KR100390872B1 - Compositions for making the Polyacrylamide gel for electrophoresis - Google Patents

Abstract

The present invention relates to a composition for producing an electrophoretic polyacrylamide gel containing an acrylamide monomer, a crosslinking agent and a photoinitiator. To the composition. The compositions of the present invention can be used to prepare homogeneous electrophoretic gels that minimize scattering of the laser beam, making them suitable for use in electrophoretic kits used in automated nucleic acid sequencing devices.

Description

Compositions for the production of electrophoretic polyacrylamide gels

The present invention relates to a composition for producing an electrophoretic polyacrylamide gel containing an acrylamide monomer, a crosslinking agent and a photopolymerization radical initiator, and more particularly, an electrophoretic polyacrylamide containing an acrylamide monomer, a crosslinking agent and a benzoin-based photopolymerization radical initiator. It relates to a composition for gel preparation.

Electrophoresis is widely used to separate protein, biomolecule mixtures such as molecular weight in order. In nucleic acid sequencing, electrophoresis using crosslinked polymer gels as a separation medium is used to separate nucleic acid fragments of various lengths in molecular order.

To date, commonly used as such separation media for electrophoresis is dextrane, agarose, crosslinked polyacrylamide gel and the like. Among them, polyacrylamide gel is separated from an electrophoresis apparatus for nucleic acid sequencing because of its ability to separate biopolymers of various lengths, chemically inert, and stable at a wide range of pH and temperature conditions. Most commonly used as a medium.

The polyacrylamide gel is a form in which a polyacrylamide linear polymer chain is crosslinked by a crosslinking agent, and is usually polymerized using a thermal polymerization radical initiator or a photopolymerization radical initiator.

When producing a polyacrylamide gel using a thermal polymerization radical initiator, the progress of a polymerization reaction is interrupted by radical scavenging of dissolved oxygen, and it is difficult to manufacture a high molecular weight polymer. As a radical initiator, when preparing a polyacrylamide gel using an initiator having a sulfate group, electroendosmosis may occur, and since the reaction composition starts to form a gel as soon as the initiator is added. There is a disadvantage in use that the reaction composition must be poured quickly into the mold.

When using ammonium persulfate (APS) as the thermal polymerization radical initiator, a long time is required for crosslinking of the monomers to form a gel, and as a result, when a laser beam is irradiated without forming a homogeneous gel as a whole. The disadvantage is that light is scattered.

Therefore, in order to solve this problem, a method of polymerizing acrylamide using a photopolymerization radical initiator has been developed. For example, riboflavin or riboflavin 5-phosphate is used as the photopolymerization radical initiator. However, when riboflavin or riboflavin 5-phosphate is used as the photopolymerization radical initiator, it takes a long time to start the polymerization reaction, and there is a problem that the conversion rate of the monomer to the polymer is lower than that of the polymerization method using the thermal polymerization radical initiator. .

In addition, cases of using a ketone compound as a photopolymerization radical initiator have been reported. When using a conventional thermal polymerization radical initiator, it takes about 2 hours to form a gel, but when using a ketone photopolymerization radical initiator, a homogeneously crosslinked gel can be obtained in a relatively short time of about 10 minutes. There is an advantage.

In the currently used automatic nucleic acid sequencing apparatus, nucleotides labeled with fluorescent material are used, and the terminal base of the separated nucleotide sequence is analyzed by irradiating a laser beam to a gel plate in which nucleic acid fragments are separated in the order of molecular weight. In this case, turbidity of the gel affects the dispersion of light, and the higher the turbidity, the lower the intensity of light that can be detected. Thus, the optical homogeneity and separation quality of the gels used in the electrophoretic kits have a significant impact on the resolution of sequencing of nucleic acid fragments.

For electrophoretic gels used in automated nucleic acid sequencing devices than for conventional electrophoretic gels, generally better separation capabilities are required. Because in general electrophoretic gels, nucleic acid sequence fragments can be analyzed only by the length of a fixed gel plate. However, in the case of automated nucleic acid sequencing devices, end nucleotide analysis by laser beam can be continued for a long time. This is because the sections can be separated and analyzed in succession.

However, such an automatic nucleic acid sequencing device also has a disadvantage in that it can not be analyzed using a laser when the nucleotide sequence appears discontinuously moved. Therefore, the automated nucleic acid sequencing apparatus is based on the premise that the nucleotide sequence ladders appear at regular intervals. This means that the current / voltage ratio applied to the electrophoretic kit must be constant. Therefore, if the gel for the automated nucleic acid sequencing device satisfies the above conditions, it is much more efficient to analyze the nucleotide sequence with the automated nucleic acid sequencing device than to analyze the nucleotide sequence by general electrophoresis.

Ketone photopolymerization radical initiators currently used include 1-hydroxycyclohexyl-phenyl-ketone, 1- [4- (hydroxyethoxy) -phenyl] -2-hydroxy-2-methyl-1-propane- 1-one (manufactured by Pharmacia).

However, the ketone-based radical initiators are very difficult to synthesize and expensive, and thus are limited to use in electrophoretic gels used in an automated nucleic acid sequencing apparatus for continuously analyzing a large amount of nucleotide sequences.

Therefore, in order to be used in an electrophoretic kit for an automated nucleic acid sequencing device, a gel is formed faster than a conventional ketone-based photopolymerization radical initiator, and it is required to develop a photopolymerization radical initiator that is easy to use and inexpensive. When used in a nucleic acid sequencing apparatus, there is a need for the development of a composition for producing a polyacrylamide gel containing a new photopolymerizable radical initiator that enables more accurate sequencing.

Accordingly, it is an object of the present invention to provide an electrophoretic polyacrylamide containing a novel photopolymerizable radical initiator, an acrylamide monomer and a crosslinking agent, which can produce a more homogeneous gel, which takes less time for gel formation than conventional photopolymerizable radical initiators. It is to provide a composition for producing a gel.

It is still another object of the present invention to provide a method for producing an electrophoretic polyacrylamide gel prepared by irradiating ultraviolet rays to a composition containing a benzoin-based photopolymerization radical initiator in a mixture of an acrylamide monomer and a crosslinking agent.

It is another object of the present invention to provide a composition kit for producing an electrophoretic polyacrylamide gel comprising a composition containing an acrylamide monomer, a crosslinking agent, a benzoin-based photopolymerization radical initiator and a buffer solution.

1 is a result of analyzing the base sequence by electrophoresis using a conventional gel.

Figure 2 shows the results of the base sequence analysis by electrophoresis using a 6% polyacrylamide gel prepared using the benzoin ethyl ether photopolymerization radical initiator of the present invention.

Figure 3 shows the results of the base sequence analysis by electrophoresis using a 6% polyacrylamide gel prepared using the benzoin photopolymerization radical initiator of the present invention.

Figure 4 shows the results of the base sequence analysis by electrophoresis using a 7% polyacrylamide gel prepared using the benzoin photopolymerization radical initiator of the present invention.

FIG. 5 shows the results of base sequence analysis using electrophoresis using an 8% polyacrylamide gel prepared using the benzoin photopolymerization radical initiator of the present invention.

The above object of the present invention is achieved by a composition for producing an electrophoretic polyacrylamide gel containing an acrylamide monomer, a crosslinking agent and a benzoin-based photopolymerization radical initiator. The photopolymerization radical initiator of the present invention is a compound having the formula

In the above formula, R is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , CH ₂ CH (OH) CH ₂ OH, (CH ₂ ) n-CONH ₂ , or n is an integer of 1 to 5 An integer of 1 to 3, and R 'is H or an alkyl group (CH ₂ CH ₂ O) n-R'.

The composition for producing an electrophoretic polyacrylamide gel of the present invention contains the benzoin-based photopolymerization radical initiator in a mixture of an acrylamide monomer and a crosslinking agent.

As said acrylamide type monomer, Preferably acrylamide, methacrylamide, or these alkyl, hydroxyalkyl derivatives, for example, N, N- dimethyl acrylamide, N-hydroxypropyl acrylamide, N- Derivatives such as hydroxymethylacrylamide can be used.

As the crosslinking agent, methylenebisacrylamide, N, N'-diallyl tardiamide, N, N'-1,2-dihydroxyethylene-bisacrylamide, N, N'-bisacrylylcitamine, trisacryl Royl-hexahydrotriazine, N, N'-diacryloylpiperazine and the like can be used.

The composition for producing a gel of the present invention is preferably prepared in an aqueous solution of 50 to 60% (w / v), more preferably 53 to 56% (w / v). In this aqueous solution, preferably 3 to 15% (w / v), more preferably 4 to 8% (w / v) is an acrylamide monomer, and the crosslinking agent is preferably 0.1 to 1% (w / v). , More preferably 0.2 to 0.5% (w / v), and the amount of such crosslinker is preferably 2 to 15% (w / v), more preferably 3 to 8% (w / v) of the total composition solution. Corresponds to

As the ratio of the crosslinking agent to the acrylamide-based monomer increases, the degree of crosslinking of the gel increases. The higher the degree of crosslinking of the gel increases the time required for separation of nucleic acid fragments by electrophoresis, while the separation resolution is higher. Therefore, the ratio of the acrylamide monomer and the crosslinking agent is usually appropriately adjusted in consideration of the separation efficiency.

The photopolymerization radical initiator contained in the composition of the present invention is added to the gel preparation composition in an amount of 0.05 to 5 mM, preferably 0.1 to 1 mM, more preferably 0.1 to 0.2 mM. Some of the photopolymerizable radical initiators of the present invention are water-soluble and can be added directly to an aqueous solution composition, but may be dissolved in an organic solvent and used. In this case, dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) may be used as the organic solvent, and a minimum amount necessary for dissolving the photopolymerization radical initiator is preferably used.

After the organic solution in which the photopolymerization radical initiator of the present invention is dissolved is mixed with the acrylamide monomer and the crosslinking agent aqueous solution composition, the mixture is poured into a gel generating plate and irradiated with ultraviolet rays to prepare a polyacrylamide gel. In this case, the ultraviolet rays are irradiated for 10 to 20 minutes at a distance of 10 to 20 cm from the gel generating plate, and ultraviolet rays of 300 to 400 nm are used.

Preferred photopolymerization radical initiators of the present invention include 2-hydroxy-2-phenylacetophenone (benzoin) having the formula:

2- (2,3-dihydroxy) propoxy-2-phenylacetophenone having the formula:

Ethoxy-phenylacetophenone (benzoin ethyl ether) which has a following formula is mentioned.

The benzoin-based photopolymerization radical initiator used in the preparation of the electrophoretic polyacrylamide gel of the present invention forms a more homogeneous gel than when the gel is prepared using other conventional photopolymerization radical initiators. Therefore, in the nucleic acid sequencing as shown in the examples and drawings of the present invention, the nucleotide sequence of the longer-chain nucleic acid fragment can be analyzed at a higher resolution, so that the nucleic acid nucleotide sequence of the long-chain such as human cDNA can be analyzed. The analysis can save significant time and money.

In addition, the benzoin-based photopolymerization radical initiator used in the composition of the present invention is cheaper than conventional photopolymerization radical initiators, and can be easily used because it is easy to purchase.

Hereinafter, the structure of the present invention will be described in more detail with reference to Examples, but the general scope of the present invention is not limited to the following Examples.

Example 1

Preparation of Gel Solution and Initiator Solution

Acrylamide was used as an acrylamide monomer in this embodiment, and methylenebisacrylamide or N, N'-diacryloylpiperazine was used as a crosslinking agent. Both crosslinkers were used in a weight ratio of 19 to 1 with respect to acrylamide, distilled water in 108 g of Trisma base, 55 g of borate, and 40 ml of 0.5 mol of ethylenediaminetetraacetic acid (EDTA) solution. Tris borate ethylenediaminetetraacetic acid (TBE) buffer diluted to a volume of 1 L was used.

The benzoin-based photopolymerization radical initiator used in this example is a benzoin (Benzoin), benzoin ethyl ether (Benzoin ethyl ether) compounds as shown in Table 1, and the conventional benzil dimethyl ketal and the like as a control Used. The initiators were dissolved in an organic solvent of dimethyl formamide (DMF: dimethyl formamide) and dimethyl sulfoxide (DMSO: dimethyl sulfoxide) in a concentration of 10 to 20% (w / v).

Types of Photopolymerization Radical Initiators Used in Examples of the Present Invention name synonym Molecular Weight constitutional formula Properties Benzoin 2-hydroxy-2-phenylacetophenone 212.2476 Melting Point: 137 ℃ Boiling Point: 344 ℃ Gray White Crystal Benzoin ethyl ether Ethoxyphenylacetophenone 240.3012 Melting Point: 56-59 ℃ Benzyldimethyl ketal 2,2-dimethoxy-2-phenylacetophenone 256.3006 Melting Point: 67-70 ℃ Light sensitive Conventional Initiator (Pharmacia) R ₁ , R ₂ = CH ₃ , C ₂ H ₅ , C ₃ H ₇ , or cycloaliphatic with 4 to 8 C R ₃ = H, CH ₃ , C ₂ H ₅ , or n is 1 -O (CH ₂ CH ₂ ) n-OH receptivity

480 g of urea and 100 ml of 10X triethylene ethylenediaminetetraacetic acid (TBE) buffer solution were added to an aqueous solution of an acrylamide monomer and a crosslinking agent in a weight ratio of 19 to 1% (w / v), and dissolved in water. 6 to 8% (w / v) of an aqueous solution composition for preparing a gel was prepared by adding primary distilled water to a total volume of 1 L. The aqueous solution composition was filtered with a filter of 0.45 mu m. The composition of the present invention was prepared by adding 8 to 24 µl of benzoin-based photopolymerization radical initiator solution per 10 ml of filtered aqueous solution composition for gel preparation.

Example 2

Gel plate production

The composition of the present invention prepared in Example 1 was poured between glass plates for gel formation. Thereafter, ultraviolet rays having a wavelength of 300 to 400 nm were irradiated for 10 to 20 minutes at a distance of 10 cm from the glass plate. At this time, the thickness of the gel was 0.3 mm.

Table 2. Difference in Crosslinking Time with Initiator Amount (concentration of initiator used 10% (w / v))

Amount of initiator (μl / 10ml gel solution) Crosslinking time (min.) 8 20 16 10 to 15 24

Example 3

Sequencing

An electrophoretic device consisting of the gel prepared in Example 2 was mounted on an automated nucleic acid sequencing device. NucleoScan 2000 as an automated nucleic acid sequencing device^TM(NucleoTech Co., Ltd.) was used. For nucleic acid sequencing, 1.5pm of plasmid DNA (pUC19) and 7pmoles of primer (24merForward universal primer for sequencing) with fluorescent material attached to the 5 'end were used.AccuPower ^TM Polymerase chain reaction (PCR) instrument (MyGenie) using DNA Sequencing Kit (Bionia Co., Ltd.)^TM, Bionic Co., Ltd.) was heat-modified at 94 ° C. for 5 minutes, and then reacted 40 times at 94 ° C. for 30 seconds, at 55 ° C. for 30 seconds, and at 72 ° C. for 60 seconds and finally at 72 ° C. for 5 minutes. Analyzed. Gel plates prepared under different conditions were subjected to NucleoScan 2000.^TMThe sample reacted after being mounted on the was electrophoresed for 90 minutes under the same electrophoresis conditions (1100V, 40 ° C). As a control used in the present invention, a conventional electrophoretic gel (ReproGel High Resolution, manufactured by Pharmacia Biotech) was used. The results are shown in FIGS. 1 to 5.

1 is a result of analyzing the base sequence by electrophoresis using a conventional gel (Pharmacia Biotech).

Figure 2 shows the results of the base sequence analysis by electrophoresis using a 6% polyacrylamide gel prepared using the benzoin ethyl ether photopolymerization radical initiator of the present invention.

Figure 3 shows the results of the base sequence analysis by electrophoresis using a 6% polyacrylamide gel prepared using the benzoin photopolymerization radical initiator of the present invention.

Figure 4 shows the results of the base sequence analysis by electrophoresis using a 7% polyacrylamide gel prepared using the benzoin photopolymerization radical initiator of the present invention.

FIG. 5 shows the results of base sequence analysis using electrophoresis using an 8% polyacrylamide gel prepared using the benzoin photopolymerization radical initiator of the present invention.

Comparing the nucleic acid sequencing results of these examples, it can be seen that the nucleic acid band is most clearly observed when using a polyacrylamide gel prepared from the composition of the present invention containing a benzoin-based photopolymerization radical initiator (FIG. 3). To 5).

The resolution was higher when electrophoresed using a gel made from the composition of the present invention than when a conventional gel (product of Pharmacia) using a conventional Benzil dimethyl ketal initiator was used (see FIG. 1). ). In particular, when using a polyacrylamide gel prepared using a benzoin photopolymerization radical initiator, it is equivalent to about 100 base pairs in the second half of the base sequence due to the improvement in resolution under the same conditions as compared with the case of using a conventional photopolymerization radical initiator. It was possible to further analyze the base. Therefore, the gel prepared from the composition of the present invention is more homogeneous than the gel prepared using the conventional photopolymerization radical initiator, and it is confirmed that the gel is more suitable for use in an automated nucleic acid sequencing apparatus because it reduces scattering of the laser beam. .

In addition, as an organic solvent for dissolving the photopolymerization radical initiator, dimethylformamide (DMF) was better than dimethyl sulfoxide (DMSO), and gel using methylenebisacrylamide as a crosslinking agent showed better resolution. . In addition, the spacing between nucleic acid base fragments was changed depending on the concentration of the aqueous solution of the composition for preparing a polyacrylamide gel. When the concentration of the aqueous solution of the composition is more than 8% (w / v), the interval between the nucleic acid base fragments is wide, it was impossible to observe a large number of bases (see Fig. 5), the interval between the nucleic acid base fragments when the concentration of the composition is less than 6% This narrowed, making it difficult to observe the band (see FIG. 3). Therefore, it was confirmed that the concentration of 7% of the aqueous solution of the composition forms the most suitable gel for observing nucleic acid base fragments (see FIG. 4).

As described above, the production of electrophoretic polyacrylamide gel using the composition of the present invention can minimize the scattering of the laser beam, shorten the gel formation time, to obtain more effective results during electrophoresis and experiment The time can also be shortened. In addition, the electrophoretic polyacrylamide gel made of the composition of the present invention is more homogeneous than conventional gels, and thus has high resolution and resolution during electrophoresis, so that it is possible to analyze long-chain sequences and to use them in an automated nucleic acid sequencing apparatus. More effective. Electrophoretic polyacrylamide gels composed of the compositions of the present invention are suitable for use in various sequencing methods using conventional isotopes, silver staining and fluorescence as well as automated nucleic acid sequencing devices.

Claims (11)

A composition for producing an electrophoretic polyacrylamide gel, comprising an acrylamide monomer, a crosslinking agent, and a photopolymerization radical initiator of the formula: In the above formula, R is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , CH ₂ CH (OH) CH ₂ OH, n is 1 to 5 (CH ₂ ) n-CONH ₂ , which is an integer, or (CH ₂ CH ₂ O) n-R ', wherein n is an integer of 1 to 3 and R' is H or an alkyl group. The composition for producing an electrophoretic polyacrylamide gel according to claim 1, wherein the photopolymerization radical initiator is a compound having the following formula. The composition for producing an electrophoretic polyacrylamide gel according to claim 1, wherein the photopolymerization radical initiator is a compound having the following formula. Aqueous solution of urea and 10X triethylene ethylenediaminetetraacetic acid (TBE) was added to an aqueous solution in which the acrylamide monomer and the crosslinking agent were mixed in a weight ratio of 19 to 1, and distilled water was added so that the total volume was 1 L. Preparing a composition; Preparing a composition for preparing a polyacrylamide gel by adding a benzoin-based photopolymerization radical initiator solution of the following formula to the aqueous solution composition; In the above formula, R is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , CH ₂ CH (OH) CH ₂ OH, n is 1 to 5 (CH ₂ ) n-CONH ₂ , which is an integer, or (CH ₂ CH ₂ O) n-R ', wherein n is an integer of 1 to 3 and R' is H or an alkyl group. Electrophoretic polyacrylamide gel production method comprising the step of irradiating the ultraviolet ray of 300 to 400nm to the composition for producing a polyacrylamide gel. The method for producing an electrophoretic polyacrylamide gel according to claim 4, wherein the photopolymerization radical initiator is a compound having the following formula. The method of claim 4, wherein the photopolymerization radical initiator is a compound having the formula: An electrophoretic gel production kit, wherein a composition for preparing a polyacrylamide gel containing an acrylamide monomer, a crosslinking agent, and a benzoin-based photopolymerization radical initiator of the following formula is filled in a sealed container. In the above formula, R is H, CH ₃ , C ₂ H ₅ , C ₃ H ₇ , CH ₂ CH (OH) CH ₂ OH, n is 1 to 5 (CH ₂ ) n-CONH ₂ , which is an integer, or (CH ₂ CH ₂ O) n-R ', wherein n is an integer of 1 to 3 and R' is H or an alkyl group. 8. The kit according to claim 7, wherein the photopolymerization radical initiator is a compound having the following formula. 8. The kit according to claim 7, wherein the photopolymerization radical initiator is a compound having the following formula. 8. The kit according to claim 7, wherein the polyacrylamide gel preparation composition further contains a buffer solution. 11. The electrophoretic gel preparation kit according to claim 10, wherein the buffer solution is ethylenediaminetetraacetic acid (TBE) buffer solution.