JPH1194796A - Gel for electrophoresis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gel capable of compressing the band width on the side of a low molecular size not receiving the effect of the high-order structure of DNA molecules, on the side of a high molecular size characteristically observed in DNA sequencing in an electrophoretic method separating in a molecular size and a compsn. thereof. SOLUTION: An electrophoretic gel consists of a crosslinking agent for preparing an electrophoretic gel composed of a compd. obtained by introducing an acryloyl group or a methacryloyl group into at least two hydroxyl groups possessed by cyclodextrin or a cyclodextrin derivative, a compsn. for preparing the electrophoretic gel containing the crosslinking agent and acrylamide, methacrylamide or derivatives of them and a polymer obtained by copolymerizing the compsn. By this constitution, an electrophoretic gel for DNA sequencing drastically increasing data quantity read by one migration can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel electrophoresis gel, and more particularly to an electrophoresis gel useful for DNA sequencing.

[0002]

2. Description of the Related Art Electrophoresis has been used for a long time as one of the analysis methods for biopolymers such as proteins and DNA because of its high resolution, various separation modes, simplicity and economy. , Greatly contribute to the development of the biotechnology field. Conventionally, polysaccharides represented by agarose and hydrophilic polymers have been widely used as gels for electrophoresis.

As a hydrophilic polymer, polyacrylamide gel obtained by using acrylamide as a monomer and cross-linking with methylenebisacrylamide is mainly used. Polyacrylamide gels, despite the fact that the monomers are toxic,
Widely used due to high separation performance. These preparation methods include adding an oxidation catalyst or a photocatalyst to a solution containing acrylamide and methylenebisacrylamide,
It is polymerized and molded in a support composed of a glass plate or the like. Depending on the analysis mode, various additives are added to these polyacrylamide gels. For example, when separating DNA by molecular size, a denaturant such as a buffer and urea is added to the solution before polyacrylamide polymerization.

[0004] DNA sequencing is a technique for determining the nucleotide sequence of DNA by separating DNA fragments having various molecular sizes on an electrophoresis gel.
In the analysis mode in which separation is performed by molecular size, the mobility of the band on the gel is proportional to the logarithm of the molecular size of the band. This means that D has the same base number difference.
Even in the NA fragment, the interval between the bands is widened on the low molecular size side, and the interval between the bands is narrow on the high molecular size side, making it difficult to read. In addition, high molecular size DNA is DN
Since the A molecule has a higher-order structure, a phenomenon in which the apparent molecular size is reduced is observed, and the band on the high molecular size side becomes narrower, which makes reading difficult. This is a serious problem due to the limited gel length.

[0005]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for electrophoresis in which separation is performed by molecular size, whereby the bandwidth on the low molecular size side can be compressed, and the high molecular size characteristic of DNA sequencing. An object of the present invention is to provide a gel free from the influence of the higher-order structure of the DNA molecule on the side and a composition thereof, and in particular, to provide an electrophoresis gel for DNA sequencing in which the amount of information that can be read by one electrophoresis is dramatically increased.

[0006]

According to the present invention, there is provided an electrophoretic gel preparation comprising a compound in which an acryloyl group or a methacryloyl group is introduced into at least two hydroxyl groups of a cyclodextrin or a cyclodextrin derivative. Is a cross-linking agent.

Further, the present invention is a composition for preparing an electrophoresis gel, comprising acrylamide, methacrylamide or a derivative thereof and the above-mentioned crosslinking agent.

Further, the present invention is an electrophoresis gel comprising a polymer obtained by copolymerizing the above composition with an oxidation catalyst or a photocatalyst.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The crosslinking agent of the present invention is a compound in which an acryloyl group or methacryloyl is introduced into at least two hydroxyl groups of a cyclodextrin or a cyclodextrin derivative. The cyclodextrin is α-cyclodextrin having 6 to 8 glucose units, β-cyclodextrin, γ-cyclodextrin, or the like, and is a compound having usually 18 to 24 hydroxyl groups. Further, the cyclodextrin derivative,
Part of the hydroxyl group of the cyclodextrin is an alkyl group,
Compounds substituted with an acyl group or an acetonide (isopropylidene) group, and other saccharides, such as maltosylcyclodextrin substituted with a maltosyl group.

More specifically, diacryloyl-α-cyclodextrin, triacryloyl-α-cyclodextrin, dimethacryloyl-α-cyclodextrin,
Trimethacryloyl-α-cyclodextrin, diacryloyl-β-cyclodextrin, triacryloyl-β-cyclodextrin, dimethacryloyl-β
-Cyclodextrin, trimethacryloyl-β-cyclodextrin, diacryloyl-γ-cyclodextrin, triacryloyl-γ-cyclodextrin,
Examples include dimethacryloyl-γ-cyclodextrin, and trimethacryloyl-γ-cyclodextrin.
Also, diacryloylmaltosyl-α-cyclodextrin, triacryloylmaltosyl-α-cyclodextrin, dimethacryloylmaltosyl-α-cyclodextrin, trimethacryloylmaltosyl-α-cyclodextrin, diacryloylmaltosyl-β -Cyclodextrin, triacryloyl maltosyl-β-
Cyclodextrin, dimethacryloyl maltosyl-
β-cyclodextrin, trimethacryloylmaltosyl-β-cyclodextrin, diacryloylmaltosyl-γ-cyclodextrin, triacryloylmaltosyl-γ-cyclodextrin, dimethacryloylmaltosyl-γ-cyclodextrin, trimetaacryloyl Maltosyl-γ-cyclodextrin and the like.

The crosslinking agent of the present invention has physical properties such as solubility.
Alternatively, in order to adjust the degree of hydrophobicity of the polymer chain after polymerization by the crosslinking agent, a functional group is introduced into the cyclodextrin. Examples of the functional group include ketals and acetals such as an alkyl group, an acyl group, an isopropylidene group and a benzylidene group.

Examples of the method for synthesizing the crosslinking agent include general chemical methods, for example, a method in which a compound having an acryloyl group or a methacryloyl group is allowed to act on cyclodextrin or a cyclodextrin derivative in the presence of a base catalyst, an active esterification method. Etc. can be used. Examples of the compound having an acryloyl group include acryloyl chloride and acryloyl bromide. Examples of the compound having a methacryloyl group include methacryloyl chloride and methacryloyl bromide.

The reaction conditions include a cyclodextrin or a compound having an acryloyl group or a methacryloyl group in a cyclodextrin derivative in a molar ratio of 1: 2 to 1: 2.
React at 1:10. The reaction molar ratio varies depending on reaction conditions such as temperature and solvent, and the acryloyl group or methacryloyl group to be introduced into one molecule of the cyclodextrin skeleton. The acryloyl group or methacryloyl group needs to be introduced into at least two, preferably two to three hydroxyl groups of the cyclodextrin or the cyclodextrin derivative. As the base catalyst, an organic base such as pyridine or triethylamine, or a hydroxide of an alkali metal such as sodium hydroxide, sodium carbonate, lithium hydroxide or lithium carbonate, a hydroxide of an alkaline earth metal or salts thereof are used. be able to. Depending on the combination of the base catalyst and the reaction solvent, a suitable solubilizing agent such as a crown ether can be added.

The reaction system can be a homogeneous system or a heterogeneous system.
As the reaction solvent, an aqueous or organic solvent such as water, methanol, ethanol, acetone, DMF, THF, chloroform, and ethyl acetate can be used. The reaction temperature is-
The temperature is 40 to 100 ° C, and specifically, -10 to 10 ° C is preferable. The reaction time is preferably from 10 minutes to 8 hours.

In the activated ester method and the activated amide method, acrylic acid is previously converted into an ester or amide with an aromatic phenol or aromatic amine such as p-nitrophenol or imidazole, and acetone, THF,
Reaction with cyclodextrin or a cyclodextrin derivative in an organic solvent such as DMF, chloroform or ethyl acetate. The reaction can be carried out at a temperature of -40 to 100 ° C, and specifically, preferably at -20 to 30 ° C. The reaction time is preferably 1 hour to 48 hours.

Alternatively, the enzyme can be synthesized by an enzymatic method using an immobilized lipase as a catalyst in a non-aqueous solvent. As the non-aqueous solvent, methanol, ethanol, acetone, THF, DMF, chloroform, ethyl acetate and the like can be used. As the immobilized lipase, the origin of the enzyme and the type of the immobilized carrier are not limited. For example, a commercially available one such as Toyozyme Lip (manufactured by Toyobo) may be used. The reaction system is a heterogeneous system because the immobilized lipase is insoluble in the reaction solvent. In addition, since the lipase activity is affected by a very small amount of water, it is preferable that a desiccant such as molecular sieve coexist. The reaction temperature is preferably around the optimum temperature of the enzyme, and varies depending on the origin of the enzyme.
It is preferred that The reaction time is preferably from 1 hour to 48 hours.

As a cross-linking agent for electrophoresis, a crude product can be used as it is, but when purification is required, it can be purified by silica gel column chromatography.

In the present invention, in order to use it as a crosslinking agent, it is necessary to introduce at least two molecules of an acryloyl group or a methacryloyl group into the cyclodextrin skeleton. The amount introduced can be adjusted by reaction conditions such as the ratio of raw materials to be reacted and the temperature, and can be confirmed by a physicochemical measurement method such as NMR. In the case of using the crude product of the above reaction as it is, the average number of acryloyl groups or methacryloyl groups introduced is 2 mol or more per mol of cyclodextrin or cyclodextrin derivative molecule because of the mixture.

Introducing an alkyl group, an acyl group or an isopropylidene group into the cyclodextrin skeleton is
It is possible before or after introducing the acryloyl group or methacryloyl group. Usually, the cross-linking agent obtained by these reactions is a mixture in which the number and positions of acryloyl groups or methacryloyl groups introduced into the cyclodextrin skeleton are different. Although purification can be performed using a purification means such as column chromatography, it is often possible to withstand the use of the mixture as an electrophoresis gel composition. When the mixture is used as it is, it is advantageous in terms of cost.

When used as an electrophoresis gel, acrylamide or methacrylamide is used as a monomer,
The above crosslinking agent is added, and polymerization is carried out using an oxidation catalyst or a photocatalyst. A part of acrylamide or methacrylamide is converted to another acrylamide derivative or methacrylamide derivative, for example, N-substituted derivative, N-hydroxymethylacrylamide, N-hydroxymethylmethacrylamide, N, N-dimethylacrylamide, N, N-dimethylmetha It may be substituted with acrylamide or the like. The solution of acrylamide or methacrylamide and the crosslinking agent before polymerization can be stored as a composition.

The gel composition for electrophoresis of the present invention contains 0.01 to 50 mol of a crosslinking agent per 1 mol of acrylamide or methacrylamide or a derivative thereof. When the composition of the present invention is used for, for example, DNA sequencing, the composition can be dissolved in a buffer solution, and formaldehyde, urea, and the like can be further added as a DNA denaturant.

The composition of the present invention can be further polymerized by adding an oxidation catalyst or a photocatalyst to obtain a gel composition. As the oxidation catalyst, ammonium persulfate and N,
N, N ', N'-tetramethylethylenediamine and the like. Examples of the photocatalyst include riboflavin. DN
For A sequencing, it is convenient to use an oxidation catalyst.

As in the case of ordinary polymerization of acrylamide gel, the composition of the present invention is preferably degassed under reduced pressure before polymerization because the presence of oxygen is not preferable for the polymerization. Although it depends on the mixing ratio of the composition, it gels within a few minutes from the start of the polymerization. Therefore, before the gelation, the solution is poured into a support such as a glass plate and allowed to stand until gelation.

When the gel composition for electrophoresis of the present invention is used for DNA sequencing, the bandwidth of the DNA fragment on the low molecular size side is smaller than that of a conventionally used acrylamide-methylenebisacrylamide gel. On the high molecular size side, there is no compression of the bandwidth due to the influence of the higher-order structure of DNA, which is characteristically observed in DNA electrophoresis, so that the information obtained by one electrophoresis increases dramatically. Since the physical properties of the gel are not much different from other conventionally used gels for electrophoresis, a conventional device can be used as it is and can be substituted.

[0025]

Example 1 (Crosslinking agent) Synthesis of acryloylated α-cyclodextrin 10 g of α-cyclodextrin (manufactured by Nacalai Tesque) was dissolved in 50 ml of DMF, and triethylamine was added at 0 ° C.
ml was added. Further, acryloyl chloride 4.5
ml was added dropwise at 0 ° C., and the temperature was returned to room temperature with stirring. After stirring for 4 hours, the reaction solution was suction-filtered with filter paper. Acetone 300m
The precipitate was collected by suction filtration and thoroughly dried under reduced pressure to obtain 8 g of a crude product.

Purification of acryloylated α-cyclodextrin The elution solvent was ethyl acetate / methanol / water (volume ratio: 7:
2: 1), the desired fractions were collected, the organic solvent was concentrated under reduced pressure, and the residual aqueous solution was lyophilized to obtain 5 g of a white powder.

Analysis of Acryloylated α-Cyclodextrin The number of acryloyl groups introduced into the α-cyclodextrin skeleton was confirmed by NMR. Measurement solvent is deuterated DM
F or heavy water can be used. NMR is 200 MHz
Was used. δ value is 5.9-6.6p
Integral value of proton derived from acryloyl group of pm and δ
The value was calculated from the ratio of integral values of protons at the anomeric position of the glucose unit skeleton having a value of 4.7 to 5.5 ppm. As a result, two hydroxyl groups of α-cyclodextrin have
A compound into which an acryloyl group was introduced was obtained.

Example 2 (Crosslinking agent) Synthesis of acryloylated α-cyclodextrin using immobilized lipase 10 g of α-cyclodextrin (manufactured by Nacalai Tesque) was dissolved in 50 ml of DMF. 1 g of immobilized lipase (trade name: Toyoteam LIP Toyobo) and 8 g of molecular sieve 3A were added, followed by stirring for 2 hours. 2 ml of vinyl acrylate was added, and the mixture was stirred at room temperature for 24 hours. further,
1.5 ml of vinyl acrylate was added and stirred for 24 hours.
After completion of the reaction, the reaction mixture was filtered through a high flow super cell (manufactured by Nacalai Tesque), and then filtered through a cartridge filter (MINISA
RT SRP15, manufactured by Sartorius). Ethyl acetate (300 ml) was added for precipitation, and the precipitate was collected by suction filtration and dried under reduced pressure to obtain 8 g of a white powder. A compound in which an acryloyl group was introduced into two hydroxyl groups of α-cyclodextrin was obtained.

Example 3 (Cross-linking agent) Synthesis of acryloylated maltosylcyclodextrin Maltosylcyclodextrin (manufactured by Salt Water Port Sugar Co., Ltd.) 10 g
Was dissolved in 50 ml of DMF, and triethylamine 1 was added at 0 ° C.
0 ml was added. 4.5 ml of acryloyl chloride was added dropwise at 0 ° C., and the temperature was returned to room temperature with stirring. After stirring for 4 hours, the reaction solution was suction-filtered with filter paper. A precipitate was added by adding 300 ml of acetone, and the precipitate was collected by suction filtration and thoroughly dried under reduced pressure to obtain 7.5 g of a crude product. A compound in which an acryloyl group was introduced into two hydroxyl groups of maltosylcyclodextrin was obtained.

Example 4 (Electrophoresis gel) Preparation of a composition containing acrylamide and acryloylated α-cyclodextrin 38 g of acrylamide and 8 g of the acryloylated α-cyclodextrin obtained in Example 1 were dissolved in distilled water. The total volume was 100 ml. Then, the pore size is 0.45μ
Was filtered through a membrane filter (manufactured by Sartorius) and stored in a brown bottle.

Preparation of 6% Electrophoresis Gel for DNA Sequencing 5.25 ml of the prepared composition containing acrylamide and acrylated α-cyclodextrin, 2.35 ml of 10 times TBE buffer and 16.8 g of urea were dissolved in distilled water. The total volume was adjusted to 35 ml, and the mixture was filtered through a membrane filter (manufactured by Sartorius) having a pore size of 0.45 μm.
After degassing under reduced pressure until no bubbles appeared, 210 μl of 10% ammonium persulfate and 9.45 μl of N, N, N ′, N′-tetramethylethylenediamine were added and mixed. Glass plate tilted at about 45 degrees (100 × 106
× 1 mm), a comb was inserted into the upper surface of the liquid, and the mixture was allowed to stand until gelling, to prepare a 6% electrophoresis gel for DNA sequencing.

Example 5 (Measurement Example) Analysis Example of DNA Molecular Weight Marker The 6% electrophoresis gel for DNA sequencing obtained in Example 4 was immobilized on an electrophoresis apparatus (manufactured by Nippon Eido), and TBE was used.
The buffer was poured into the buffer tank so that air did not enter the gel-exposed surface. Sample marker, φX174 / Ha
Apply 5 μl of eIII digest (manufactured by Toyobo), and
Electrophoresis was performed at a constant voltage of V for 150 minutes. After the electrophoresis, the gel was peeled off from the glass plate, the gel was stained with an ethidium bromide solution, and the FAS (Fluorescence Analyzer Syste
m) (Toyobo sales) confirmed the band. FIG. 1 shows the results of plotting the molecular size (bps) of each molecular weight marker and the mobility on the gel. FIG. 2 shows the results obtained by changing the molecular size to logarithmic (log bps).

Comparative Example 1 (Measurement Example) Preparation of 6% Acrylamide Gel 38 g of acrylamide and methylenebisacrylamide 2
g was dissolved in distilled water to make a total volume of 100 ml. The solution was filtered through a membrane filter (manufactured by Sartorius) having a pore size of 0.45 μm. 5.25 ml of prepared acrylamide and methylenebisacrylamide solution, 10 times T
2.35 ml of BE buffer and 16.8 g of urea were dissolved in distilled water to make a total volume of 35 ml. Pore size is 0.4
The solution was filtered through a 5 μm membrane filter (manufactured by Sartorius). After degassing until the bubbles disappear under reduced pressure, 10%
210 μl of ammonium persulfate and N, N, N ′,
9.45 μl of N′-tetramethylethylenediamine was added and mixed. Glass filter (1
(00 × 106 × 1 mm), a comb was inserted into the upper surface of the liquid, and the mixture was left until gelling.

Analysis of DNA Molecular Weight Marker The above 6% acrylamide gel was immobilized on an electrophoresis apparatus (manufactured by Nippon Eido), and a TBE buffer solution was poured into a buffer tank so that air did not enter the gel-exposed surface. DNA molecular weight marker as sample, φXZ174 / Ha
Apply 5 μl of eIII digest (manufactured by Toyobo), and
Electrophoresis was performed at a constant voltage of V for 160 minutes. After the electrophoresis, the gel was peeled off from the glass plate, the gel was stained with an ethidium bromide solution, and the FAS (Fluorescence Analyzer Syste
m) (Toyobo sales) confirmed the band. FIG. 1 shows the results of plotting the molecular size (bps) of each molecular weight marker and the mobility on the gel. FIG. 2 shows the results obtained by changing the molecular size to logarithmic (log bps).

As is clear from FIGS. 1 and 2, the acrylamide-acryloylated α-cyclodextrin of the present invention has a smaller DNA fragment bandwidth on the low molecular size side than the conventional acrylamide-methylenebisacrylamide. On the polymer side, there is no compression of the bandwidth due to the influence of the higher-order structure of DNA, which is characteristically observed in DNA electrophoresis.

Example 6 (measurement example) Influence of electrophoresis time on separation performance The 6% electrophoresis gel for DNA sequencing obtained in Example 4 was subjected to the same method as in Example 5 (measurement example) to obtain a DNA molecular weight marker. 5 μl of φX174 / HaeIII digest (manufactured by Toyobo) was applied and electrophoresed. However, the migration time is 110, 130, 150, 170, 190 minutes,
The results of plotting the logarithm (log bps) of each molecular weight marker and the mobility on the gel are shown in FIG.

In FIG. 3, the separation curve does not change and the gel performance is constant irrespective of the migration time, that is, the developing position on the gel.

Comparative Example 2 (Measurement Example) The 6% acrylamide gel obtained in Comparative Example 1 was subjected to the same method as in Example 6 (Measurement Example) using a DNA molecular weight marker, φX174.
5 μl of HaeIII digest (manufactured by Toyobo) was applied and electrophoresed. However, the migration time was 120, 14
FIG. 4 shows the results of plotting the logarithm (log bps) of each molecular weight marker and the mobility on the gel for 0, 160, 180, and 200 minutes.

In FIG. 4, the separation curve does not change irrespective of the electrophoresis time, that is, the developing position on the gel, the interval between the bands is wide on the low molecular size side, and the high-order structure of the DNA molecule is high on the high molecular size side. , The band spacing is narrow and reading is difficult.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between the molecular size of a molecular weight marker and the mobility on a gel when a DNA molecular weight marker is electrophoresed.

FIG. 2 is a diagram showing the relationship between the logarithm of the molecular size of a molecular weight marker and the mobility on a gel when a DNA molecular weight marker is electrophoresed.

FIG. 3 is a graph showing the relationship between the logarithm of the molecular size of a molecular weight marker and the mobility on a gel when a DNA molecular weight marker is electrophoresed.

FIG. 4 is a graph showing the relationship between the logarithm of the molecular size of a molecular weight marker and the mobility on a gel when a DNA molecular weight marker is electrophoresed.

Claims (10)

[Claims] 1. A cross-linking agent for preparing an electrophoresis gel, comprising a compound in which an acryloyl group or a methacryloyl group is introduced into at least two hydroxyl groups of cyclodextrin or a cyclodextrin derivative. 2. An acryloylated or methacryloylated cyclodextrin having at least two acryloyl or methacryloyl groups in one molecule or 1
A cross-linking agent for preparing an electrophoresis gel which is an acryloylated or methacryloylated maltosylcyclodextrin having at least two acryloyl groups or methacryloyl groups in a molecule. 3. The crosslinking agent according to claim 1, which is an acryloylated α-cyclodextrin having at least two acryloyl groups in one molecule. 4. The crosslinking agent according to claim 1, which is an acryloylated maltosylcyclodextrin having at least two acryloyl groups in one molecule. 5. A composition for preparing a gel for electrophoresis, comprising acrylamide, methacrylamide or a derivative thereof and the crosslinking agent according to claim 1 or 2. 6. The composition according to claim 5, comprising 0.01 to 50 mol of the crosslinking agent according to claim 1 or 2 based on 1 mol of acrylamide or methacrylamide or a derivative thereof. 7. The composition according to claim 5, wherein the acrylamide derivative or methacrylamide derivative is an N-substituted derivative. 8. An electrophoresis gel comprising a polymer obtained by copolymerizing the composition according to claim 5 with an oxidation catalyst or a photocatalyst. 9. The gel for electrophoresis according to claim 8, wherein the oxidation catalyst is ammonium persulfate and N, N, N ′, N′-tetramethylethylenediamine. 10. The photocatalyst is riboflavin.
The gel for electrophoresis according to the above.