KR20060081575A - Dehydrated pcr premixes - Google Patents

Abstract

The present invention is dehydrated by drying a PCR reaction mixture comprising a reaction buffer containing MgCl ₂ , four dNTPs (dATP, dGTP, dCTP, dTTP) and a DNA polymerase (Polymerase). Dried Evaporated PCR Premix and its use.

The present invention is a liquid that is not dehydrated even with a PCR premix dehydrated by drying rather than a lyophilization (freeze-dry) method commonly used to maintain protein activity. Compared to the PCR premix, we can obtain PCR results with no detectable difference and are stable for at least 20 days at room temperature (about 25 degrees Celsius). Therefore, the dehydrated PCR Premix of the present invention maximizes the simplicity in preparation and ease of PCR reaction and facilitates storage at room temperature, thereby providing advantages in delivery to a long distance.

Description

Dehydrated PCR Premixes

1 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of the reaction buffer (Reaction Buffer) in PCR.

Figure 2 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of the reaction buffer in PCR premix dehydration.

Figure 3 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of (NH ₄ ) ₂ SO ₄ and KCl in PCR.

Figure 4 is a photograph of the PCR reaction electrophoresis analysis in 1% Agarose Gel to show the effect of dehydration on the PCR in the PCR Premix.

Figure 5 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of sedimenting Reagents (Sedimenting Reagents).

Figure 6 is a photograph of the PCR reaction electrophoresis analysis on 1% Agarose Gel to show the effect of the tracking dye (Tracking Dyes).

Figure 7 is a picture of the PCR reactions electrophoresis analysis on 1% Agarose Gel to show the stability to heat.

Figure 8 is a photograph of the PCR reaction electrophoresis analysis on 1% Agarose Gel to show the effect of the drying temperature.

The present invention relates to dehydrated PCR premixes and their use, and more particularly, to dry rather than a lyophilization method commonly used to maintain protein activity. Dehydration (Drying; Evaporation) PCR premixes and their use.

PCR (Polymerase Chain Reaction) refers to amplifying specific DNA sequences using heat-stable DNA polymerases (Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis). KB and Erlich HA.Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.Science, 239: 487-491 (1988)). DNA polymerases used in PCR are known in a wide variety (eg, Taq, Pfu, Klen-Taq, Kod, Pwo, Vent, Tfl, etc.), but Taq Polymerase is the most widely used. In addition to DNA polymerase, PCR is generally performed by using a reaction buffer containing MgCl ₂ , a template DNA containing a specific DNA sequence, a primer set for specifically amplifying a specific DNA sequence, and a DNA polymerase. It requires the mixing of reactants, including dNTPs (dATP, dGCT, dCTP and dTTP), which are converted into constituents of DNA by enzymatic action. The reaction mixture is subjected to a primer set (sense sequence and anti-sense of specific DNA sequences) through a series of cycles (Denaturation, Annealing, and Synthesis) that are repeated continuously after the initial denauration process (usually 2-5 minutes at 94 degrees Celsius or higher). By limiting the sequence, the product of the PCR limited by the element that determines the final product of the PCR is amplified. PCR is performed in an amplified manner until the time when the primer set or dNTP used in the reaction mixture acts as a limiting factor. Depending on the purpose of the experiment, PCR is usually performed about 25-40 cycles. After the PCR is completed, PCR products are analyzed by various methods.

PCR is currently used in a number of ways, including diagnostics, experimentation, and use (Bartlett JMS and Stirling D. PCR Protocols, 2 ^nd Ed. (2003). Humana Press, Totowa, NJ). Diagnostic uses include detection of viruses (such as hepatitis virus, HPV, HIV, etc.) or bacteria in blood, body secretions, or biological tissues, mutation testing of genes associated with genetic diseases, and detection of diagnostic labels. . Experimental examples include amplification of specific genes, amplification of specific DNA sequences, rapid amplification of cDNA ends (RACE) to identify gene ends, genetic mapping, genotyping, sequencing, and mutagenesis for specific bases. . Investigations include amplification of specific DNA sequences from internship samples (e.g., blood, body secretions, hair, biological tissues, or interns containing body secretions), and the like.

In performing PCR, the reaction buffer, template DNA, primer set, dNTPs, and DNA polymerase (eg, Taq Polymerase) are mixed in sequence or other components except template DNA and primer set are pre-mixed and dispensed, respectively. Prepare the PCR by adding template DNA and primer set to the aliquot. However, when dealing with many samples at the same time, it takes longer to prepare for PCR, and experimenters sometimes make mistakes such as contamination or no template DNA or primer set in making PCR mixtures. PCR premixes have been developed to alleviate these pain points for PCR users, and there are currently two types of products. One type is a mixture of reaction buffers, dNTPs and DNA polymerases (e.g. Taq Polymerase) in 2x (X) liquid phase (Epicentre Catalog (2004) .FailSafe PCR 2X Premix.EPICENTRE, Madison, WI) PCR was performed by adding the template DNA, primer set, and distilled water to 1x (X). This double liquid phase also adds sediment and tracking dyes, depending on the method to be analyzed after PCR. The other type is mixed with reaction buffer, dNTPs, DNA polymerase (e.g. Taq Polymerase), sedimenting Reagents and tracking dye in 1x (X) liquid phase and dispensed into PCR tubes in advance. And then lyophilized (Freeze-dry) (Park HO and Kim JJ.Lyophilized reagent for polymerase chain reaction.US Patents 5861251 (1999) and 6153412 (2000); Bioneer Catalog (2004). AccuPower PCR PreMix. BIONEER, Daejeon, Korea) The experimenter can perform PCR by adding only a mixture of distilled water, template DNA and primer set. Since the lyophilized PCR premix x contains sediment and tracking dyes, the PCR results can be analyzed by immediately loading the gel without subsequent processing for gel loading after PCR.

Two types of PCR premixes currently on the market provide extreme convenience for experimenters in performing PCR, but lyophilized PCR premixes have several advantages over liquid phase PCR premixes. First, lyophilized PCR premixes are much more thermally stable than liquid phase PCR premixes. The liquid phase PCR premix is made twice, so its stability is inferior, but the existence of the solution itself is basically unstable. Second, since the lyophilized PCR premix is heat stable, it is far more advantageous for delivery over long distances than liquid phase PCR premixes. Liquid phase PCR premixes are usually shipped under ice packs because they need to be refrigerated at 4-8 degrees Celsius, while lyophilized PCR premixes can be shipped remotely at room temperature without further action. Third, since the lyophilized PCR premix is pre-dispensed in the PCR container, it provides better convenience for the experimenter in the pre- and post-PCR processing compared to the liquid PCR premix that is not. However, even though lyophilized PCR premixes have this advantage over comparable liquid phase PCR premixes, they also have many advantages in performing PCR compared to conventional non-premixed forms. Also has a disadvantage that cannot be ignored in manufacturing.

In general, lyophilization (Freeze-dry) is one method of removing water from an active substance with the goal of maximizing storage or storage while minimizing damage to its activity (Rey L and May J C. Freeze-Drying / Lyophilization of Pharmaceutical and Biological Products, 2 ^nd Ed. (2004) .Marcel Dekker, Inc., New York, NY. It is well known that lyophilization can be used for this purpose and is very commonly applied to pharmaceuticals and the like. Lyophilization is accomplished by freezing the sample and then removing it by sublimation. Sublimation refers to gasification directly from the solid state without going through the liquid state. Therefore, sublimation requires low pressure and the sample must be continuously frozen throughout the lyophilization period. Therefore, lyophilization requires a large-scale equipment equipped with a freezing device and a decompression device, has a long manufacturing time, and has an expensive manufacturing cost. In particular, large-scale manufacturing requires large-scale investments because of the corresponding facilities. On the other hand, dehydration (drying; evaporation) is a process of removing moisture by applying proper heat, so there is no need to freeze the sample or lower the pressure. This has the advantage of saving. In addition, in terms of equipment, much simpler equipment can remove moisture as much as desired compared to lyophilization, and thus there is a great advantage in terms of investment for equipment. Therefore, if a PCR premix such as a lyophilized PCR premix can be produced in terms of properties and functions through dehydration by drying rather than freeze drying, various advantages of lyophilized PCR premixes are maintained intact while being manufactured. In U.S. would be able to produce PCR premixes much more advantageously.

Therefore, the inventors have found that all DNA polymerases used in PCR are heat stable, and thus, by optimizing the reaction buffer, the inventors found that PCR premixes can be prepared without compromising detectable activity even through dehydration by drying. The invention has been completed.

It is a primary object of the present invention to provide a PCR premix dehydrated by drying without detectable damage to activity.

The present invention is a dehydrated PCR premix prepared by drying a PCR reaction mixture comprising a reaction buffer containing MgCl ₂ , four dNTPs (dATP, dGTP, dCTP, dTTP) and a DNA polymerase (Polymerase). Provide (Premix).

In the present specification, the PCR premix refers to ready-to-use reagents which are prepared in advance before the PCR reaction, and can immediately put the sample of interest into the PCR reaction.

In the PCR premix of the present invention, the reaction buffer is characterized in that it does not contain potassium chloride (KCl) or ammonium sulfate ((NH ₄ ) ₂ SO ₄ ). Potassium chloride (KCl) or ammonium sulphate ((NH ₄ ) ₂ SO ₄ ) is present in a typical PCR reaction buffer, but these may be concentrated upon drying and degrade the activity of DNA polymerase. As shown in the embodiment of the present invention, it was confirmed that the activity of the DNA polymerase is sufficiently maintained even when the potassium chloride (KCl) or ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) is removed from the reaction buffer.

In the PCR premix of the present invention, the DNA polymerase includes any heat-stable DNA polymerase used in a conventional PCR reaction, but preferably, Taq DNA Polymerase or super thermophilic bacteria derived from thermophilic calm bacteria Derived Pfu DNA Polymerase, or a mixture of both.

In the present invention, the drying means evaporating the liquid from the liquid reaction mixture by applying appropriate heat, and is clearly distinguished from conventional lyophilization in that it is not in a frozen state and a reduced pressure environment. That is, the dehydration process provided by the present invention is not a lyophilization (Freeze-dry) method commonly used for maintaining the activity in the protein having activity (Dehydration; Drying; Evaporation).

In the PCR premix of the present invention, the drying is preferably characterized by drying in a ventilated environment at a temperature of 30-70 degrees Celsius. If the drying temperature is less than 30 degrees Celsius, the drying time to dehydration takes too long, and if the drying temperature is greater than 70 degrees Celsius, the reaction mixture may be denatured.

Specifically, the drying may be performed in a dry oven or an incubator. Dry ovens are typically used to dry clean superfluous or laboratory equipment by circulating dry hot air. An incubator is used to raise or cultivate an organism, and thus can be used for drying of the present invention because the temperature or humidity can be constantly adjusted.

In the PCR premix of the present invention, the PCR reaction mixture is characterized in that it further comprises sedimenting agents (Sedimenting Reagents) to facilitate the gel loading (loading). By adding the sediment to the reaction mixture composition, gel loading can be performed immediately without further processing after the PCR reaction. Precipitating materials that can be used in the present invention include, for example, Ficoll, glycerol (Glycerol), Glucitol (Glucitol), Sorbitol (Sorbitol) and Sucrose (Sucrose).

In the PCR premix of the present invention, the PCR reaction mixture is characterized in that it further comprises a tracking dye (Tracking Dyes) to easily observe the movement on the gel. The tracking dye makes it easy to know the degree of dissolution of the PCR premix in the process of rehydration when performing PCR using the dehydrated PCR premix as well as loading the gel in the gel loading which will be after the PCR reaction. There is an advantage to facilitate tracking (Tracking) in the movement on the gel of the PCR reaction. Tracking dyes that may be used in the present invention include, for example, xylene cyanole, bromophenol blue, cresol red, bromocresol red, or mixtures thereof. Can be mentioned.

In order to stabilize DNA polymerase and dNTP, gelatin, bovine serum albumin (BSA), ammonium sulfate ((NH ₄ ) ₂ SO ₄ )), and Thesit can be used. Nonionic surfactants such as NP40 and Tween20 can be added to the reaction mixture (Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB and Erlich HA.Primer-directed enzymatic amplification). of DNA with a thermostable DNA polymerase.Science, 239: 487-491 (1988).

The PCR premix of the present invention may be subjected to a PCR reaction after rehydration by adding a template DNA, a primer set and a distilled water of interest to the PCR premix according to the purpose to be used. The PCR premix of the present invention may be included in a sequencing or disease diagnosis kit that requires a PCR reaction, and may be used to determine sequencing or diagnose infection of a disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

(MATERIALS AND METHODS)

1) Heat-stable DNA Polymerases

As Taq Polymerase, Ace-Taq Polymerase purchased from Genenmed (Seoul, Korea) and Taq Polymerase purchased from Roche Applied Science (Pensberg, Germany) were used. Pfu Polymerase was purchased from Stratagene (La Jolla, CA, USA). Taq Polymerase was purchased from AB Peptides (St. Louis, MO, USA).

2) Chemicals

All Chemical Reagents were purchased from Sigma (St. Louis, Mo., USA) except Heat-stable DNA Polymerase.

3) PCR reaction

PCR reactions followed the manufacturer's protocol for each heat-stable DNA polymerase. However, PCR was performed 25 cycles to avoid the point of time when primers or dNTPs act as a limiting factor of the reaction. Template DNA and Primer were respectively expressed in pCMVdwSEC (CV) ILZTRAIL plasmid (Kim MH, Billiar TR and Seol DW.The secretable form of trimeric TRAIL, a potent inducer of apoptosis.Biochem.Biophys.Res.Commun. 2004)) (1ng / 100ul PCR reaction solution) and Sense primer (5'-GTGAGGGAAAGAGGTCCTCAG-3 '; 500ng / 100ul PCR reaction solution) derived from human TRAIL gene and Anti-sense primer (5'-TTAGCCAACTAAAAAGGCCCCG-3'); 500ng / 100ul PCR reaction solution) was used, and the PCR reaction was performed at 95 degrees Celsius for 5 minutes for denaturation, followed by 1 minute at 95 degrees Celsius, 25 seconds at 55 degrees Celsius, and 30 seconds at 72 degrees Celsius for 25 cycles. Was done. PCR reactions consisted of 100ul or 15ul and PCR reactions were analyzed by electrophoresis under TBE buffer on 1% Agarose Gel.

4) Dehydration of PCR Premix

PCR premixes were prepared according to the composition shown in the Examples of the present invention and were aliquoted in 15 ul and dehydrated in an incubator fixed at the appropriate temperature.                     

5) Rehydration of PCR Premixes

The dehydrated PCR premix was rehydrated with 15ul of reactant (1ng template plasmid / 100ul PCR reaction solution, 500ng sense primer / 100ul PCR reaction solution, 500ng anti-sense primer / 100ul PCR reaction solution, and distilled water).

Example 1 Influence of Reaction Buffer on PCR

In the dehydration of the PCR premix, for the purpose of obtaining a higher efficiency of PCR reactivity, the composition of the reaction buffer (Reaction Buffer) in PCR in the liquid phase condition was first investigated how Taq Polymerase (Fig. 1). For this experiment, the inventors used Buffer A (50 mM Tris-Cl (pH 9.1), 16 mM (NH ₄ ) ₂ SO ₄ , 3.5 mM MgCl ₂ , 150 ug / ml BSA) provided by Genenmed for Ace-Taq and Ace-Taq. And a comparative experiment was performed using Taq Polymerase (Roche Taq) and Roche Taq Buffer R (10mM Tris-HCl, 15mM MgCl ₂ , 50mM KCl, pH 8.3) provided by Roche. Ace-Taq showed very good PCR results in Buffer A, a magnetic buffer composition (lane 2), but Roche Taq failed to detect a detectable result in Buffer R, a magnetic buffer composition (lane 5). However, Roche Taq showed a PCR reaction that was detectable in Buffer A (lane 4). Although not as much as by Buffer A, Ace-Taq showed satisfactory PCR results for Buffer R (lane 3). These results show that Ace-Taq is more reactive than Roche Taq and that the PCR reaction occurs more efficiently in Buffer A than in Buffer R. Figure 1 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of the reaction buffer (Reaction Buffer) in PCR. Lane 1 shows the fx174 / HaeIII Marker, and A and R indicate that PCR was performed using Buffer A and Buffer R, respectively.

Example 2 Influence of Reaction Buffer on PCR Premix Dehydration

To determine the effect of dehydration on the PCR reaction, PCR premixes were prepared (1x reaction buffer, 200uM dNTPs, 5U / 100ul PCR reaction solution), and 15 μl aliquots were first dehydrated at 37 degrees Celsius. 15 ul of reactant (1 ng template plasmid / 100 ul PCR reaction solution, 500 ng sense primer / 100 ul PCR reaction solution, 500 ng anti-sense primer / 100 ul PCR reaction solution, the rest of the distilled water) was added to the dehydrated PCR premix, followed by rehydration. PCR reaction was performed as in Example 1 (FIG. 2). Dehydration significantly reduced the reactivity of PCR regardless of the reaction buffer used for both Ace-Taq and Roche Taq. This result implies that dehydration seriously impairs the activity of Taq Polymerase used under these reaction buffer compositions. This result also shows that even if the liquid phase shows very good PCR reactivity, dehydrating the liquid phase immediately does not preserve the PCR reactivity in the liquid phase after dehydration. The premix is believed to be the reason why it could not be produced by methods other than the lyophilization method. Figure 2 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of the reaction buffer in PCR premix dehydration. Lane 1 shows the fx174 / HaeIII Marker, and A and R indicate that PCR was performed using Buffer A and Buffer R, respectively. Lanes 2, 4, and 6 are the result of PCR reaction using liquid phase PCR premix, and lanes 3, 5, and 7 have the same composition as the liquid phase PCR premix used in lanes 2, 4, and 6, respectively, but PCR using dehydrated PCR premix. Reaction result.

Example 3 Influence of (NH ₄ ) ₂ SO ₄ and KCl in PCR

In general, the reaction buffer is formulated to optimize the enzyme action. However, as observed in Example 2 above, Buffer A and Buffer R did not preserve the activity of Ace-Taq and Roche Taq during the dehydration process. Therefore, in this embodiment, to find out which components of each reaction buffer composition are directly related to the inactivation of Taq Polymerase in the dehydration process in order to optimize the reaction buffer in dehydration, The effect of key components on PCR reactivity was investigated.

Roche Taq's Buffer R shows the composition of reaction buffers that most Taq Polymerase producers typically offer for Taq Polymerase, while Ace-Taq's Buffer A is slightly different. That is, Buffer A has a pH of 9.1 in Tris buffer and (NH ₄ ) ₂ SO ₄ instead of KCl. (NH ₄ ) ₂ SO ₄ is well known as a substance that reacts with and stabilizes proteins. However, although the (NH ₄₎ during the _second known is even then dehydration function of the SO ₄ (NH ₄₎ a concentration of ₂ SO ₄ could adversely affect the rather structure of Taq Polymerase by gradually increases with the progress of the dehydration process, The effect of (NH ₄ ) ₂ SO ₄ on the PCR reaction was investigated (FIG. 3, Left Panel). Buffer A which does not contain (NH ₄ ) ₂ SO ₄ (hereinafter referred to as Buffer O; composition of Buffer O is 50mM Tris-Cl (pH 9.1), 3.5mM MgCl ₂ , 150ug / ml BSA) compared with Buffer A There was no detectable difference in the PCR reactivity of Ace-Taq (lanes 2 and 3). Roche Taq showed lower PCR reactivity in Buffer O than in Buffer A (lanes 4 and 5). Nevertheless, Buffer O provided better PCR reactivity for Roche Taq compared to Buffer R (lane 5 of FIG. 1 and lane 5 of FIG. 3). This result shows that when using Buffer A, the PCR reactivity of Ace-Taq is hardly affected by the presence or absence of (NH ₄ ) ₂ SO ₄ .

On the other hand, Ace-Taq using Buffer A showed significantly superior results in PCR reactivity compared to Buffer R, which can be seen as a reaction buffer for Roche Taq and general Taq Polymerase, as seen in the above examples. In the experiment, the experiment was conducted using the composition of Ace-Taq and its reaction buffer, Buffer A (or Buffer O).

Buffer R, Roche Taq's reaction buffer, contains KCl (50mM) contained in most Taq Polymerase reaction buffers. Therefore, KCl was also considered to have some effect on the structure of Taq Polymerase due to the increase in concentration that occurs during the dehydration process, we examined the effect of KCl on the PCR reaction (Fig. 3, Right Panel). Buffer A and Buffer O both show comparable results for Ace-Taq (Fig. 3, lanes 2 and 3), but for Buffer A, once dehydrated (Fig. 2, lanes 2 and 3), Significantly lost activity, and Buffer R does not ensure sufficient PCR reactivity in either liquid phase or after dehydration in both Ace-Taq and Roche Taq, so the effect of KCl on Ace-Taq is due to Buffer O, a variant of Buffer A. Investigated. Importantly, increasing the concentration of KCl in Buffer O significantly reduced the PCR reactivity of Ace-Taq (FIG. 3, lane 6-9). These results show that KCl, which is basically included in most Taq Plymerases, has a negative effect on Buffer O composition rather than a positive effect on PCR, while Roche Taq has a lower PCR reactivity in Buffer R than in Buffer A. This may mean that the cause is probably KCl in the Buffer R. Figure 3 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of (NH ₄ ) ₂ SO ₄ and KCl in PCR. Lane 1 shows the fx174 / HaeIII Marker, and A and O indicate that PCR was performed using Buffer A and Buffer O, respectively.

Example 4 Effect of Dehydration on PCR in PCR Premixes

As shown in Example 3 above, Buffer O, which is a buffer composition containing (NH ₄ ) ₂ SO ₄ in Buffer A, showed no detectable difference in the liquid phase PCR reaction in Ace-Taq compared to Buffer A. So, to see how dehydration affects the reaction of Ace-Taq in Buffer O, we made a PCR premix (1x Buffer O, 200 uM dNTPs, 5U / 100ul PCR reaction solution), and then aliquoted in 15ul each to 37 degrees Celsius. Dehydrated at. 15 ul of reactant (1 ng template plasmid / 100 ul PCR reaction solution, 500 ng sense primer / 100 ul PCR reaction solution, 500 ng anti-sense primer / 100 ul PCR reaction solution, the rest of the distilled water) was added to the dehydrated PCR premix, followed by rehydration. PCR reaction was performed as in Example 2 (FIG. 4). Lane 1 is the result of PCR reaction using liquid phase PCR premix and Lane 2 is the result of PCR reaction using dehydrated PCR premix having the same composition as liquid phase PCR premix used in Lane 1. Unlike dehydration using Buffer A (FIG. 2, lanes 2 and 3), dehydration using Buffer O did not cause any detectable damage to Ace-Taq. When dehydration was performed by including KCl in Buffer O, and the PCR reaction was carried out as shown in Example 3, the degradation of the PCR reaction was observed similarly to that shown in Example 3 (Fig. 3, lane 7-9). These results show that the significant inactivation of Ace-Taq and Roche Taq in dehydration with Buffer A was due to (NH ₄ ) ₂ SO ₄ contained in Buffer A (FIG. 2, lanes 2, 3, and 6). And 7), the significant deactivation of Ace-Taq in dehydration using Buffer R (Fig. 2, lanes 4 and 5) may be due to KCl. Importantly, as mentioned above, since KCl is basically included in most Taq Plymerase reaction buffers, it means that KCl cannot be dehydrated while preserving Taq Polymerase activity as long as it is included in the reaction buffer. Therefore, the results of this example are currently lyophilized PCR premix (Bioneer Catalog (2004). AccuPower PCR PreMix.BIONEER, Daejeon, Korea) is made using a reaction buffer containing KCl, so lyophilization is not applied. It would be shown that it could not have been manufactured properly without.

In this embodiment, Pfu Polymerase, Klen-Taq Polymerase, and a mixture of the two (Klen-Taq: Pfu = 19: 1 (v / v)) are dehydrated using Buffer O as in Ace-Taq, and dehydrated on Buffer O. The effect of dehydration on their PCR reactivity was also investigated (Figure 4, lanes 3-8). When dehydrated with Buffer O, Pfu Polymerase, Klen-Taq Polymerase and the mixture of both showed no detectable degradation of PCR reactivity as in Ace-Taq. The results show that Buffer O can dehydrate a wide variety of heat-stable DNA polymerases without serious activity loss. Figure 4 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of dehydration on the PCR in the PCR premix. O means that PCR is performed using Buffer O. Lanes 1, 3, 5 and 7 are the result of PCR reaction using liquid phase PCR premix, and lanes 2, 4, 6 and 8 have the same composition as liquid phase PCR premix used in Lanes 1, 3, 5 and 7, respectively, PCR reaction results using PCR premix.

Example 5 Influence of Sedimenting Reagents

We investigated the effect of addition of sediment on the dehydration of Buffer O as a basic reaction buffer composition for Ace-Taq in dehydration so that gel loading can be done immediately after the PCR reaction without further treatment. Ficoll, Glycerol, Sucrose, etc. are widely used as the sedimentation material. In this embodiment, the effect of decollation was investigated using Ficoll and Glyceol in various concentrations (FIG. 5). Figure 5 is a photograph of the PCR reaction by electrophoresis on 1% Agarose Gel to show the effect of sedimenting Reagents (Sedimenting Reagents). Make a PCR premix as in Example 4 above, but with a settling material (1x Buffer O, 200uM dNTPs, 5U / 100ul PCR reaction solution, total volume (v / v) 0-2% Ficoll or 2-4% Glycerol) 15ul was dispensed at first and dehydrated at 37 degrees Celsius. 15 ul of reactant (1 ng template plasmid / 100 ul PCR reaction solution, 500 ng sense primer / 100 ul PCR reaction solution, 500 ng anti-sense primer / 100 ul PCR reaction solution, the rest of the distilled water) was added to the dehydrated PCR premix, followed by rehydration. PCR reaction was performed as in Example 2. The precipitate did not have any detectable effect on PCR reactivity regardless of the concentration used. This result implies that the dehydration process provided by this patent can be applied by further including the settling material in the PCR premix, and the dehydrated PCR premix can omit the treatment in the gel loading which will be after the PCR reaction. This means that it is possible to provide users with a breakthrough simplicity in the PCR reaction.

Example 6 Influence of Tracking Dyes

The tracking dye not only makes it easy to know the degree of dissolution of the PCR premix during the rehydration process when the PCR is performed using the dehydrated PCR premix, but also the gel phase of the loaded PCR reaction in the gel loading that will be present after the PCR reaction. There is an advantage of facilitating tracking in movement. Therefore, dehydration by adding the tracking dye to the PCR premix allows the user to provide simplicity in the PCR reaction by omitting the processing such as gel loading by separately adding the tracking dye in the gel loading which will be after the PCR reaction. do.

Xylene cyanole, Bromophenol blue, Cresol red and Bromocresol red are known as tracking dyes. In this embodiment, Xylene cyanole and Bromophenol blue are used in a single or mixed manner to affect the dehydration of the PCR premix provided by this patent. Investigated (FIG. 6). Figure 6 is a photograph of the PCR reaction electrophoresis analysis on 1% Agarose Gel to show the effect of the tracking dye. X and B refer to Xylene cyanole and Bromophenol blue respectively, Lane 1-4 contains 2% Ficoll to total volume (v / v) when making PCR premix and Lane 5-8 to total volume when making PCR premix (v / v) 4% Glycerol. Make a PCR premix by including the settling material as in Example 5 above, but additionally by adding a single or mixed tracking dye (1x Buffer O, 200uM dNTPs, 5U / 100ul PCR reaction solution, total volume ratio (v / v)) 15% of 2% Ficoll or 4% Glycerol, total volume (w / v) 0.005% X or 0.0025% B or 0.0025% X and 0.0025% B) was first dehydrated at 37 degrees Celsius. 15 ul of reactant (1 ng template plasmid / 100 ul PCR reaction solution, 500 ng sense primer / 100 ul PCR reaction solution, 500 ng anti-sense primer / 100 ul PCR reaction solution, the rest of the distilled water) was added to the dehydrated PCR premix, followed by rehydration. PCR reaction was performed as in Example 2. The tracking dye had no detectable effect on PCR reactivity regardless of the type and concentration used. The results show that the inclusion of tracking dyes in the PCR premix allows the dehydration process provided by this patent to be applied.                     

Example 7 Stability Against Heat

Although Taq Polymerase is thermally stable, delivery is usually carried out at low temperatures using dry ice or ice packs. In actual solution, Taq Polymerase is inactivated to a considerable extent when left at room temperature (about 25 degrees Celsius) for about a week. This is probably due to the result of Taq Polymerase's structural reaction by constant reaction with water in solution. Removing water, either lyophilized or dehydrated, is well known as a very important process for improving sample stability. Thus, commercially available lyophilized PCR premixes are much more stable to heat than aqueous solutions, as expected. Therefore, in this Example, we investigated how dehydration affects the heat stability of the PCR premix provided by this patent. PCR premixes were prepared as described in Example 6 (1x Buffer O, 200 uM dNTPs, 5U / 100ul PCR reaction solution, total volume (v / v) 2% Ficoll or 4% Glycerol, total volume (w / v) A mixture of 0.0025% X and 0.0025% B) 15ul each was dehydrated first at 37 degrees Celsius. Store the dehydrated PCR premix at room temperature (approximately 25 degrees Celsius) with 15 ul of reactant (1 ng template plasmid / 100 ul PCR reaction, 500 ng sense primer / 100 ul) at 5, 10 and 20 days. PCR reaction solution, 500ng anti-sense primer / 100ul PCR reaction solution, the rest was distilled water) and then rehydrated to perform a PCR reaction as in Example 2 (Fig. 7). Figure 7 is a picture of the PCR reactions electrophoresis analysis on 1% Agarose Gel to show the stability to heat. Lanes 1-4 contain 2% Ficoll to total volume (v / v) when making PCR premixes and Lane 5-8 contain 4% Glycerol to total volume (v / v) when making PCR premixes.

As the results of this example show, the dehydration PCR premix provided by this patent was stable for at least 20 days at room temperature. The results not only offer advantages in the delivery of PCR premixes, but also provide researchers with the convenience of storing PCR premixes.

Example 8 Influence of Drying Temperature

To investigate the effect of drying temperature on the dehydration of the PCR premix, a PCR premix was prepared as in Example 7 (1x Buffer O, 200 uM dNTPs, 5U / 100ul PCR reaction solution, 2% Ficoll to total volume). 15 μl of the total volume (w / v) of 0.0025% X and 0.0025% B was dispensed first to dehydrate at various temperatures (30, 37, 40, 45, 50, 55, 60, 70 degrees Celsius). 15 ul of reactant (1 ng template plasmid / 100 ul PCR reaction solution, 500 ng sense primer / 100 ul PCR reaction solution, 500 ng anti-sense primer / 100 ul PCR reaction solution, the rest of the distilled water) was added to the dehydrated PCR premix, followed by rehydration. PCR reaction was performed as in Example 2 (Fig. 8). Figure 8 is a photograph of the PCR reaction electrophoresis analysis on 1% Agarose Gel to show the effect of the drying temperature. As shown in this example, PCR reactivity showed no detectable difference over the entire range of drying temperatures investigated. This result means that it can be dehydrated in the PCR premix without compromising detectable activity over the entire range of these temperatures. The drying time was 6 hours when dried at 37 degrees Celsius or more, and when dried at 37 degrees Celsius, there was no detectable difference in PCR reaction between drying for 6 hours and drying for 12 hours. The elapsed drying time does not seem so important.

When removing water while maintaining the activity of the protein (Integrity) in general, Lyophilization (Freeze-dry) method is used, but the present invention is prepared by dehydration (Drying; Drying; Evaporation) by drying It is shown that even with a PCR premix, PCR results with no detectable difference can be obtained compared to a non-dehydrated liquid phase PCR premix and are stable for at least 20 days at room temperature (about 25 degrees Celsius). Therefore, the dehydrated PCR premix of the present invention maximizes the simplicity in preparation and ease in PCR reaction and facilitates storage at room temperature, thereby providing advantages in delivery over long distances.

Claims (8)

Prepared by drying a PCR reaction mixture containing a reaction buffer containing MgCl ₂ , four dNTPs (dATP, dGTP, dCTP, dTTP) and a heat-stable DNA polymerase (Polymerase). Dehydrated PCR Premix. The PCR premix according to claim 1, which does not contain potassium chloride (KCl) or ammonium sulfate ((NH ₄ ) ₂ SO ₄ ). The PCR premix according to claim 1, wherein the thermally stable DNA polymerase is Taq DNA Polymerase, Pfu DNA Polymerase, or a mixture of the two. The PCR premix according to claim 1, wherein the drying is performed in a ventilation environment at a temperature of 30-70 degrees Celsius. The PCR premix of claim 1, wherein the PCR reaction mixture further comprises sedimenting reagents to facilitate gel loading. The PCR premix according to claim 5, wherein the sediment is Ficoll or Glycerol. The PCR premix of claim 1, wherein the PCR reaction mixture further comprises a tracking dye for easily observing the rehydration state and the movement on the gel. 8. The PCR premix according to claim 7, wherein the tracking dye is Xylene cyanole or Bromophenol blue, or a mixture of the two.

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