, the tube is brought into close contact with the block and heat transfer from the block to the tube is prevented. The device characterized in that well done. 2. The apparatus of claim 1, wherein said block has a well of a type into which a slide is inserted. The apparatus of claim 1, wherein a hole is formed in a bottom surface of one of the blocks to insert an optical fiber, and a detector is mounted to detect a fluorescence emitted by projecting light onto the optical fiber. .