KR100436126B1 - Enzyme Reaction Pre-Mixture with Improved Stability - Google Patents

Abstract

The present invention relates to an enzyme reaction pre-mixture with improved stability, and more particularly, an enzyme stabilizer, buffer, selected from the group consisting of neotrehalose, isotrehalose, raffinose, melezitose and combinations thereof. The present invention relates to an enzyme reaction premix including a solution and an enzyme, and a kit for DNA amplification, a sequence analysis kit, and a disease diagnosis kit using the same, wherein the enzyme reaction premix of the present invention improves the stability of the enzyme and reacts with the enzyme. By pre-mixing and storing them, it is possible to solve the problem of mutual contamination that occurs when mixing each reactant separately, and greatly shorten the step of the enzymatic reaction work, simplifying the operation, and mixing a predetermined amount of the reactants in advance. This helps reduce errors that can occur when mixing multiple reactants separately. Increasing the reproducibility of the test result, thereby increasing the reliability of the results.

Description

Enzyme Reaction Pre-Mixture with Improved Stability}

The present invention relates to an enzyme reaction pre-mixture having improved stability, and more particularly, to an enzyme reaction premix including a specific stabilizer and a kit for DNA amplification including the same.

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 a 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 pre-mixtures of enzyme reaction solutions in advance, store them stably for days or months, and then use them for enzyme reactions whenever necessary. In the preparation of the enzyme reaction premix, the most important consideration is the use of suitable stabilizers.

Such stabilizers are generally added to create an environment similar to the natural environment in order to maintain the activity of the enzyme isolated from the cell.

Examples of such stabilizers include glycerol (Gekko, K. et al., Biochemistry , 20: 4677 (1981)), glucose, sucrose, fructose, sorbitol, and the like (Smith, MB et al., Proc. Aust. Biochem. Soc. , 11: 4 (1978)).

Throughout this specification, numerous papers and patent documents are referred to and their citations are shown in parentheses. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

Accordingly, it is an object of the present invention to provide a premix of enzyme reactions.

Another object of the present invention is to provide a DNA polymerase premix.

Still another object of the present invention is to provide a kit for DNA amplification, a sequencing kit, and a kit for diagnosing a disease, comprising a prepolymer mixture prepared according to the present invention.

Other objects and advantages of the present invention will be more clearly understood through the following detailed description and drawings.

1 is a gel photograph showing the stability of the premix of the enzyme reaction of the present invention in a liquid phase;

Figure 2 is a gel photograph showing the stability of the enzyme reaction premix of the present invention lyophilized;

Figure 3 is a gel photograph comparing the stability of the pre-enzyme reaction mixture each containing the stabilizers of the present invention; And

Figure 4 is a gel photograph showing the stability of the lyophilized reverse transcriptase reaction premix of the present invention for RT-PCR.

According to one aspect of the invention, the invention is an enzyme reaction pre-mixture comprising an enzyme stabilizer, a buffer and an enzyme selected from the group consisting of neotrehalose, isotrehalose, raffinose, melezitose and combinations thereof ).

Surprisingly, the inventors have found that Neotrehalose, Isotrehalose, Raffinose, Melezitose and combinations thereof exhibit much better stabilizing properties than other conventional sugar stabilizers. The above-mentioned sugars belong to non-reducing disaccharides (neotrehalose and isotrehalose) and trisaccharides (rapinose and melezitose).

Neotrehalose and isotrehalose in the stabilizers of the present invention are α, β-trehalose and β, β-trehalose, respectively, which are different anomers than α, α-trehalose, which is commonly referred to as trehalose in nature. . Two types of trehalose anomers selected as stabilizers in the present invention are excellent in the degree of stabilization in the liquid phase.

In the present invention, the stabilizer not only stabilizes the activity of the enzyme, but also performs the role of a settling agent at the same time, there is an advantage that does not need to add a separate settling agent during electrophoresis of the reaction product.

According to a preferred embodiment of the present invention, the amount of the enzyme stabilizer described above is 1-15% by weight based on the premix of the enzyme reaction. If the amount of the enzyme stabilizer exceeds 15% by weight, the viscosity is too high, there is a problem in drying and dissolving again in water, if less than 1% by weight of the stabilizer is insufficient to stabilize the enzyme effectively There is a problem that can not.

Enzymes included in the enzyme reaction premix of the present invention may be any enzyme known in the art, more preferably, DNA polymerase, reverse transcriptase and RNA polymerase or RNAase H using DNA or RNA as a substrate, Most preferably DNA polymerase. The enzymes described above are enzymes commonly used in gene related experiments, which are particularly advantageous when the reaction mixture is prepared in advance.

The buffer included in the premix of the present invention can be used in any buffer commonly used in the art, and the type of buffer is generally determined according to the type of enzyme as known to those skilled in the art. For example, Na ₂ HPO ₄ -NaH ₂ PO ₄ , MOPS-KOH, HEPES-NaOH, tris (hydroxymethyl) aminomethane-HCl, borate and glycine-NaOH.

The premix of the present invention may further include other components required for the reaction, such as substrates, cofactors, coenzymes, and the like.

The premixes of the present invention may be present in liquid or dried (vacuum dried or lyophilized) form. Drying is accomplished by vacuum drying or lyophilizing the prepared liquid premix in accordance with methods known in the art.

The premix of the present invention may preferably be stored or stored in each of several tubes so that each component has a certain amount. Therefore, in the case of using the tube containing the premix of the present invention in each experiment, it is possible to solve the inconvenience of preparing the reaction mixture in each experiment, to avoid the deviation, and finally from the constant conditions for each experiment. Results can be obtained, thereby improving the reproducibility of the experimental results.

On the other hand, the storage temperature of the pre-mixture of the present invention is low temperature (about 4 ℃ to about -20 ℃) storage if the pre-mixture is liquid, and low temperature (about 4 ℃ to about -20 ℃) storage if the dry state It is also possible to store at room temperature.

In addition, most reaction premixes containing enzymes are generally prepared in a lyophilized state, but the premixture of the present invention exhibits excellent stability even in a liquid phase, and thus a sufficient effect can be achieved even with a liquid premix. .

According to another aspect of the present invention, the present invention provides a DNA polymerase reaction premix comprising an enzyme stabilizer, a buffer and a DNA polymerase selected from the group consisting of neotrehalose, isotrehalose, raffinose, melezitose and combinations thereof. To provide.

In the DNA polymerase reaction premix of the present invention, the amount of the enzyme stabilizer is 1-15% by weight relative to the DNA polymerase reaction premix. If the amount of the enzyme stabilizer exceeds 15% by weight, the viscosity is too high, there is a problem in drying and dissolving again in water, if less than 1% by weight of the stabilizer is insufficient to stabilize the enzyme effectively There is a problem that can not.

The DNA polymerase reaction premix of the present invention preferably further includes MgCl ₂ and four dNTPs which are generally included in the DNA polymerase reactant.

In addition, according to a preferred DNA polymerase reaction premix of the present invention, it further comprises xylene cyanol, bromophenol blue, bromocresol red or cresol red used as a dye during electrophoresis to identify the product of the DNA polymerization reaction. do.

The DNA polymerase reaction premix of the present invention is particularly useful for PCR (polymerase chain reaction), and in the case of applying to PCR, the DNA polymerase is preferably a DNA polymerase isolated from Thermophiles. Such DNA polymerases are, for example, thermally stable DNA polymerases separated by Thermus aquaticus (US Pat. No. 4,889,818) and Thermococcus litoralis (US Pat. No. 5,322,785).

In the DNA polymerase reaction premix of the present invention, the enzyme reaction premix preferably further comprises a primer for the DNA polymerase. Due to the nature of the DNA polymerase itself, a primer having a sequence complementary to some sequences of the template is added to the reaction solution to proceed. In particular, when applied to PCR, two kinds of primers are required, namely a sense primer and an antisense primer.

Generally, the DNA polymerase reaction premix is prepared in the state of freeze-drying in the art, but the DNA polymerase reaction premix of the present invention is not only freeze-dried but also shows excellent stability in the liquid phase. Effect can be achieved.

The above DNA polymerase reaction premix of the present invention can be used as a kit for DNA amplification. That is, it can be used as a reaction kit for DNA amplification prepared by the PCR technique currently used in general.

In addition, the above DNA polymerase reaction premix of the present invention can be used as a kit for sequencing. Determination of DNA sequences using PCR is known in the art (Gyllensten U., Biotechniques , 7: 700 (1989)), wherein the DNA polymerase premix of the present invention can be used as a reaction kit. .

On the other hand, PCR and reverse transcriptase (RT-PCR) is a technique widely used in the diagnosis of diseases, especially cancer, and is used to identify biomarkers of known cancers (eg, HER-2 / neu). The DNA polymerase reaction premix of the present invention can be used as a kit for diagnosing diseases when performing the above PCR and RT-PCR.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example 1 Stability of Enzymatic Premixes of the Invention in Liquid

2 μl of buffer solution (100 mM Tris-Cl, pH 8.3, 500 mM KCl and 15 mM MgCl2) in a 200 μl PCR tube, 2 μl of 250 μM dNTP (dATP, dGTP, dCTP and dTTP), 5 μl of 10% raffinose, 0.1 0.5 μl of% xylene cyanol and 2.5 units of Taq DNA polymerase (Perkin Elmer, USA) were mixed and then stored at 4 ° C. with distilled water to a final volume of 10 μl. Then, after 7 days of storage, PCR was performed under the following conditions:

Genome DNA isolated from humans was used as a template, and the primers were sense primers GATGATACCCACTTCAGGAAG and antisense primers GATGTGTAGGAATTAGCCAGG. After filling with genome DNA and primer, the distilled water was filled to a final volume of 20 μl, and then reacted for 35 cycles once at 94 ° C. for 5 minutes, 30 seconds at 94 ° C., 40 seconds at 60 ° C., and 45 seconds at 72 ° C., 72 The reaction was carried out at 5 ° C. for 5 minutes. After the PCR reaction was completed, the PCR product was electrophoresed on a 2% agarose gel to confirm the product, and the results are shown in FIG. 1.

In FIG. 1, lane M is a 100 bp ladder loaded as a size marker, lane 1 is a negative control, and a template-free PCR reaction is loaded, and lane 2 is a PCR reaction immediately after preparing a PCR reaction of the present invention. Lane 3 is the result of PCR after storing the PCR reaction product of the present invention and refrigerated at 4 ° C. for 7 days. As can be clearly seen in the band pattern and intensity of Figure 1, the PCR reaction mixture of the present invention shows the same PCR results as the PCR reaction immediately after preparation even after 7 days refrigerated storage. That is, it can be seen that the PCR reaction mixture of the present invention shows improved stability.

Example 2: Stability of Enzymatic Reaction Premixes of the Present Invention

2 μl of buffer solution (100 mM Tris-Cl, pH 8.3, 500 mM KCl and 15 mM MgCl2) in a 200 μl PCR tube, 2 μl of 250 μM dNTP (dATP, dGTP, dCTP and dTTP), 5 μl of 10% raffinose, 0.1 0.5 μl of% xylene cyanol and 2.5 units of Taq DNA polymerase (Perkin Elmer, USA) were mixed and then lyophilized (Telstar, Spain). Then, stored at room temperature for 7 days and PCR was performed under the following conditions.

Genome DNA isolated from humans was used as a template, and the primers were sense primers GATGATACCCACTTCAGGAAG and antisense primers GATGTGTAGGAATTAGCCAGG. After filling with genome DNA and primer, the distilled water was filled to a final volume of 20 μl, and then reacted for 35 cycles once at 94 ° C. for 5 minutes, 30 seconds at 94 ° C., 40 seconds at 60 ° C., and 45 seconds at 72 ° C., 72 The reaction was carried out at 5 ° C. for 5 minutes. After the PCR was terminated, the PCR product was electrophoresed on a 2% agarose gel to confirm the product, and the results are shown in FIG. 2.

In FIG. 2, lane 1 is a PCR reaction mixture of the present invention immediately after freeze-drying, lane 2 is a PCR result by the PCR reaction mixture of the present invention refrigerated and stored at 4 ° C. for 7 days after freeze-drying, Lane 3 is a PCR result by the PCR reaction mixture of the present invention stored at room temperature for 7 days after freeze-drying, lane 4 is a result of PCR reaction immediately after mixing the reaction mixture, respectively. As can be seen in Figure 2, the PCR reaction mixture of the present invention does not change the stability even when the freeze-drying treatment, and maintained the stability for 7 days at room temperature, not 4 ℃. That is, it can be seen that the PCR reaction mixture of the present invention subjected to lyophilization shows improved stability.

Example 3 Comparison of Stabilizers Contained in Enzymatic Premixes of the Invention

2 μl of buffer (100 mM Tris-Cl, pH 8.3, 500 mM KCl and 15 mM MgCl2), 2 μl of 250 μM dNTP (dATP, dGTP, dCTP and dTTP) in 4 200 μl PCR tubes, 0.1% xylenecyanol 0.5 μl and 2.5 units of Taq DNA polymerase (Perkin Elmer, USA) are respectively mixed, and then 10% raffinose, 10% melezitose, 10% isotrehalose, 10% neotrehalose and 10% α, α- in each tube. 5 μl of each trehalose was stored at 4 ° C. for 7 days and PCR was performed under the following conditions.

Genome DNA isolated from humans was used as a template, and the primers were sense primers GATGATACCCACTTCAGGAAG and antisense primers GATGTGTAGGAATTAGCCAGG. After filling with genome DNA and primer, the distilled water was filled to a final volume of 20 μl, and then reacted for 35 cycles once at 94 ° C for 5 minutes, 30 seconds at 94 ° C, 40 seconds at 60 ° C, and 45 seconds at 72 ° C. The reaction was carried out at 5 ° C. for 5 minutes. After the PCR was terminated, the PCR product was electrophoresed on a 2% agarose gel to confirm the product, and the results are shown in FIG. 3.

In FIG. 3, lane M is a 50 bp ladder loaded as a size marker, lane 1 is a PCR result by the PCR reaction mixture of the present invention containing raffinose, lane 2 is of the present invention containing melezitose. PCR result by the PCR reaction mixture, lane 3 is the PCR result by the PCR reaction mixture of the present invention containing neotrehalose, lane 4 is the PCR result by the PCR reaction mixture of the present invention containing isotrehalose. , Lane 5 is a PCR result by the PCR reaction mixture containing α, α-trehalose, lane 6 is the result of the PCR reaction by mixing the reaction mixture immediately without the addition of a stabilizer, respectively. As can be seen in Figure 3, the PCR reaction mixture of the present invention can be seen that there is no change in stability even when stored at 4 ℃, and also compared to α, α-trehalose, raffinose, melezitose, neotrehalose and iso Both trehalose showed better performance as stabilizers.

Example 4 Stability of Lyophilized Reverse Transcriptase for RT-PCR

2 μl of buffer solution (500 mM Tris-Cl, pH 8.3, 1 M KCl and 100 mM MgCl 2, 100 mM DTT) in a 200 μl PCR tube, 2 μl, 2.5 mM dNTP (dATP, dGTP, dCTP and dTTP), 10% raffinose 5 20 μl and 20 units of AMV reverse transcriptase (Promega, USA) were respectively mixed and then lyophilized (Telstar, Spain). Subsequently, it was stored at 4 ° C. for 7 days and subjected to reverse transcription under the following conditions: 5 μl of RNA isolated from humans and 1 μl of 0.5 μg / μl oligo-dT primer were mixed and distilled water was brought to a final volume of 20 μl. Was added. Subsequently, the mixture was allowed to react at 70 ° C for 10 minutes, and placed on ice for 10 minutes, and then transferred to the tube where the reverse transcriptase was freeze-dried, and the dried material was completely dissolved. The reaction was carried out at 42 ° C. for 1 hour, and PCR was performed as in Example 2 using 1 μl of the reaction product as a DNA template. After the PCR was terminated, the PCR product was electrophoresed on a 2% agarose gel to confirm the product, and the results are shown in FIG. 4.

In FIG. 4, lane M is loaded with a 50 bp ladder as a size marker, and lane 1 is a result of a reverse transcription reaction using a reverse transcriptase that is not lyophilized, followed by a PCR reaction with a DNA polymerase that is not lyophilized. , Lane 2 is the result of reverse transcription using a reverse transcriptase that is not lyophilized, and then PCR is performed with a freeze-dried PCR reaction product of the present invention, and lane 3 is a reverse transcriptase freeze-dried with the present invention. After the reverse transcription reaction, PCR is performed by the PCR reaction product of the present invention. The reverse transcription reaction mixture of the present invention had no change in stability even when subjected to lyophilization treatment as in the PCR reaction mixture of the present invention, and the stability was maintained even in long-term storage. In other words, it can be seen that the reverse transcription reaction mixture of the present invention subjected to lyophilization shows improved stability.

The present invention provides an enzyme reaction premix. The present invention also provides a DNA polymerase reaction premix. Meanwhile, the present invention provides a kit for DNA amplification, a sequencing kit, and a kit for diagnosing a disease, including a DNA polymerase reaction premix. Enzyme pre-mixture of the present invention can improve the stability of the enzyme, and by pre-mixing and storing the reaction mixture of the enzyme to solve the problem of mutual contamination that occurs when mixing each reaction separately, and the step of the enzyme reaction operation Not only does it dramatically shorten the operation, but also a certain amount of reactants are pre-mixed, which reduces the errors that can occur when mixing several reactants separately, thereby increasing the reproducibility of the experiment and eventually increasing the reliability of the results. In addition, the pre-mixture of the present invention exhibits long-term stability in cold storage even in a liquid state, and when dried, shows long-term stability in normal temperature or cold storage, and thus can be conveniently used for storage and transportation.

Claims (15)

(a) an enzyme stabilizer selected from the group consisting of neotrehalose, isotrehalose, raffinose, melezitose and combinations thereof; (b) buffers; And (c) Enzymatic reaction premixes comprising DNA polymerase or reverse transcriptase. The pre-enzyme reaction mixture according to claim 1, wherein the amount of the enzyme stabilizer is 1-15% by weight based on the pre-enzyme reaction mixture. delete The pre-enzyme reaction mixture according to claim 1, wherein the enzyme is a DNA polymerase. A DNA polymerase reaction premix comprising an enzyme stabilizer, a buffer and a DNA polymerase selected from the group consisting of neotrehalose, isotrehalose, raffinose, melezitose and combinations thereof. 6. The prepolymer mixture of claim 5, wherein the amount of the enzyme stabilizer is 1-15% by weight based on the prepolymer mixture of the DNA polymerase reaction. 6. The DNA polymerase reaction premix according to claim 5, wherein the DNA polymerase reaction premix further comprises MgCl ₂ and four dNTPs. 6. The DNA polymerase reaction of claim 5, wherein the DNA polymerase reaction premix further comprises a dye selected from the group consisting of xylene cyanol, bromophenol blue, bromocresol red and cresol red. mixture. 8. The prepolymer mixture of claim 7, wherein the prepolymer mixture further comprises a primer for the DNA polymerase. 6. The prepolymer mixture of claim 5, wherein the DNA polymerase is a thermostable DNA polymerase isolated from pyrogenic bacteria. The pre-enzyme reaction mixture according to claim 1, 2 or 4, wherein the pre-enzyme reaction mixture has a liquid phase, vacuum dried or lyophilized state. 11. The DNA polymerase reaction premix according to any one of claims 5 to 10, wherein the DNA polymerase reaction premix has a liquid phase, vacuum dried or lyophilized state. Claim 12 DNA amplification kit comprising a DNA polymerase pre-mix. Claim 12 DNA sequence analysis kit comprising a prepolymer mixture of the reaction. A disease diagnostic kit comprising the DNA polymerase reaction premix of claim 12.