KR100730364B1 - A method for preparing PCR premixtures by drying at room temperature or warm temperature - Google Patents

Abstract

In view of the fact that the DNA polymerase is a heat-resistant enzyme, the present invention solves the disadvantages such as increase in production cost, need for separate centrifugation, and delay in production time, which are problems caused by relying on lyophilization. According to the present invention, by adding DNA polymerase, reaction buffer, dNTP, and gelling buffer to room temperature or warm-drying, an excellent or at least equivalent effect can be obtained.

Room temperature drying, warming drying, PCR reaction mixture

Description

Method of preparing PCR premixtures by room temperature or warm drying method {A method for preparing PCR premixtures by drying at room temperature or warm temperature}

1 is an electrophoretic photograph of a PCR reaction using a reaction mixture according to a conventional protocol and a reaction mixture according to the present invention.

2 is an electrophoresis result of PCR reaction using a reaction mixture (including a stabilizer and a settling agent) and a reaction mixture according to the present invention used in the conventional freeze-drying method,

Figure 3 is a electrophoresis result of the PCR reaction using the conventional lyophilization method, the reaction mixture by room temperature drying and warm drying according to the present invention,

Figure 4 is an electrophoresis result of the PCR reaction using the reaction mixture according to the warm dry conditions according to the present invention,

Figure 5 is an electrophoresis result of the PCR reaction to confirm the stability in various storage conditions to see whether the stability in the storage conditions of the reaction mixture prepared by the conventional lyophilization method, the same even in the reaction mixture according to the present invention .

The present invention relates to a method for preparing a PCR reaction mixture, and more particularly, to a PCR reaction mixture obtained by heating the PCR reaction mixture at room temperature or under a specific condition, a method for preparing the same, and a room temperature or warm dried PCR reaction mixture as an active ingredient. The present invention relates to a kit for sequencing analysis, a kit for diagnosing a disease comprising a room temperature or warm-dry PCR reaction mixture as an active ingredient.

Currently, various products related to biotechnology are prepared by lyophilization. The freeze drying method is a method of sublimating ice by freezing an aqueous solution or a material containing a large amount of water and depressurizing it to remove moisture to obtain a dried product. The purpose of most freeze drying can be preserved for a long time through the ease of storage. It aims at obtaining long-term storage at room temperature, and obtaining a product excellent in re-dissolution to water.

According to the Republic of Korea Patent No. 162270, "Method of preparing DNA polymerase reaction mixture", a method of preparing a reaction mixture of stabilized DNA polymerase by adding a stabilizing material to the DNA polymerase reaction mixture in an aqueous state and lyophilizing Is open to the public. Specifically, to the DNA polymerase reaction mixture, that is, a mixture containing a reaction buffer solution, MgCl 2, four dNTPs and DNA polymerase, a stabilizer, a precipitant and a water-soluble dye are added and lyophilized, and the DNA polymerase reaction is performed. A method of making a mixture is disclosed.

In the case of preparing the DNA polymerase reaction mixture using the above-described lyophilization method, in terms of productivity, an expensive lyophilizer is required, the complete lyophilization time takes about 3 to 5 hours, and it is contained in the liquid during lyophilization. Since the bubbles are splashed by decompression, they need to be lyophilized by separate degassing and centrifugation and difficult to mass-produce due to the size limitation of the lyophilizer. In the application of freeze-drying technology of enzymes, the content of water and the amount of enzyme stabilizing material thus affect the enzyme activity. If the ratio is not appropriate, the enzyme activity is generally lowered. In this case, it is pointed out that it affects the degradation of enzyme activity. In particular, during lyophilization, freezing is performed according to the water content, and a suitable stabilizer (preservative) is searched for a water content by searching for a suitable stabilizer (preservative) to compensate for the problem that the activity of the DNA polymerase is lowered due to ice crystals. The fact that it should be added during lyophilization in proportion to the ratio is indicated as a practical difficulty.

The present invention has been studied to solve the above problems, and as a result, the DNA polymerase (Taq DNA Polymerase or pfu DNA Polymerase) is a heat-resistant enzyme, focusing on the application of room temperature drying or warming drying without depending on the lyophilization method While showing excellent results in terms of enzymatic activity during lyophilization, in terms of productivity, the problem of the lyophilization method was solved by introducing a room temperature or warming step after dispensing the solution without the need for centrifugation.

The present invention is a step of dispensing a mixture of DNA polymerase, reaction buffer, dNTP and gel-loading buffer in a tube, and warming or drying at room temperature for 1 hour at 50 ~ 60 ℃ Comprising the step of drying at room temperature for 4 to 5 hours, the PCR reaction mixture according to the present invention shows the same or superior technical results compared to the reaction mixture prepared by the conventional lyophilization method.

An object of the present invention is to dispense a solution containing a DNA polymerase, a reaction buffer, a dNTP and a gel-loading buffer into a tube, and to dry for 1 hour at 50 to 60 ° C. Or it provides a PCR reaction mixture manufacturing method comprising the step of drying at room temperature for 4-5 hours.

Another object of the present invention is to provide a PCR reaction mixture prepared above, and another object is to provide a PCR reaction mixture prepared for sequencing and disease diagnosis kit.

The present invention will be described in detail.

The reaction mixture applied to the present invention is composed of DNA polymerase (Taq DNA Polymerase or Pfu DNA Polymerase), reaction buffer, dNTP solution, and conventional gelling buffer, in particular the core drying method of the present invention. Since is a room temperature or warm-drying method, a stabilizer (preservative) added to prevent the degradation of the DNA polymerase activity required in the conventional lyophilization is not used separately.

In the present invention, the method comprises dispensing a mixture of a DNA polymerase, a reaction buffer, a dNTP, and a gel-loading buffer into a tube, followed by room temperature or warm drying. 4 hours in the case of room temperature drying, 1 hour in the case of 50 ~ 60 ℃ warming drying can be left to dry without a separate degassing step or centrifugation step.

PCR reaction mixture according to the present invention is not only the efficiency due to the simplification of the process and the reduction of production time, but also the reaction mixture by room temperature or warm drying stabilized reaction to ensure the stability according to the storage temperature appearing at the time of DNA polymerase freeze drying To provide a mixture.

Hereinafter, the present invention will be described in detail with reference to examples, but these examples are for explaining the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1

The inventors of the present invention first carried out PCR by inserting DNA polymerase and other reactants (reaction buffer, four dNTPs and MgCl 2) in a manual manner, and the DNA polymerase (Taq DNA Polymerase) and reaction buffers mentioned in the present invention. After mixing only a solution (reaction buffer), dNTP solution and gelling buffer, the PCR experiment was performed as follows after warming at 50-60 ° C. for 1 hour.

Total RNA was extracted from the SNU-5 human cell using the easy-BLUETM Total RNA Extraction Kit (Cat.No.17061) and cDNA was made using the Power cDNA Synthesis Kit (Cat.No.25011). After diluting the prepared cDNA by 1/2 step, the sensitivity was compared by PCR using GAPDH primer. In Figure 1, Panel A is a result of PCR by inserting DNA polymerase and other reactants (reaction buffer, 4 dNTP and MgCl2) by manual method, and Panel B is the DNA polymerase (Taq DNA) mentioned in the present invention. Polymerase), reaction buffer, dNTP solution, and gelling buffer were mixed together, followed by PCR for 1 hour warm-drying at 50-60 ℃. At this time, Lane M is 100bp Ladder Molecular Weight DNA Marker (Cat. No. 24012); Lanes 1 to 5 show electrophoresis results for (1/2)-(1/2) -5 diluted cDNA.

As shown in FIG. 1, as a protocol commonly used in a laboratory, DNA polymerase, a reaction buffer solution, 4 dNTPs, and MgCl ₂ were added to each other, followed by PCR without heating and using only the reaction mixture shown in the present invention. 1 hour warm-drying result does not affect the enzyme activity and can be seen that the equivalent results in the sensitivity section. Therefore, it can be seen that only the reaction mixture of the present invention is suitable for PCR.

Example 2

The present inventors performed the following experiments in order to prepare a PCR result using a reaction mixture (including a stabilizer and a settling agent) according to the conventional freeze-drying method and the warm-dry reaction mixture according to the present invention.

Total RNA was extracted from the SNU-1 human cell using the easy-BLUETM Total RNA Extraction Kit (Cat.No. 17061) and cDNA was made using the Power cDNA Synthesis Kit (Cat.No. 25011). After diluting the prepared cDNA by 1/2 step, the sensitivity was compared by PCR with the reaction mixture and the reaction mixture of the present invention using the GAPDH primer. Panel A is a case using the mixture mentioned in the prior patent, Panel B is a case where the reaction mixture according to the present invention (1 hour warm-drying) is applied. At this time, Lane M is 100bp Ladder Molecular Weight DNA Marker (Cat.No. 24012), lanes 1 to 5 is the result of electrophoresis corresponding to (1/2)-(1/2) -5 diluted cDNA.

As can be seen in Figure 2, in contrast to the reaction mixture of the conventional patent (including stabilizers, settling agents), the reaction mixture according to the present invention is equivalent to the activity of the sensitivity and the like without the stabilizers or sedimentants and other ancillary materials included in the conventional patent The result was obtained.

Example 3

The inventors conducted a sensitivity experiment by PCR using the reaction mixture mentioned in the prior patent (prepared by freeze drying), the reaction mixture dried at room temperature for 4 hours, and the reaction mixture dried at 50 to 60 ° C for 1 hour. . Lambda DNA was diluted 1/10 by 600pg and then subjected to a sensitivity comparison experiment by PCR to obtain a result as shown in FIG. 3. At this time, Panel A is a PCR result for the reaction mixture mentioned in the conventional patent, Panel B is a mixture of DNA polymerase (Taq DNA Polymerase), reaction buffer, dNTP solution and conventional gel-buffing buffer After 4 hours of room temperature drying, PCR was performed, and Panel C was mixed with DNA polymerase (Taq DNA Polymerase), reaction buffer, dNTP solution, and a conventional gelling buffer. Lane M is 1 Kb Ladder Molecular Weight DNA Marker (Cat. No. 24032); lane 1, 600 pg DNA; lane 2, 60 pg DNA; lane 3, 6 pg DNA; lane 4, 600fg DNA; This picture corresponds to lane 5, 60fg DNA.

In comparison with the conventional freeze-drying method in Figure 3, it was confirmed that the room temperature drying (4 hours), warm drying (1 hour) according to the present invention does not affect the enzyme activity and showed an equivalent result in the sensitivity portion. Therefore, as a result of this experiment, it was found that the room temperature or warm-drying method disclosed in the present invention did not affect the maintenance of DNA enzyme activity.

Example 4

The present inventors experimented with DNA polymerase activity change during 1 hour to 4 hours warm drying at 50 ~ 60 ℃ to confirm the stability of the reaction mixture according to the warm drying time.

Total RNA was extracted from the SNU-5 human cell using the easy-BLUETM Total RNA Extraction Kit (Cat.No. 17061), and cDNA was made using the Power cDNA Synthesis Kit (Cat.No. 25011). After diluting the prepared cDNA by 1/2 step, the sensitivity was compared by PCR using the GAPDH primer and shown in FIG. 4. Panel A was PCR after 1 hour of warm drying, Panel B was PCR after 2 hours of warming drying, Panel C was PCR after 3 hours of warming drying, and Panel D was PCR after 4 hours of warming drying. to be. Lane M, on the other hand, was 100 bp Ladder Molecular Weight DNA Marker (Cat. No. 24012); Lanes 1 to 5 correspond to (1/2)-(1/2) -5 diluted cDNA.

As can be seen in Figure 4, even though the warming was carried out for 1 to 4 hours, it was found that the activity and sensitivity of the DNA polymerase according to the warming had no effect at all, thereby confirming the stability of the reaction mixture. there was.

Example 5

The present inventors compared the stability of the reaction mixture according to the storage temperature of the conventional lyophilization method with the stability of the reaction mixture according to the present invention 1 month at room temperature, 6 months after refrigeration and freezing, 12 months after After storage until stability was measured by PCR as follows.

Total RNA was extracted from the SNU-1 human cell using an easy-BLUETM Total RNA Extraction Kit (Cat.No. 17061), and cDNA was prepared using a Power cDNA Synthesis Kit (Cat.No. 25011). After diluting the prepared cDNA by 1/2 step, the sensitivity was compared by PCR using the GAPDH primer, and the result of the sensitivity test by PCR of the 1Kb PCR Product after the step dilution of Lambda DNA from 600pg for 1/10 step is shown in FIG. 5. Shown in Panel A was tested immediately after drying (GAPDH confirmed), Panel B was PCR tested after 1 month after warming (GAPDH), Panel C was PCR tested 6 months after warming (1 Kb PCR Product) and Panel D is the result of PCR after 12 months of warming drying (checking 1 Kb PCR Product). 5 is (1) frozen storage (2) cold storage (3) room temperature storage experimental data, each lane M in Panel A, B is 100bp Ladder Molecular Weight DNA Marker (Cat. No. 24012); Lanes 1 to 5 are the results of electrophoresis corresponding to (1/2) (1/2) -5 diluted cDNA, and Lane M in Panels C and D is 1Kb Ladder Molecular Weight DNA Marker (Cat.No. 24032) ; lane 1, 600 pg DNA; lane 2, 60 pg DNA; lane 3, 6 pg DNA; lane 4, 600fg DNA; The result of electrophoresis corresponding to lane 5, 60fg DNA.

As shown in Figure 5 it can be seen that after 1 hour warm-drying, even after room temperature storage or refrigeration, freezing storage, the activity change of DNA polymerase can be seen the same stability by storage temperature as the conventional freeze-drying method.

In view of the fact that the DNA polymerase is a heat-resistant enzyme, the present invention solves the disadvantages such as increase in production cost, need for separate centrifugation, and delay in production time, which are problems caused by relying on a conventional freeze-drying method. Alternatively, by heating and drying, it is possible to achieve a better or at least equivalent effect as compared to the conventional method, and at the same time, to achieve technical effects such as a reduction in production cost, no need for centrifugation, and a reduction in production time. In addition, the conventional freeze-drying method contains a variety of samples in the reaction mixture, according to the present invention by adding DNA polymerase, reaction buffer, dNTP, and gelling buffer to reduce the production cost and simplify the process It is a useful invention that can be obtained.

Claims (5)

Dispensing a mixture of DNA polymerase, reaction buffer, dNTP and Gel-Loading buffer in a tube, and the step of 50 ~ 60 ℃ warm drying or room temperature drying, PCR reaction mixture preparation method. The method of claim 1, wherein the warming is carried out for 1 hour or the room temperature drying for 4 hours. delete delete delete

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