KR101008335B1 - Reaction Mixtures for Nucleic Acid?Involving Reaction Catalyzing Enzymes - Google Patents

Abstract

The present invention comprises the steps of (i) drying a reaction solution comprising a nucleic acid-involving reaction catalyzing enzyme under dehumidification or (i ') a reaction comprising a nucleic acid-involving reaction catalytic enzyme. The present invention relates to a method for preparing a reaction mixture of nucleic acid-associated reaction catalytic enzymes, and to a reaction mixture of nucleic acid-associated reaction catalytic enzymes. According to the present invention, it is possible to produce a reaction mixture (particularly an amplification mixture) product of a stable nucleic acid-associated reaction catalytic enzyme without a change in activity (enzyme activity or reaction activity). When the present invention is carried out under humidity control, it is possible to produce a finished product of a certain quality within a certain time regardless of season and place. According to the present invention it is possible to minimize the manufacturing time and cost. Only the dehumidifier and the humidity control device allow mass production of the product within a certain space. In addition, the reaction mixture of the present invention is very excellent in room temperature or high temperature storage stability.

Enzyme, reaction mixture, amplification, humidity, dehumidification, low temperature

Description

Reaction Mixtures for Nucleic Acid-Involving Reaction Catalyzing Enzymes

The present invention relates to a kit comprising a process for preparing a reaction mixture of nucleic acid-associated reaction catalyzed enzymes, a reaction mixture of nucleic acid-associated reaction catalyzed enzymes, and a reaction mixture of nucleic acid-associated reaction catalyzed enzymes.

Enzymatic reactions generally carried out in the prior art have been carried out through a multi-step operation of separately mixing the components required for the reaction. This process is not only inconvenient, but also requires the preparation of the reaction solution for each enzyme reaction, and thus the components are not likely to be mixed in a constant amount each time. In particular, if a small amount of the enzyme should be added, the above-mentioned deviation is more severe, which may cause the experiment result to be inconsistent. In addition, the possibility of cross contamination of the components in the mixing of the components is also very high, which may lead to undesirable results.

Thus, attempts have been made to prepare a mixture of enzyme reaction solutions in advance and store them stably for several days or months, and then use them for enzyme reactions whenever necessary. Among these, many attempts have been made to develop reaction mixtures for DNA polymerase, which are used for DNA amplification reactions.

For example, Korean Patent No. 162270 adds primers, stabilizers, precipitants, and water-soluble dyes to a DNA polymerase reaction mixture in an aqueous solution containing reaction buffer, MgCl ₂ , four dNTPs, and DNA polymerase. And lyophilization to prepare a DNA synthase reaction mixture.

Republic of Korea Patent No. 730364 is a step of dispensing a mixture of DNA polymerase, reaction buffer, dNTP and gel-loading buffer in a tube and 50 ~ 60 ℃ warm drying or room temperature drying Disclosed is a method for preparing a PCR reaction mixture, comprising the steps of:

However, in the case of the lyophilization method disclosed in the Republic of Korea Patent No. 162270, (i) the need for expensive equipment, power station, installation space, etc .; (ii) a problem of three hours being consumed in the preheating stage of the shelf and maintaining the vacuum, and at least five hours of drying time; (iii) the problem of ejecting the sample while the vacuum is released; And (iv) cost increases too much for the outcome.

In addition, the room temperature or warm drying method disclosed in Korean Patent No. 730364 is (i) the condition of humidity varies depending on the season or place, and when drying at room temperature, at least 7 hours to dry 10 μl of the reactant to a volume of 2 μl or less. And (ii) indoor humidity is maintained at an average of 50% in January to 85% in July (Kwang Kwang-Woo et al., Building Environment Planning Theory , Taerim Culture History (1998)); (iii) The polymerase itself is stable, but there is a problem that activity decreases when dried in a mixed state with the reaction buffer and other components, and (iv) in the case of warm drying, the activity decreases by about 30-50% compared to the actual activity. (v) In the case of warm-drying, there is a problem in that it is not suitable for mass production due to the problem of the internal space of the apparatus, like freeze-drying.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to improve the problems of the prior arts and to develop a method for producing enzyme reaction mixtures more efficiently. As a result, the present inventors have found that, when producing the enzyme reaction mixture under dehumidification or concentration, the present inventors can produce an enzyme reaction mixture which has more simplified equipment and improved activity in a shorter time at lower cost. The invention was completed.

It is therefore an object of the present invention to provide a process for the preparation of reaction mixtures of nucleic acid-associated reaction catalytic enzymes.

Another object of the present invention is to provide a reaction mixture of nucleic acid-associated reaction catalytic enzymes.

It is another object of the present invention to provide a kit comprising a reaction mixture of nucleic acid-associated reaction catalytic enzymes.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the invention comprises (i) drying a reaction solution comprising a nucleic acid-involving reaction catalyzing enzyme under dehumidification or (i ') a nucleic acid- It provides a method for preparing a reaction mixture of nucleic acid-associated reaction catalyst enzyme comprising the step of concentrating and drying the reaction solution comprising the reaction catalyst catalyst involved.

According to another aspect of the present invention, the present invention provides a reaction mixture of the nucleic acid-associated reaction catalyst enzyme prepared by the above-described method of the present invention.

The present inventors have sought to improve the problems of the prior arts and to develop a method for producing enzyme reaction mixtures more efficiently. As a result, the inventors have found that when the enzyme reaction mixture is prepared under dehumidification or concentration, it is possible to produce an enzyme reaction mixture which has more simplified equipment and improved activity in a shorter time at lower cost.

The present invention employs two strategies largely: (i) dehumidification and (ii) concentration.

As used herein, the term “dehumidification” refers to the removal of moisture or the reduction of humidity, which means to reduce the humidity of the environment (space) from which the reaction mixture is made. Humidity control for the dehumidification can be achieved by installing the dehumidifier in a closed space or the outside air space and maintaining a suitable humidity to circulate the air inside. In general, laboratories have a confined space, so if only a dehumidifier capable of controlling humidity is installed in this space, the humidity can be controlled sufficiently.

According to a preferred embodiment of the present invention, the dehumidification has a relative humidity of 50% or less, more preferably 40% or less, even more preferably 30% or less, even more preferably 20% or less, Most preferably by keeping it at 15% or less. The lower limit of the numerical value indicated while referring to relative humidity in this specification is 0% relative humidity, preferably 5%.

Such dehumidification greatly shortens the time required for drying the reaction mixture, and further maintains almost the same activity of the enzyme contained in the reaction mixture.

The second strategy of the present invention is the concentration of the reaction mixture. As used herein, the term "concentration" refers to increasing the molar concentration of the components contained in the reaction mixture than conventionally used reaction mixtures, in which case the volume of the reaction mixture is generally reduced than that of the commonly used reaction mixtures. .

Concentration in the present invention is 2-100 times, preferably 2-50 times, even more preferably 4-30 times, even more preferably 5 compared to the molar concentration of the components in the reaction mixtures commonly used. By -20 times, most preferably 5-15 times.

For example, a typical PCR reaction mixture is a reaction mixture comprising 40 mM Tris.Cl (pH 8.8), 100 mM KCl, 4 mM MgCl ₂ and 400 μM dNTP. Applying the strategy of the present invention to this conventional PCR reaction mixture, a PCR reaction comprising 80-4000 mM Tris.Cl (pH 8.8), 200-10000 mM KCl, 8-400 mM MgCl ₂ and 0.8-40 mM dNTP. A mixture is prepared.

Concentration in the present invention can also be expressed as a volume reduction of the reaction mixture. 20-95% volume reduction, preferably 30-95% volume reduction, and more preferably 40-90% volume reduction, compared to the reaction solution (eg PCR reaction solution) finally added to the reaction Preferably the concentration is effected through a volume reduction of 60-90%.

This concentration can prevent the salt concentration stress received by the enzyme by minimizing the rapid change in the salt concentration, it is possible to achieve the maintenance of the enzyme activity by reducing the drying time.

The present invention can be applied to enzyme reaction mixtures of various nucleic acid-associated reactions. As used herein, the term “nucleic acid-associated reaction” refers to catalysis by enzymes using nucleic acids as substrates, templates or primers. Nucleic acid-associated enzyme reactions result in modification of nucleic acid molecules. As used herein, the term “nucleic acid” refers to a short or double chain deoxynucleotide or ribonucleotide polymer.

The present invention can be used to prepare enzyme reaction mixtures of various kinds of nucleic acid-associated reactions. Preferably, the present invention comprises amplification reactions, reverse transcription, DNA ligation, nuclease-mediated reactions, DNA methylation, topoisomerase-mediated reactions or telomerase-mediated reactions. Applied to the reaction.

As used herein, the term "amplification reaction" means a reaction that amplifies a nucleic acid molecule. Various amplification reactions have been reported in the art, which are called polymerase chain reaction (hereinafter referred to as PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcriptase-polymerase chain reaction (hereinafter referred to as RT-PCR). (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329, 822), ligase chain reaction (LCR) (17, 18), Gap- LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) (WO 88/10315), self-sustained sequence replication sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction; CP-PCR (US Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (US Pat. Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification (nucleic) acid sequence based amplification; NASBA (US Pat. Nos. 5,130,238, 5,4) 09,818, 5,554,517, and 6,063,603), strand displacement amplification (21, 22) and loop-mediated isothermal amplification (LAMP) (23) It is not limited. Other amplification methods that can be used are described in US Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and US Pat. No. 09 / 854,317.

As used herein, the term "reverse transcription" refers to a reaction that synthesizes DNA in an RNA template. For example, the reverse transcription process involves RT-PCR. The term “DNA ligation” refers to the covalent bond formation reaction between two DNA molecules. For example, the DNA ligation process is included in the LCR.

The term “nuclease-mediated reaction” described herein refers to a reaction catalyzed by nucleases that cleave phosphodiesteric bonds in nucleic acid molecules. Many kinds of nucleases are known to those skilled in the art. For example, nucleases include endonucleases (eg, FEN- 1 endonucleases, restriction enzymes, DNase I, DNase II, and RNase I) and exonucleases (snake phosphodiesterase and liver). Phosphodiesterase], but is not limited thereto.

The term “DNA methylation” described herein refers to a reaction that methylates a base at a specific position on DNA. The “topoisomerase-mediated reaction” described herein is directed to topoisomerase (eg, type I and II topoisomerases) that converts one phase isomer of covalently closed cyclic DNA into another. It refers to the reaction catalyzed by. As used herein, a "telomerase-mediated reaction" refers to a reaction catalyzed by telomerase that results in the production of one strand of repeating units in telomeres.

According to the most preferred embodiment, the present invention is used to prepare a PCR reaction mixture. PCR is the best known nucleic acid amplification method, and many modifications and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR, and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction (inverse) Polymerase chain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR), and multiplex PCR have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

According to a preferred embodiment of the invention, the nucleic acid-involved reaction catalytic enzyme is a polymerase, reverse transcriptase, DNA ligase, nuclease, phosphodiesterase, DNA methylase, topoisomerase or telomerase to be.

According to a preferred embodiment of the present invention, the nucleic acid-associated reaction catalyzed enzyme is a polymerase or reverse transcriptase, more preferably a polymerase, even more preferably a thermophilic polymerase.

Examples of polymerases include the Klenow fragment of E. coli DNA polymerase I, thermophilic DNA polymerase and bacteriophage T7 DNA polymerase. Thermophilic polymerases are thermophilic eubacteria or aquibacteria, for example Thermus aquaticus , T. thermophilus , T. bockianus , T. flavus , T. rubber , Thermococcus litoralis , Pyrococcus furiousus , P. wosie , Pyrococcus spec . KGD , Thermatoga maritime, Thermoplasma acidophilus , and Sulfolobus spec . Can be separated from. Preferably, the thermophilic polymerase is Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase and modified thermostable polymerase which are inert at room temperature.

According to a preferred embodiment of the present invention, the process of the present invention is carried out by concentrating a reaction solution comprising a nucleic acid-associated reaction catalytic enzyme and then drying under dehumidification.

According to a preferred embodiment of the present invention, the drying in the present invention is low temperature drying at room temperature or 4-20 ° C. When low temperature drying is performed, it is preferably performed at 5-18 ° C, more preferably 8-18 ° C, and most preferably 10-17 ° C.

The reaction mixture prepared by the present invention may include not only enzymes but also other reactants.

Preferably, the reaction solution to be dried further includes a buffer solution. As the buffer solution included in the reaction solution, any buffer solution commonly used in the art may be used, and the type of buffer is generally determined according to the type of enzyme as known to those skilled in the art. For example, available buffers include tris (hydroxymethyl) aminomethane-HCl (Tris.Cl), Na ₂ HPO ₄ -NaH ₂ PO ₄ , MOPS-KOH, HEPES-NaOH,, borate and glycine-NaOH. .

More preferably, the reaction solution to be dried further comprises a substrate, cofactor, stabilizer, dye or mixtures thereof.

For example, when preparing a DNA polymerase reaction mixture, a DNA template, a primer and / or dNTP (a mixture of dATP, dTTP, dGTP and dCTP) may be included as a substrate. For example, when preparing a DNA polymerase reaction mixture, MgCl ₂ may be additionally included as a carrier.

Examples of stabilizers include glycerol, glucose, mannitol, galactitol, glycitol and sorbitol.

Examples of dyes include dyes used during electrophoresis to identify the product of the reaction, and include, for example, xylene cyanol, bromophenol blue, bromocresol red and cresol red.

When the present invention is applied to prepare a DNA polymerase reaction mixture, the reaction solution to be dried includes a buffer solution, MgCl ₂ and dNTP, in addition to the polymerase. More preferably, the reaction solution to be dried includes a polymerase, a buffer solution, MgCl ₂ , dNTP and KCl, even more preferably a polymerase, a buffer solution, MgCl ₂ , dNTP, KCl, stabilizer, dye And primers.

The primer is a primer that is specifically annealed to the target sequence to be amplified and / or a housekeeping gene (eg, GAPDH (glyceraldehyde phosphate dehydrogenase) gene, β-actin gene, PGK1 (phosphoglycerate kinase 1) gene, β-) Globin gene or 28S RNA gene].

According to another aspect of the invention, the invention comprises a kit comprising a reaction mixture of the nucleic acid-associated reaction catalyst enzyme of the invention described above.

For example, when the reaction mixture contains a DNA polymerase, the kit of the present invention can be prepared as a kit for gene amplification, gene analysis and gene sequencing.

The features and advantages of the present invention are summarized as follows:

(i) According to the present invention, it is possible to produce a reaction mixture (particularly an amplification reaction mixture) product of a stable nucleic acid-associated reaction catalytic enzyme without a change in activity (enzyme activity or reaction activity).

(Ii) When the present invention is carried out under humidity control, finished products of a certain quality can be produced within a certain time regardless of season and place.

(Iii) According to the present invention, manufacturing time and cost can be minimized.

(Iii) Only the dehumidifier and the humidity control device allow mass production of the product within a certain space.

(Iii) As demonstrated in Example 7 below, the reaction mixture of the present invention is very excellent in room temperature or high temperature storage stability.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: primer sequence and reaction solution, reaction conditions

Using E. coli genome DNA as a template, primers Sbf and Sbr, which can obtain a 500 bp reaction product, were synthesized and used as comparative reactions in subsequent experiments. Primer sequences are as follows: Sbf: 5 'ATG GCC AGC AGA GGC G 3'; Sbr: 5 'TCA GAA CGG AAT GTC ATC 3'.

The reaction mixture consisted of 1X PCR buffer (20 mM TrisCl (pH 8.8), 50 mM KCl and 2 mM MgCl ₂ ), 200 μM dNTP mixture, 0.1 pmol primer, 1 U Taq DNA polymerase based on 20 μl total volume. was, the reaction conditions were 10 seconds at 94 ^o C for 30 cycles 5 min preincubation, 30 seconds at 55 ^o 72 ^o C 20 sec at C to react at 94 ^o C. The final extension reaction was carried out at 72 ^o C for 10 minutes, and the PCR reaction was confirmed by EtBr staining by electrophoresis on 1% agarose gel (FIG. 1).

1, M is a DNA size marker, lanes 1 and 2 are reaction results using a liquid phase PCR master mix (control group in the following experiment), and lanes 3 and 4 are PCR master mixes vacuum-dried and lanes 5 and 6 Drying at room temperature for 10 hours, lanes 7, 8 are reaction results using a PCR master mix dried at 50 ° C. for 2 hours. 5 μl each of the results, and compared to lanes 1 and 2, the activity increased in the order of room temperature drying, oven drying, lyophilization.

The preparation method of the PCR reaction membrane composition of the following example is summarized in Table 1 below.

Drying method Drying time (hr)  Starting volume (μl) % Dryness ^* Vacuum freeze drying 5 10 85 Room temperature 10 10 85 Oven (50 ℃) 2 10 85 Concentrated room temperature 5 3 50 Dehumidifying room temperature 3 3 50 Concentrated dehumidifying room temperature 2 3 50 Concentrated Dehumidification Refrigeration 2.5 3 50

^* Drying rate (%) = (starting mass-final mass) / starting mass X 100 (%)

Example  2: Preparation using vacuum freeze drying

40 mM TrisCl (pH 8.8), 100 mM KCl, 4 mM MgCl ₂ , 400 with a 10 μl solution using a vacuum freeze dryer (manufactured by Ilsin Lab, model name: PVTFD10R, volume: 10 L, production year: 2004) μM dNTP mixed solution and 0.5 Unit Taq DNA polymerase were included, and 5% glycerol and 0.2 mg / ml xylene cyanool were dried for 5 hours. The drying conditions were based on the initial mass of the 8 PCR tubes after the sample was added and the mass after drying and the mass after drying to be 3 mg per tube based on the amount of water evaporated. In the same reaction conditions as in Example 1 prepared by vacuum freeze drying, the template was added to each primer and concentration in distilled water, and then reacted with a volume of 20 μl. The resulting product was confirmed by electrophoresis on agarose gel (10 μl of FIG. 1). 3 and 4), the dried reactants were used in subsequent comparative experiments.

Example  3: Influence on the activity of the concentrated reaction liquid at room temperature and high temperature drying

To confirm the degree of drying at room temperature, 10 μl (40 mM TrisCl (pH 8.8), 100 mM KCl, 4 mM MgCl ₂ , 400 μM dNTP mixture and 0.5 μm of the solution of the above example, at a humidity of about 65% at room temperature). Unit Taq DNA polymerase) was placed in a PCR tube and placed at 22-23 ^o C. It was left to dry for about 10 hours or more. In the same manner, oven drying was performed at 50 ^° C. for 3 hours under two conditions, a general warm air dryer and a drying oven. The drying condition was set as the termination condition where the total amount of water evaporated over 90% based on the initial volume. That is, it was based on the point that about 2 mg (16-18 mg by 8 tubes) of each tube by mass basis (Table 1). PCR products were carried out according to the conditions of Example 1, and the resultant dried two conditions were confirmed by electrophoresis on the agarose gel 10 μl of the result (lane 5-8 of Fig. 1).

As a result of the experiment, drying at room temperature takes a lot of time, and in general, the time required to evaporate more than 90% of the total amount of moisture under 60% room humidity and 20 ^° C. is 10 hours or more (Table 1).

Reaction activity was found to be more than 50% of the original activity in the case of room temperature drying, 30% or more activity loss in the case of high temperature or oven drying (Fig. 1).

Example  4: room temperature drying through sample concentration

The prepared solution concentrated to 3 μl of 20 μl of the reaction solution was placed in a PCR tube and allowed to stand at room temperature. The composition of the concentrate is as follows. Instead of 10X buffer composition, 10 μL samples could be reduced to 3 μL by applying 20X PCR buffer (400 mM TrisCl (pH 8.8), 1 M KCl, 40 mM MgCl ₂ ) and 10X dNTP mixtures (25 mM each). . The reaction solution thus prepared was dispensed into 3 μl of each PCR tube and then dried under the following conditions.

First, the mixture was left to dry at 20-22 ° C. until the mass was reduced by 50% or more, and a PCR reaction was performed under the same conditions as in Example 1 and the result was analyzed (FIG. 2).

In FIG. 2, M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 were dried for 10 hours without concentration, and lanes 5 and 6 were concentrated to 3 μl at room temperature. Dry for 5 hours. PCR results were developed by 5 μl. As can be seen in Figure 2, when concentrated according to the strategy of the present invention it can be seen that the activity for the PCR polymerization is well maintained. As can be seen, the concentration / drying at room temperature according to the present invention almost no decrease in activity, in the case of concentrated liquids to minimize the rapid change in salt concentration to reduce the salt concentration stress conditions enzymatically received, shortening the drying time Maintenance of activity was achieved.

Example  5: drying by humidity control (concentration vs . Non-concentrated )

After installing a dehumidifier in an enclosed space or an outside air space, the inside air was circulated by maintaining 15% humidity. The dried sample was dried at 20-22 ° C. under two conditions, each containing 10 μl of Example 2 and 3 μl of Example 4 in a PCR tube. The drying time was 2.5-3 hours for the 10 μl sample and about 1 hour for the 3 μl sample. Samples prepared above were subjected to PCR under the conditions of Example 1, followed by electrophoresis on agarose gel, followed by EtBr staining (FIG. 3).

In FIG. 3, M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 are dehumidified and dried, and lanes 5 and 6 are concentrated and dehumidified. As can be seen in Figure 3, it can be seen that the activity is maintained relatively excellent during dehumidification drying.

Example  6: low temperature treatment

In the case of Example 5, the temperature of the internal space rose to 35 ℃ due to the heat generated in the dehumidifier in the drying process, Example 5 while operating by setting to 15 ℃ using a cooler outside the closed space to offset this The process of repeating. The time required for drying was not significantly different, and it was found that the original enzyme activity was maintained intact in the analysis by PCR reaction (FIG. 4).

In FIG. 4, M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 are dried in a dehumidified state at room temperature, and lanes 5 and 6 are dried in a refrigerated state and dried. When dried while refrigerated, the activity was not different from the control group.

Example  7: Humidity control, reaction liquid concentration, low temperature maintenance

The sample was concentrated to 3 μl while maintaining the humidity at 15% and 20 ° C. in a PCR tube, followed by drying. The time required was about 1 hour and 30 minutes, which was compared with each sample prepared by freeze drying, oven drying, dehumidification concentrated drying, and dehumidification freeze drying through PCR reaction (FIG. 5). The prepared samples were left for 5 days or 10 hours in an oven at 50 ° C. for 3 days or 1 day at room temperature. This was compared through a PCR reaction (FIG. 5).

In FIG. 5, M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 are control groups refrigerated and dried, and lanes 5 and 6 are left at room temperature for 3 days, and lanes 7 and 8 are at room temperature. When left for 1 week, lanes 9 and 10 show PCR results when left for 5 hours at 50 ° C. and lanes 11 and 12 for 10 hours at 50 ° C. FIG. Interestingly, it can be seen that dehumidification and drying of the concentrated sample maintains good activity without being greatly influenced by the external environment.

Example  8: Primer  Put Drying

10 pmol of SBf and 0.5 µl of SBr were added to the sample concentrated to 3 µl while maintaining a humidity of 15% at 20 ° C, followed by drying for 2 hours in a PCR tube. This was compared with the refrigerated sample of Example 6 by a PCR reaction (Fig. 6).

In FIG. 6, M is a DNA size marker, lanes 1 and 2 are cases where the primers are reacted after drying, and lanes 3 and 4 are cases when the primers are combined and dried. In both cases there was no significant difference in activity.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is a result of the amplification reaction using a conventional DNA amplification reaction mixture prepared by various methods. M is a DNA size marker, lanes 1 and 2 are reaction results using a liquid phase PCR master mix (control group in the following experiments), lanes 3 and 4 are PCR freeze-dried PCR master mixes and lanes 5 and 6 at room temperature. Drying for 10 hours, lanes 7, 8 are the results of the reaction using a PCR master mix that was dried for 2 hours in a 50 ° C. oven.

Figure 2 is an amplification reaction result carried out using the concentrated and room temperature dried PCR reaction mixture. M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 were dried at 10 μl for 10 hours without concentration and lanes 5 and 6 were concentrated to 3 μl at room temperature for 5 hours. While dry.

Figure 3 is the result of the amplification reaction carried out using a PCR reaction mixture prepared under humidity control. M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 are dehumidified and dried, and lanes 5 and 6 are concentrated and dehumidified.

Figure 4 is the result of the amplification reaction carried out using a PCR reaction mixture prepared under low temperature. In FIG. 4, M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 are dried in a dehumidified state at room temperature, and lanes 5 and 6 are dried in a refrigerated state and dried.

5 shows the results of amplification reactions using PCR reaction mixtures prepared under various conditions. M is a DNA size marker, lanes 1 and 2 are control groups, lanes 3 and 4 are control groups refrigerated and dehumidified, and lanes 5 and 6 are left at room temperature for 1 week when lanes 5 and 6 are left at room temperature for 3 days. In one case, lanes 9 and 10 show PCR results when the cells were left at 50 ° C. for 5 hours, and lanes 11 and 12 were left at 50 ° C. for 10 hours.

Figure 6 is the result of the amplification reaction carried out using the reaction mixture of the present invention dried by adding a primer. M is a DNA size marker, lanes 1 and 2 are cases where the primers are reacted after drying, and lanes 3 and 4 are cases where the primers are combined and dried.

Claims (15)

(i) concentrating the reaction solution containing a nucleic acid-involving reaction catalyzing enzyme, and then drying at room temperature or 4-20 ° C. under dehumidifying conditions maintained at a relative humidity of 20% or less. Or (i ') concentrating the reaction solution comprising the nucleic acid-associated reaction catalyzed enzyme and then drying the reaction mixture of the nucleic acid-associated reaction catalyzed enzyme at room temperature or at low temperature at 4-20 ° C. How to manufacture. The method of claim 1, wherein the nucleic acid-involvement reaction is an amplification reaction, reverse transcription reaction, DNA ligation, nuclease-mediated reaction, DNA methylation reaction, topoisomerase-mediated reaction or telomerase (telomerase) -mediated reaction. The method of claim 1, wherein the nucleic acid-involvement reaction catalytic enzyme is a polymerase, reverse transcriptase, DNA ligase, nuclease, phosphodiesterase, DNA methylase, topoisomerase or telomerase How to. 4. The method of claim 3, wherein the nucleic acid-associated reaction catalytic enzyme is a polymerase or reverse transcriptase. delete delete delete delete delete delete The method of claim 1, wherein the reaction solution further comprises a buffer solution. The method of claim 11, wherein the reaction solution further comprises a substrate, cofactor, stabilizer, dye, or mixtures thereof. The method of claim 12, wherein the reaction solution comprises a polymerase, a buffer solution, MgCl ₂ and dNTP. The method of claim 13, wherein the reaction solution further comprises a primer, dye or stabilizer. A reaction mixture of a nucleic acid-associated reaction catalyst enzyme prepared by the method of any one of claims 1 to 4 or 11 to 14.

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