KR0162270B1 - Method for preparing dna-polymerase - Google Patents

Abstract

The present invention provides a method for preparing a reaction mixture of stabilized DNA polymerase by adding a stabilizing material to the DNA polymerase reaction mixture including a reaction buffer solution, MgCl ₂ , four dNTPs and a DNA polymerase, and lyophilizing. It is about. The freeze-dried DNA polymerase reaction mixture prepared by the method of the present invention simplifies the multi-step operation in which each component for the reaction of the DNA polymerase should be mixed for each sample, increases the stability of the reaction mixture, Increase the reliability in reproducibility. When the lyophilized DNA polymerase reaction mixture prepared above is used as a sequencing kit or a disease diagnosis kit, highly reliable results can be obtained in a short time.

Description

Method for preparing DNA polymerase reaction mixture

FIG. 1A shows base sequences of primers P1 (20mer) and primers P2 (18mer) synthesized using λ DNA as a template.

Figure 1b is a photograph showing the result of agarose gel electrophoresis of the PCR reaction product amplified using the primers described in Figure 1a.

Figure 2 is a photograph showing the result of agarose gel electrophoresis of the amplified reaction product by PCR reaction of the reaction mixture of the DNA polymerase to which the stabilizing material is added.

FIG. 3 is a photograph showing the result of agarose gel electrophoresis of the amplified reaction product by lyophilizing the reaction mixture in the aqueous solution used in FIG. 2, dissolving it in distilled water and PCR reaction.

FIG. 4 is a photograph showing the result of agarose gel electrophoresis of the amplified reaction product by leaving the lyophilized reaction mixture used in FIG. 3 at 50 ° C. for 62 hours and then PCR reaction.

FIG. 5 is a photograph showing the result of agarose gel electrophoresis of the amplified reaction product after the reaction mixture in a freeze-dried state and a control group to which the stabilizing material was added was left at 50 ° C. for a predetermined time.

FIG. 6 is a photograph showing the result of agarose gel electrophoresis of the amplified reaction product, which was left to react at 60 ° C. for 62 hours in a freeze-dried reaction mixture to which polyol was added.

7 is a photograph showing the result of agarose gel electrophoresis of the amplified reaction product by dissolving the reaction mixture in a lyophilized state to which a water-soluble dye is added in distilled water and PCR reaction.

8 is an electrophoresis photograph showing the result of determining the nucleotide sequence after leaving the lyophilized reaction mixture to which the stabilizing material is added at a predetermined temperature for 12 hours.

The present invention relates to a method for preparing a DNA polymerase reaction mixture. More specifically, the present invention relates to a reaction mixture of a DNA polymerase by adding a stabilizing material to the DNA polymerase reaction mixture and lyophilized, and a method of preparing the same.

DNA polymerase chain reaction (hereinafter, referred to as 'PCR reaction' for convenience) is two oligonucleotides that are chemically synthesized by using the sock end sequence of the portion to be amplified on the genome as a template. And DNA polymerase to amplify a specific DNA sequence, wherein the two oligonucleotides complementary to each other in the double chain of the DNA double chain act as a primer for DNA synthesis. Amplify. Each cycle of the PCR reaction consists of denaturation that separates the DNA double chain, annealing that hybridizes primers and complementary DNA sequences, and an extension that synthesizes DNA. Proceed 40 times to amplify the desired DNA.

After all, a PCR reaction is a reaction that allows only a specific DNA fragment from an organism's genomic DNA to be selectively cloned and amplified in vitro, thereby enabling the search and acquisition of a specific gene in a short time. This response is based on the search for genetic disease related genes (Suzuki, Y. et al., Anal. Biochem., 192: 82-84 (1991); Gibbs, RA et al., Nucleic Acids Res., 17: 2374-2448 (1989); Ballabio A. and Gibbs, RA, Nature, 343: 220 (1990), search and expression of mRNA through amplification of cDNA (RT-PCR, RACE) (Rappolee, DA et al., Science, 241: 708-712 (1991); Frohman, MA et al., Proc. Natl, Acad. Sci., USA, 85: 8998-9002 (1988)), direct sequencing from amplified DNA (direct sequencing (Gyllensten, UB and Erlich, HA, Proc. Natl. Acad. Sci., USA, 85: 7652-7657 (1988)), variable number of tandem repeat (VNTR) analysis (Ali, S. and Wallace, RB, Nucleic Acids Res., 16: 8487-8496 (1988) and preparation of genetic maps (Nelson, DL et al., Proc. Natl. Acad. Sci., USA, 86: 6686-6690 (1989)) is widely used in the life sciences. In addition, human T-cell lymphoma / leukemia virus type I (HTLV-I) (Kwok, S. et al., Blood, 72: 1117-1123 (1988)), human immunodeficiency virus (HIV) (Ou , CY et al., J. Infect. Dis., 158: 1170-1176 (1988)), hepatitis B virus (HBV) (Kaneko, S. et al., Proc. Natl. Sci., USA, 86) : 312-316 (1989)) is also used in the diagnosis of various diseases, such as PCR reactions, the use of the situation continues to expand.

In addition, the development of DD-PCR (differential display-PCR) and Immuno-PCR (Hong, Z. et al., Nucleic Acids Res., 21: 6038-6039 (1993)) using an electric PCR reaction was developed. Therefore, it is also used to detect a very small amount of samples that could not be detected by conventional methods.

In general, amplification of DNA by PCR reaction is performed by template DNA (paired DNA), a pair of primers, reaction buffer, MgCl ₂ , KCl, four dNTPs (dATP, dCTP, dGTP and dTTP) and DNA polymerase, respectively. It is made by mixing the reactive components of, and then reacting the DNA polymerase. Therefore, there is a problem in that the amount of troublesome use and mixing of trace amounts of each reactive component to be mixed for each sample occurs. In particular, when a large amount of samples are to be analyzed in a short time, the experimental hassle and error is an important problem to be solved. In addition, PCR. When the reaction product is to be analyzed, a sample loading buffer should be mixed with the PCR reaction product. The aerosol generated at this time (Kwok, S. and Higuchi, R., Nature, 339: 237-238 (1989)), carry over contamination caused by PCR reaction products, in particular, false positives in the diagnosis of the disease, therefore must be resolved in the diagnosis of the disease using the PCR reaction It's the most important thing to do.

Accordingly, the inventors of the present invention, in order to solve the above problems, the DNA polymerase is added to the DNA polymerase reaction mixture containing a PCR reaction buffer, MgCl ₂ , four dNTPs and DNA polymerase and lyophilized DNA polymerase When the reaction mixture was prepared and reacted with distilled water, template DNA and primers, it was confirmed that the DNA could be synthesized immediately without any experimental error. Also, a precipitant and a water-soluble dye were added to the electric DNA polymerase reaction mixture. The reaction mixture of the added and lyophilized DNA polymerase was found to be capable of preventing contamination by PCR reaction products, thus completing the present invention.

After all, the main object of the present invention is to provide a method for preparing a reaction mixture of stabilized DNA polymerase by adding a stabilizing material to the DNA polymerase reaction mixture in an aqueous solution and lyophilized.

Another object of the present invention is to provide a DNA polymerase reaction mixture prepared in the former.

Still another object of the present invention is to provide a DNA polymerase reaction mixture prepared as above for the sequencing and disease diagnosis kit.

Hereinafter, the present invention will be described in more detail.

The DNA polymerase reaction mixture of the present invention is a lyophilized DNA polymerase reaction mixture including a reaction buffer solution, MgCl ₂ , four dNTPs and a DNA polymerase, or a primer, or a primer and a template to the electric reaction mixture. It is prepared by lyophilizing a DNA polymerase reaction mixture including DNA. The DNA polymerase reaction mixture of the present invention may be subjected to various DNA polymerase reactions such as adding distilled water, primer and template DNA; distilled water and template DNA; or distilled water only to the electric DNA polymerase reaction mixture. Can be. For example, when a DNA polymerase reaction mixture is to be used for diagnosis of HIV, HBV, TB (tuberculosis), a reaction mixture is prepared to include a primer complementary to a genomic DNA portion of an electric virus, and a DNA sequence If a DNA polymerase reaction mixture is to be used for the determination, the reaction mixture may be prepared to include a universal primer or an optional primer.

On the other hand, gelatin, bovine serum albumin (BSA), ammonium sulfate, and Thesit can be used to stabilize DNA polymerase and dNTP.Non-ionic surfactants such as NP40 and Tween 20 are added to the reaction mixture to improve reactivity. (Saiki, RK et al., Science, 239: 487-491 (1988)). Thus, in the present invention, as a stabilizing material of the DNA polymerase reaction mixture, a polyhydric alcohol (polyol) such as gelatin, bovine serum albumin, Thesit, PEG-8000, and glycerol, glucose, mannitol, galactitol, glycitol, sorbitol, etc. Most preferably, polyhydric alcohol is used. At this time, the use of the polyhydric alcohol as a stabilizing material has the advantage that the polyhydric alcohol is also used as a settling agent, there is no need to add a settling agent separately.

On the other hand, the DNA polymerase reaction mixture of the present invention is added to the DNA polymerase reaction mixture in the aqueous solution state without the addition of the electrostabilizing material, or to the DNA polymerase reaction mixture in the aqueous solution state to which the stabilizing material is added, a precipitant or water-soluble dye is added. It may be prepared by lyophilization. The lyophilized reaction mixture thus prepared can easily confirm the complete mixing of the reaction solution during the mixing of the sample to which the dye is added, and the mixing process of the sample injection buffer required for the analysis of the PCR reaction product can be omitted. In this way, contamination by PCR reaction products can be avoided. At this time, in the present invention, a polyhydric alcohol is used as the precipitation agent, and most preferably, glutitol is used. In addition, bromophenol blue, xylene cyanole, bromocresol red, cresol red, and the like may be used as the water-soluble dye.

The DNA polymerase reaction mixture of the present invention prepared as described above has the following advantages: Firstly, a multi-step operation in which each component for the reaction of the DNA polymerase must be mixed for each sample; Second, to increase the stability of the reaction mixture; Third, it is possible to prevent contamination by the PCR reaction product by omitting the step of adding the sample injection buffer required for the analysis of the reaction product; and Fourth, in the diagnosis of disease and reproducibility of repeated experiments, By eliminating mistaken experimental errors, the reliability of the DNA polymerase reaction is increased.

The reaction mixture of the DNA polymerase of the present invention can be used as a sequencing kit or a kit for diagnosing a disease, thereby determining sequencing or diagnosing infection with a disease.

Hereinafter, the present invention will be described in detail by way of examples. These examples are only for illustrating the present invention, 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 Preparation of DNA Polymerase Reaction Mixture

First, primers P1 (20mer) and primers P2 (18mer) capable of obtaining a 1 kb reaction product using λ DNA as a template were synthesized (see FIG. 1a), 10 mM Tris-HCl (ph 8.3), and 40 mM KCl. A DNA polymerase reaction mixture for a general PCR reaction was prepared, which consisted of 1.5 dM MgCl ₂ , 1 mM DTT, 50 mg / ml BSA, four types of dNTP, 250 μM each, 50 pmoles primer P1, 50 pmoles primer P2, and 1 ng DNA. At this time, the concentration and amount of each component represents the final concentration and amount of each component contained in 0.05ml of the reaction solution. The PCR reaction amplified DNA by 30 cycles of denaturation (1 min at 94 ° C.), binding (1 min at 54 ° C.), and extension (1 min at 72 ° C.). 1% (w / v) agarose gel electrophoresis of the PCR reaction product thus amplified is shown in Figure 1b. In Figure 1b, the first lane is the result of electrophoresis of the reaction product obtained by the PCR reaction of the reaction mixture, the second lane is the result of electrophoresis of the standard molecular weight DNA of 1kb ladder (ladder). As shown in FIG. 1b, only about 1 kb of DNA was correctly amplified in the PCR reaction of the reaction mixture.

Example 2 Influence of Stabilizing Material on DNA Polymerase Chain Reaction

In order to examine the effect of the stabilizing material on the DNA polymerase chain reaction of the PCR reactions described in Example 1, the reaction mixture of Example 1, respectively, the final concentration of 20mM ammonium sulfate, 4% glycerol, 20mM glucitol, 20 mM ammonium sulfate, 4% glycerol, 0.1% PEG-8000 (polyethylene glycol-8000), and 0.1% Thesit (Boeringer Mannheim, Germany) were added, and then PCR reactions were performed under the same conditions as in Example 1. The PCR reaction product thus prepared was electrophoresed, and the results are shown in FIG.

In Figure 2, lane 1 is 20mM ammonium sulfate, lane 2 is 4% glycerol, lane 3 is 20mM glutathol, lane 4 is 20mM ammonium sulfate and 4% glycerol, lane 5 is 0.1% PEG- 8000 and 6th lane represent DNA polymerase reaction mixtures for PCR reaction added 0.1% Thesit, respectively, lane 7 is a control group without addition of stabilizing material, and lane M represents a standard molecular weight DNA of 1 kb ladder, respectively. . As shown in FIG. 2, DNA of the desired size was identified from all reaction mixtures except for the reaction mixture containing 20 mM ammonium sulfate and 4% glycerol of lane 4. Thus, it was found that 20 mM ammonium sulfate, 4% glycerol, 20 mM glutitol, 0.1% PEG-8000 and 0.1% Thesit did not inhibit the DNA polymerase chain reaction of the PCR reaction mixture.

Example 3 Effect of Lyophilization on DNA Polymerase Chain Reaction

In order to examine the DNA polymerase reaction after lyophilization of the reaction mixture described in Example 2, 20 μl of each reaction mixture of Example 2 was placed in a 0.5 ml test tube and rapidly frozen at −70 ° C., followed by a lyophilizer (ILSIN Engineering, Dried in Korea). 20 μl of distilled water was added to each of the lyophilized samples to completely dissolve the dried reaction mixture. Then, the PCR reaction was carried out under the same conditions as in Example 1, and the PCR reaction product was electrophoresed. Degree).

In FIG. 3, the sample injected into each lane is the same as the sample injected into each lane of FIG. As shown in FIG. 3, DNA of the desired size was identified from all reaction mixtures except the reaction mixtures of the first and fourth lanes to which ammonium sulfate was added. Thus, it was found that lyophilization did not inhibit the DNA polymerase chain reaction of the reaction mixture without stabilizer and the reaction mixture with 4% glycerol, 20 mM glutitol, 0.1% PEG-8000 and 0.1% Thesit. Could.

Example 4 Measurement of Stability of Each Lyophilized Reaction Mixture

In order to measure the stability of each lyophilized reaction mixture, each lyophilized reaction mixture holding DNA polymerase activity in Example 3 was left at 50 ° C. for 62 hours, and then subjected to PCR reaction under the same conditions as in Example 1. PCR reaction products were electrophoresed (see Figure 4). In FIG. 4, DNA polymerase reaction mixture for PCR reaction was added with 4% glycerol in the first lane, 20 mM glutitol in the second lane, 0.1% PEG-8000 in the third lane, and 0.1% Thesit in the fourth lane. The 5th lane shows the control bacteria and the M'th lane without addition of the stabilizing substance, respectively, and the standard molecular weight DNA of the 1 kb ladder was shown. As shown in FIG. 4, only the reaction mixture of the second lane to which 20mM Glucitol was added could clearly identify the DNA of the desired size, and although it was weak, the bands were also identified in the lanes 1, 3 and 4. Therefore, it was found that the reaction mixture to which the lyophilized 20 mM glutitol was added was most stabilized to maintain DNA polymerase activity for a long time.

[Example 5: Checking the stability of the reaction mixture in the freeze-dried state to which the stabilizing material is added]

In order to check how the stability of the reaction mixture is affected by the addition of the stabilizing material and the lyophilization of the reaction mixture, the reaction mixture in the aqueous solution state without the stabilizing material added, and the aqueous solution state in which 20 mM ammonium sulfate is added as the stabilizing material. PCR reaction was carried out at 12 hours intervals while leaving the reaction mixture of, the lyophilized reaction mixture without stabilizing material and the lyophilized state with 100 mM glutitol added as stabilizing material at 50 ° C. ( See Figure 5.)

In FIG. 5, A is a reaction mixture in an aqueous solution state without added stabilizing material, B is a reaction mixture in an aqueous solution state in which 20 mM ammonium sulfate is added as a stabilizing material, and C is a reaction mixture in a lyophilized state without adding stabilizing material. And D represents a lyophilized reaction mixture to which 100 mM glutitol was added as a stabilizing material, respectively; The first lane is 12 hours, the second lane is 24 hours, the third lane is 36 hours, the fourth lane is 50 hours, the fifth lane is 62 hours, the sixth lane is 86 hours, the seventh lane is 112 hours and Eight lanes each represent a reaction mixture left for 136 hours. As shown in FIG. 5, when the stability increase of each reaction mixture is compared with the reaction mixture (A) in the aqueous solution state without the stabilizing material, the reaction mixture (B) in the aqueous solution state in which 20 mM ammonium sulfate is added is 12 hours. As described above, the reaction mixture (C) in the lyophilized state without adding the stabilizing material was found to be stable for more than 120 hours in the lyophilized state (D) in which the lyophilized state was added for 24 hours or more. Therefore, it was found that the reaction mixture in the lyophilized state to which the stabilizing material was added increased stability.

Example 6 Increasing Stability of Lyophilized Reaction Mixtures with Polyhydric Alcohols

Based on the results of Example 5, in order to confirm the increase in the stability of the reaction mixture by addition of a polyhydric alcohol (polyol), each reaction mixture to which 20 mM glucose, mannitol, galactitol, and glutitol were added was lyophilized. Thereafter, the mixture was allowed to stand at 50 ° C. for 62 hours, followed by PCR reaction under the same conditions as in Example 1, and electrophoresis of the PCR reaction product (see FIG. 6). In FIG. 6, the first lane represents 20mM glucose, the second lane 20mM mannitol, the third lane 20mM galactitol, and the fourth lane represent a DNA polymerase reaction mixture for PCR reaction with 20mM glutitol added. , Lane M represents standard molecular weight DNA of 1 kb ladder, respectively. As shown in FIG. 6, it was found that each reaction mixture containing 20 mM glucose, mannitol, galactitol, and glutitol retained the activity of the DNA polymerase. Therefore, it was found that the stability of the reaction mixture was increased by the addition of the polyhydric alcohol.

Example 7 Effect of Water-Soluble Dye on DNA Polymerase Chain Reaction

In order to confirm the reaction mixture in the lyophilized state and to easily check the complete mixing of the reaction solution during the mixing of the sample, water-soluble dyes such as bromophenol blue, xylene cyanol, bromocresol red, methyl green and methyl green Cresol red and the like were added to the reaction mixture of Example 1 so that the final concentration was 0.01% (v / v), and lyophilized, followed by PCR reaction (see FIG. 7). In FIG. 7, lyophilized state is added with bromophenol blue in the first lane, xylene cyanol in the second lane, bromocresol red in the third lane, cresol red in the fourth lane, and methyl green in the fifth lane. Reaction mixtures of As shown in FIG. 7, it was found that in all cases except the lyophilized reaction mixture to which methyl green was added, the DNA polymerase reaction did not decrease. Therefore, it was found that bromophenol blue, xylene cyanol, bromocresol red, cresol red, and the like may be added in the preparation of the reaction mixture in a lyophilized state.

From the results of Examples 1 to 7 above, the PCR of the present invention prepared by lyophilizing a DNA polymerase reaction mixture for a general PCR reaction in an aqueous solution state, or adding a stabilizing substance or a water-soluble dye to the electric reaction mixture, and lyophilizing The DNA polymerase reaction mixture for the reaction was found to maintain the DNA polymerase activity even at high temperatures due to increased stability. Therefore, Example 8 was performed to determine the nucleotide sequence using the DNA polymerase reaction mixture for the electric PCR reaction as a kit for sequencing.

[Example 8: Determination of base sequence using a reaction mixture in a freeze-dried state to which a stabilizing substance was added]

To determine the DNA sequence, 30 μM ddGTP (G reaction) and 300 μMddATP (A), respectively, were used in four test tubes consisting of 10 μm dNTP, 50 mM Tris-HCl, 1.5 mM MgCl ₂ , DNA polymerase, and 100 mM glutitol as a stabilizing agent. Reaction), 400 μM (ddTTP) (T reaction) and 200 μM ddCTP (C reaction) were mixed, lyophilized, and left at a constant temperature for 12 hours to determine the base sequence (see FIG. 8). PCR reaction for sequencing was carried out 35 times of denaturation (30 seconds at 94 ℃), binding (30 seconds at 45 ℃), extension (60 seconds at 72 ℃) using a thermocyclic method, The reaction product was electrophoresed, and then DNA bands were identified using a silver staining method (see Bassam, BJ and Anolles, GC, Applied Biochemistry and Biotechnology, 42: 181-188 (1993)).

In FIG. 8, Nos. 1 and 2 are lyophilized controls without addition of stabilizing materials, and Nos. 3 and 4 represent lyophilized reaction mixtures with addition of stabilizing materials, respectively; After the first and third lyophilization, the base sequence was determined after 12 hours at -20 ° C., the second and fourth times at 12 ° C. for 12 hours, respectively. As shown in FIG. 8, it can be seen that the lyophilized reaction mixture to which the stabilizing material was added from Nos. 2 and 4 maintained the activity of the DNA polymerase for 12 hours at 50 ° C.

On the other hand, when the reaction mixture including ddGTP, ddATP, ddTTP and ddCTP is independently lyophilized and used as a sequencing kit, the nucleotide sequence can be immediately determined by adding DNA, thereby providing reproducible experimental results quickly. .

As described and demonstrated in detail above, the present invention is stabilized DNA polymerase by adding a stabilizing material to the DNA polymerase reaction mixture comprising a reaction buffer, MgCl ₂ , four dNTPs and DNA polymerase and lyophilized It provides a method for preparing a reaction mixture. The lyophilized DNA polymerase reaction mixture prepared by the method of the present invention facilitates a multi-step operation in which each component for the reaction of the DNA polymerase should be mixed for each sample, increasing the stability of the reaction mixture, and experimenting. Increase the reliability in the reproducibility. When the lyophilized DNA polymerase reaction mixture prepared above is used as a sequencing kit or a disease diagnosis kit, highly reliable results can be obtained in a short time.

Claims (7)

DNA synthesis enzyme reaction mixture by adding a primer, a stabilizing material, a precipitant, and a water-soluble dye to a DNA synthesis enzyme reaction mixture containing a reaction buffer solution, MgCl ₂ , four dNTPs and DNA polymerase. How to prepare. The method of claim 1, wherein the stabilizing material is at least one material selected from the group consisting of Thesit PEG-8000, glycerol, glucose, mannitol, galactitol, glycitol and sorbitol. The method of claim 1, wherein the precipitation agent is at least one substance selected from the group consisting of glycerol, glucose, mannitol, galactitol, glycitol and sorbitol. The method of claim 1, wherein the water-soluble dye is at least one selected from the group consisting of bromophenol blue, xylene cyanocle, bromocrphenol red, cresol red Characterized in that it is a substance. DNA polymerase reaction prepared by adding primers, stabilizers, precipitants, and water-soluble dyes to a DNA polymerase reaction mixture containing a reaction buffer solution, MgCl ₂ , four dNTPs and DNA polymerase, and lyophilizing mixture. A base sequence analysis kit comprising the lyophilized DNA polymerase reaction mixture of claim 5 as an active ingredient. A disease diagnostic kit comprising the lyophilized DNA polymerase reaction mixture of claim 5 as an active ingredient.