KR20000075140A - DNA size marker which can be preserved and delivered at room temperature - Google Patents

Abstract

PURPOSE: Provided is a DNA size marker composition that can be preserved and transported even at room temperature and used easily. CONSTITUTION: A DNA size marker composition consists of DNA fragment, a buffer solution, a dye, a stabilizer and a synergist for specific gravity. A DNA fragment, a buffer solution and a dye of them are frozen and dried separately as a form of a freeze-dried object. Therefore, it can be preserved and transported at room temperature before and after recomposition. Also, a freeze-dried object is directly applied to electrophoresis if it is dissolved by the recomposition solution comprising 2,5-4% of ficoll and 8-20 ml of EDTA. The dye is favorably 0.01-0.1% of bromophenol blue or xylenecyanol; the stabilizer is favorably 1-50 mM EDTA; and the synergist for specific gravity is favorably, 1-5 % of ficoll or 2.5-10 % of glycerol.

Description

DNA size marker which can be preserved and delivered at room temperature}

The present invention relates to DNA size markers that can be preserved and transported at room temperature. More specifically, the present invention relates to a DNA size marker composition composed of DNA, a buffer, a dye, a stabilizer and a specific gravity enhancer.

In addition, the present invention relates to a DNA size marker composition in the form of a lyophilized lyophilized separately from the DNA, the buffer solution and the staining agent so that the DNA component is not degraded even at room temperature, the user is a reconstituting solution consisting of the remaining components at the first use It relates to a DNA size marker composition that can be used by suspending the lyophilisate.

Knowing the size of DNA is very basic and important for modern molecular biology research, medical research and genetic engineering. DNA can be separated by electrophoresis on the gel, depending on its size, and the distance is different, and the exact size of the DNA fragments can be obtained by using the formula between the molecular weight and the distance.

D = a-b (logM)

Where D is the moving distance on the gel, M is the molecular weight, and a and b are constants determined by electrophoresis conditions.

However, such a method is so complicated that recently, a size marker having a known size is used to compare the moving distance, and thus, a method of measuring a large size is used. In other words, the size marker and the DNA fragment to be measured are electrophoresed side by side on a side track on a gel to compare the moving distance. Compared to the existing method, it is very simple and widely used. Despite the presence of more accurate DNA sizing and quantitation methods, DNA size markers are widely used in conjunction with gel electrophoresis because they are fast and easy to analyze and can be analyzed with relatively small samples. Therefore, DNA size markers have been preferred to be simpler to use and simple to prepare.

Many of these size markers are fragments that cut DNA of lambda phage (phage λ) with restriction enzymes, and most commonly used fragments of λ phage digested with restriction enzyme HindIII, and λ phage fragments cut with HindIII. If you use them as a size marker can measure the size from about 125bp to 23kb. In addition, appropriate size markers are sold depending on the approximate size range of the DNA to be measured.

As such, the most frequently used material for molecular biological research is the DNA size marker, but it is not yet developed in Korea and is imported in its entirety. In addition, if the DNA size marker is left at room temperature for a long time, the most important component of the DNA is degraded and the intended purpose of measuring the size of the DNA sample cannot be achieved. Therefore, the storage and transportation environment of the DNA size marker should be kept at a low temperature. As a result, DNA size markers are stored and transported using dry ice, and the purchase cost is very high. In addition, it is common practice that users inadvertently leave DNA size markers at room temperature for a long time and discard expensive DNA size markers.

While studying a method that can be stored and transported at room temperature in order to improve the uneconomical and inconvenient use of the existing size marker as described above, the present inventors increase the stability by adding a solution of a constant composition to the DNA lyophilized The present invention was completed by discovering that the freeze-dried body can be directly electrophoresable by dissolving the freeze-dried solution in a recomposition solution.

An object of the present invention to provide a size marker composition that can be stored and transported at room temperature.

FIG. 1 is a diagram illustrating band bands of size markers in which λ phages were digested with restriction enzyme HindIII before lyophilization, respectively, using gel electrophoresis at different buffer concentrations, EDTA (ethylenediaminetetraacetic acid) concentrations, and ficoll concentrations. It is a photograph,

A): When using TE buffer

B): TEN buffer is used

M: Commercially used size marker

Lane 1: picol concentration 2.5%, EDTA concentration 8 mM

Row 2: Picol concentration 2.5%, EDTA concentration 10mM

Line 3: Picol concentration 2.5%, EDTA concentration 15mM

Row 4: picol concentration 2.5%, EDTA concentration 20mM

Row 5: picol concentration 3.3%, EDTA concentration 10mM

Line 6: Piccol concentration 3.3%, EDTA concentration 15mM

Row 7: picol concentration 3.3%, EDTA concentration 20mM

Line 8: Picol concentration 4.2%, EDTA concentration 10mM

Line 9: Picol concentration 4.2%, EDTA concentration 15mM

Row 10: picol concentration 4.2%, EDTA concentration 20mM

Figure 2 is a picture of the gel electrophoresis band of the size marker before, after lyophilization and after 90 days to investigate the stability of the size marker.

A): Size marker before freeze drying

B): Size marker reconstituted after 60 days of freeze drying

C): Size marker left for 90 days at room temperature after recomposition

M: Commercially used size marker

Line 1: Room Temperature + Light

Line 2: Room temperature + light blocking

Line 3: 4 ℃ + Light Blocking

In order to achieve the above object, the present invention provides a DNA size marker composition composed of DNA, a buffer, a dye, a stabilizer, and a specific gravity enhancer. Since the DNA size marker composition of the present invention is stored and transported by lyophilizing DNA, a buffer solution, and a dye in the above components, it can be stored at room temperature before or after recomposition.

The present invention also provides a solution for recomposing the size marker lyophilized body and a solution of the size marker lyophilized to make the size marker composition of the complete component.

Hereinafter, the present invention will be described in detail.

The DNA size marker composition of the present invention is composed of DNA fragments, buffers, stains, stabilizers and specific gravity increasing agents. In addition, DNA fragments, buffers, and dyes in the composition of the present invention can be prepared by lyophilization, and when used, they are directly electrophoresed by dissolving them in a recomposition solution [Picol 2.5-4%, 8-20 mM EDTA (ethylenediaminetetraacetic acid)]. It is configured to be possible.

As the DNA used in the present invention, any DNA included in the conventional DNA size marker can be used.

The buffer solution of the present invention is configured to increase the stability during lyophilization of the DNA size marker composition, and can be used directly for electrophoresis. The buffer used for this purpose is preferably TEN buffer (10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1 mM EDTA) and TE buffer (10 mM Tris-HCl, 1 mM EDTA), particularly TE buffer of pH 8.0 (See FIG. 1).

The dyeing agent of the present invention is configured to measure the progress of the DNA during electrophoresis and to facilitate loading on the gel, and bromophenol blue (BPB, bromophenol blue) or xylene cyanol (xylene cyanol), respectively. It is composed by adding to the buffer solution. At this time, the dyeing agent is preferably added so that the final concentration is 0.01 ~ 0.05% at the time of recomposition.

The DNA size marker composition of the present invention may further contain an antioxidant such as dithiothreitol, or may include a DNA degradation inhibitor.

The components of the DNA size marker composition of the present invention can be prepared by lyophilization by lyophilization and the lyophilized body can be stored at room temperature for a long time, and can be directly used for electrophoresis after melting with a recombination solution.

The recombination solution of the present invention is configured to increase stability when stored at room temperature after recomposition of the lyophilized DNA size marker composition, and to increase specific gravity so that the gel is correctly loaded (loaded) upon electrophoresis.

As the specific gravity increasing agent, glucose, sucrose, glycerol, ficoll, and the like may be used, but picol is most preferred, and EDTA may be used as a stabilizer. As picol and EDTA concentrations increase, the electrophoretic bands of DNA fragments appear wider, and in particular, low-molecular-weight DNA fragments become faint and difficult to distinguish. Therefore, it is necessary to optimize the concentration of picol and EDTA, and in the case of picol, the final concentration is preferably 2.5% to 4%, especially at 2.5%. A clear, low-diffusion band can be obtained.

Since the above components are contained, the lyophilized product of the size marker according to the present invention can be used simply by adding and suspending the reconstituting solution. In addition, long-term storage at room temperature in a freeze-dried state is possible, and even after long-term storage at room temperature after suspension with a recombination solution, both the quantity and quality of the size marker are maintained well.

In addition, the size marker lyophilized product of the present invention can increase the stability of the dye by blocking sunlight. The size marker lyophilized product of the present invention is not affected even if it is left at room temperature for 60 days in the state of blocking the light, and the electrophoresis result is not affected. (See Figure 2).

The present invention will be described by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited to the examples.

Example 1.Lyophilization of DNA Size Markers

1) Preparation of DNA

Bacillus bacteriophage λ cl ⁸⁵⁷ Sam7 (hereinafter referred to as λ phage) as the host of E. coli VCS (STRATAGENE, USA) and XL1 Blue MRF´ (STRATAGENE, USA), NZCYM (1% NZ amine, 0.5% NaCl, 0.1) % casimino acids, 0.5% Bacto-yeast extract, 0.2% MgSO ₄ · 7H ₂ O) and LB medium (Luria-Bertani medium).

The phage particles were purified purely according to the method of Sambrook et al. (Molecular clonging, Cold Spring Harbor Laboratory Press, 1989), followed by column chromatography (Qlum, Qiagen, Germany). About 1 mg of phage DNA was isolated from the culture. Specifically, XL1 Blue MRF´ strains were cultured in NZCYM medium for 16 hours, concentrated by centrifugation of OD ₆₀₀ = 1, suspended in 3 ml SM buffer, and then 5 × 10 ⁷ phage particles were added. Infection for 20 minutes at 37 ° C. This was added to 500 ml of NZCYM medium and shaken at 37 ° C. for 9-12 hours to separate DNA from the phage lysate obtained by chromatography.

2) Manufacture of size marker lyophilized

The DNA isolated in step 1) was treated with restriction enzyme HindIII in large quantities, and then heated at 68 ° C. for 20 minutes to inactivate the enzyme. Phage DNA treated with restriction enzymes were extracted with phenol to purely separate DNA. 50 μg of the isolated DNA was dissolved in 200 μl of TE buffer (pH 8.0) to a concentration of 50 μg DNA / 200 μl. 100 μl of the dye solution [0.3% bromophenol blue (BPB), 0.3% xylene cyanol (XC), and finally 300 μl of TE or TEN buffer were added. A μl DNA composition was prepared (final concentration: 50 μg DNA / 600 μl, 0.01-0.05% BPB, 0.01-0.05% XC), and the composition was dried in a centrifugal vacuum dryer for 3 hours to obtain a size marker lyophilized product.

3) Recomposition and use of size marker lyophilized body

The lyophilized body was dissolved by adding a reconstituting solution of the same composition as the volume before drying (600 μl) and then directly used for electrophoresis, and thus the same band shape as that of the band before lyophilization without modification of pigments. There was no change in quality and quantity.

Reconstituting solutions (1)

Picol 2.5%

8mM EDTA

H ₂ O

On the other hand, even if the electrophoresis was performed by including the antioxidant DTT (dithiothreitol) in the size marker composition, DTT did not affect the resolution.

Comparative Example 1. Reforming solution using glycerol as specific gravity increasing agent

In the same manner as in Example 1, a solution for recomposition of the following composition was added to suspend and recompose the size marker lyophilized product. As a result, there was no particular abnormality before and after lyophilization in the result of recomposition and electrophoresis.

Reconstituting solutions (2)

2.5 to 10% (w / v) glycerol

8 ~ 20mM EDTA

H ₂ O

Comparative Example 2. Determination of Buffer

In order to determine the preferred buffer solution in the size marker lyophilizer, a lyophilizer was prepared in the same manner as in Example 1, but TEN buffer (10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1 mM EDTA) was used instead of the TE buffer as a buffer solution. Used.

As a result, using the TE buffer solution was able to obtain a clearer DNA band than when using the TEN buffer solution (see Figure 1). Therefore, it was concluded that it is preferable to use TE buffer rather than TEN buffer in order to obtain higher resolution.

Comparative Example 3. Determination of Concentration of Components in Solution

In order to determine the preferred concentrations of the picol and the stabilizer EDTA among the components of the reconstituting solution containing picol and EDTA, the concentration in the reconstituting solution was changed to 2.5%, 3.3% or 4.0%, For EDTA, the size marker composition was electrophoresed in 1% agarose gel at a concentration of 1 μg / 12 μl per well, changing to 8 mM, 10 mM, 15 mM or 20 mM (see FIG. 1).

In the same manner as in Example 1, one of the reconstituting solutions of the composition shown in Table 1 was selected and added to suspend and recompose the size marker lyophilized product.

Picol concentration (%) EDTA concentration (mM) Reconstituting solutions (3) 2.5 10 Reconstituting solutions (4) 2.5 15 Reconstituting solutions (5) 2.5 20 Reconstituting solutions (6) 3.3 10 Reconstituting solutions (7) 3.3 15 Reconstituting solutions (8) 3.3 20 Reconstituting solutions (9) 4.2 10 Reconstituting solutions (10) 4.2 15 Reconstituting Solutions (11) 4.2 20

As can be seen in Figure 1, the higher the concentration of picol, the wider the DNA band spreads, so it is preferable to use an appropriate concentration of additives, preferably 2.5% to 4% in the re-formulation solution, especially when 2.5% A clear, low-diffusion band can be obtained. On the other hand, the higher the EDTA concentration, the wider the electrophoretic band of the DNA fragments, especially the fragments of less than 562bp faint became difficult to distinguish.

As a result, it is preferable to use distilled water containing 2.5% picol and 8 mM EDTA as the component of the reconstituting solution (1) as the reconstituting solution.

Comparative Example 4. Determination of the concentration of the dye

In the same manner as in Example 1, the lyophilisate was suspended and reconstituted, but the final concentration of the two dyes was changed to 0.01% instead of 0.05% during preparation of the lyophilisate. As a result, it was easier to observe the DNA present in the gel site where the dye was located. As such, even when the concentration of the dye was reduced to 0.01%, there was no inconvenience in the function of the size marker.

Example 2. Stability at Room Temperature of DNA Size Marker Compositions

In order to determine the preferred preservation conditions, the size marker lyophilized product of the present invention was allowed to stand for 60 days under three conditions of room temperature, room temperature + light blocking and 4 ° C. + light blocking. After 60 days, they were suspended in 600 µl of sterile reconstituted solution (1), respectively. After suspension, the mixture was left at room temperature for 90 days and each size marker was electrophoresed on agarose gel at a concentration of 1 µg / 12 µl per well. (See Figure 2)

As a result, BPB discolored from blue to pale yellow in the experimental group that did not block light about one week before and after storage, but the original color of BPB was maintained at both room temperature and 4 ° C under light blocking conditions.

After suspending the lyophilized DNA size marker with the reconstituting solution (1), there was no difference in quantity and quality from that before the lyophilization, and furthermore, even after 90 days after the suspending with the reconstituting solution (1) There was no change in the amount and quality of the size markers.

As a result, it is desirable to store the lyophilized size marker in the state of blocking light to maintain the properties of the pigment.

As described above, the size marker lyophilized product of the present invention obtained by lyophilization in a state of containing the size marker DNA, a buffer solution, and a dyeing agent, can be stored and transported at room temperature, and includes a stabilizer and a specific gravity increaser. It can be used immediately by simply suspending the prepared reconstituting solution, and the reconstituted DNA size marker composition can be stored at room temperature. Therefore, the use of the size marker lyophilized body and the reconstituted DNA size marker composition of the present invention can prevent the degradation of the DNA size marker due to inadvertent storage and transportation, and reduce the cost for low temperature storage and storage. As a result, DNA size markers can achieve user convenience and cost reduction.

Claims (9)

DNA size marker composition that can be stored at room temperature, characterized in that consisting of DNA, buffers, stains, stabilizers and specific gravity increase agents. The DNA size marker composition according to claim 1, comprising 1) a lyophilized product obtained by lyophilizing DNA together with a buffer solution and a staining agent, and 2) a solution for recomposition comprising a stabilizer and a specific gravity enhancer. The DNA size marker composition according to claim 1, further comprising a DNA degradation inhibitor or an antioxidant in addition to DNA, a buffer, a dye, a stabilizer, and a specific gravity enhancer. The DNA size marker composition according to claim 1, wherein the buffer is TEN buffer (10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA) or TE buffer (10 mM Tris-HCl, 1 mM EDTA). The DNA size marker composition according to claim 1, wherein the dyeing agent is 0.01 to 0.1% bromophenol blue or xylene cyanol. The DNA size marker composition according to claim 1, wherein the stabilizer is 1 to 50 mM EDTA. The DNA size marker composition according to claim 1, wherein the specific gravity increasing agent is 1-5% ficoll or 2.5-10% glycerol. The DNA size marker composition according to claim 1, wherein the buffer solution is TE buffer, the stabilizer is 5-20 mM EDTA, and the specific gravity increasing agent is 1-5% ficoll. A component other than a stabilizer and a specific gravity enhancer is separately lyophilized, stored and transported, and a method for producing a DNA size marker, wherein the freeze-dried body is dissolved and used as a reconstituting solution comprising a stabilizer and a specific gravity enhancer.