JP3077313B2 - Capillary filler - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel capillary packing material used in capillary electrophoresis, and is useful for the separation, purification, qualification and quantification of various natural or unnatural compounds mainly containing nucleic acid bases. It provides a simple analytical means.

[0002]

2. Description of the Related Art In recent years, proteins, oligopeptides,
Capillary electrophoresis (CE) is beginning to attract attention as an alternative to conventional reversed-phase high-performance liquid chromatography (HPLC) in the detection, quantification, purification, and the like of NA, RNA, and fragments thereof.

[0003] The capillary electrophoresis method has the following requirements: [1] The sample amount is as small as nanoliters, and the detection sensitivity is picogram level and high mass sensitivity. [2] The use of a capillary in the electric field dissipates Joule heat generated during electrophoresis. Is fast, and therefore has very high resolution.- Short analysis time.- Data processing similar to reversed-phase high-performance liquid chromatography is possible.- Simple operation.- Number from relatively low molecular weight This is an analytical method that has numerous advantages, such as the ability to measure up to 10,000 high molecular weight samples.

Depending on the type of capillary and packing used, various techniques such as capillary zone electrophoresis, capillary isotachophoresis, capillary isoelectric focusing, and capillary gel electrophoresis are known. I have.

[0005]

In the conventional capillary electrophoresis method, a fused silica or a polyacrylamide gel filled inside thereof is used as a capillary. However, they separate and migrate only based on the difference in charge or molecular weight in the sample substance.In addition to these separation principles, they also have a specific separation ability for the nucleobase composition in the sample. No capillary filler is known.

Accordingly, it is an object of the present invention to provide a capillary packing material having a separation ability specific to a nucleobase composition in a sample in a capillary electrophoresis method.

[0007]

SUMMARY OF THE INVENTION The present invention is a filler for a capillary comprising a polyacrylamide gel containing a polymer of 9-vinyladenine. Hereinafter, the present invention will be described in more detail. The polymer of 9-vinyladenine used in the present invention is a homopolymer of 9-vinyladenine and a copolymer with another copolymerizable comonomer.
Here, the other copolymerizable comonomer is not particularly limited, but preferably, the homopolymer is a water-soluble homopolymer, for example, acrylamide, N-methylacrylamide, N, N-dimethylacrylamide, N-acryloylmorpholine , 2-hydroxyethyl methacrylate,
Examples include N-vinylpyrrolidone, acrylic acid, methacrylic acid, maleic anhydride, itaconic acid and the like, and mixtures thereof.

[0008] Synthesis of these polymers can be easily carried out by a general polymerization method such as radical polymerization or ionic polymerization. For example, water, in a solvent such as dioxane, ammonium persulfate, in the presence of a polymerization initiator such as benzoyl peroxide, degassed conditions, or heated under an inert gas atmosphere, polymerized, and if necessary, reprecipitated, A high yield can be obtained by performing purification such as dialysis and gel filtration. In this case, the composition ratio of 9-vinyladenine in the polymer is preferably 30% by weight or more in order to exhibit sufficient performance. The molecular weight is not particularly limited, but is preferably 300 or more in order to exhibit sufficient performance.

The preparation of a capillary filled with a polyacrylamide gel containing a polymer of 9-vinyladenine is carried out by dissolving acrylamide and N, N-methylenebisacrylamide in a suitable buffer solution, for example, tris-borate-urea. After simultaneously dissolving the polymer of 9-vinyladenine, the polymer is injected into a capillary and further polymerized. The polymerization initiator used at this time is preferably a water-soluble one. Particularly, ammonium persulfate and N, N, N ', N'-
Redox polymerization initiator systems with amines such as tetramethylethylenediamine are preferred. The amount of the 9-vinyladenine polymer in the preparation for polymerization is 0.001 to 10% by weight.
Is preferred. The polymerization reaction is preferably degassed under reduced pressure or replaced with an inert gas, if necessary. The polymerization temperature is preferably 0 to 120 ° C, preferably 20 to 60 ° C, for 30 minutes to 48 hours.

The capillary used at this time is as follows:
Any material may be used as long as it is compatible with capillary electrophoresis, and is not particularly limited. For convenience, commercially available fused silica having an inner diameter of 40 to 200 μm is used. The effective length of the capillary is not particularly limited, but is preferably 1 cm to 100 cm. If it is less than 1 cm, sufficient resolving power cannot be obtained, and if it exceeds 100 cm, the electrophoresis time becomes too long, which is not preferable.

[0011]

The use of the capillary packing material specified in the present invention enables selective separation based on the nucleobase composition in a sample compound, which could not be achieved by conventional capillary electrophoresis. Separation, purification, qualification, and quantification of various natural or non-natural compounds using capillary electrophoresis have become possible.

[0012]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Preparation Example 1 Synthesis of poly (9-vinyladenine) In a three-necked flask, 20 g of 9-vinyladenine, 10 parts of distilled water
0 g, and 0.5 g of ammonium persulfate, and the mixture was stirred at room temperature for 30 minutes under a nitrogen stream, and then at 60 ° C. for 3 hours,
Stirred at C for 3 hours. This was reprecipitated in methanol to give a white powder, Mw = 1200, Mw / Mn =
2.3 (GPC method, styrene conversion) poly (9-vinyladenine) was obtained (55% yield). In addition, the obtained poly (9-vinyl adenine) had good water solubility.

Example 1 0.1 M tris-urea borate / 7 M urea buffer (pH = 8.
6) using 8% by weight of acrylamide / 5 containing 0.03% by weight of the poly (9-vinyladenine) obtained in the above preparation example.
A 10% by weight solution of N, N-methylenebisacrylamide (a reagent for electrophoresis, manufactured by Wako Pure Chemical Industries, Ltd.) was prepared. After deaeration under reduced pressure, 0.2 g of a 10% by weight aqueous solution of ammonium persulfate and 10% by weight of N, N, N ',
0.2 g of an aqueous solution of N'-tetramethylethylenediamine was added, and the mixture was stirred well. On the other hand, the inside of a commercially available capillary for electrophoresis (GL Science Co., Ltd., inner diameter 100 μm, uncoated fused silica) was washed with pure water for 10 minutes, and then sufficiently replaced with the above-mentioned acrylamide solution. This was allowed to stand at room temperature for 30 minutes and at 37 ° C. for 2 hours to complete the polymerization, thereby preparing a polyacrylamide-based gel-filled capillary.

Example 2 In the same manner as in Example 1, a polyacrylamide gel-filled capillary containing 0.1% by weight of poly (9-vinyladenine) was prepared. Example 3 In the same manner as in Example 1, a polyacrylamide-based gel-filled capillary containing 0.2% by weight of poly (9-vinyladenine) was prepared.

Example 4 0.03% by weight prepared in Examples 1 and 3 and 0.3%
After the polyacrylamide gel-filled capillary containing 2% by weight of poly (9-vinyladenine) is sufficiently rinsed with a buffer solution, these are used to obtain (a) a 12-18 mer of polydeoxyadenylic acid and (b) a polymer. Of deoxythymidylic acid
Capillary electrophoresis of the 12-18 mer was measured. The migration patterns are shown in FIGS.

The measurement was performed under the following conditions. Capillary: prepared in Examples 1 and 3. Effective length 22 cm Voltage: 214 V / cm Column temperature: 30 ° C. Buffer: 0.1 M Tris-borate / 7 M urea Buffer (pH 8.6) Sample injection: 5 KV, 1 second Detection: UV 260 nm.

When comparing FIG. 1 with FIG. 2, in the case of a polyacrylamide gel-filled capillary containing 0.03% by weight of poly (9-vinyladenine), the migration pattern of FIG. 2 is broader. Thus, by adding only 0.03% by weight of poly (9-vinyladenine), compared to the 12-18 mer of polydeoxyadenylic acid,
It can be seen that a specific interaction has occurred with the polydeoxythymidylic acid 12 to 18 mer. From FIG. 2, the polyacrylamide-based gel-filled capillary containing 0.2% by weight of poly (9-vinyladenine) does not migrate 12 to 18 mer of polydeoxythymidylic acid. It can be seen that selective detection of only the ~ 18 mer is possible.

Comparative Example 1 Using a polyacrylamide gel-filled capillary prepared in the same manner as in Example 1 except that poly (9-vinyladenine) was not used, (a) 12 to 18 parts of polydeoxyadenylic acid Body, (b) 12-18 of polydeoxythymidylic acid
Capillary electrophoresis of a 1: 1 mixture of the dimer, (c) 12-18 mer of polydeoxyadenylic acid and 12-18 mer of polydeoxythymidylic acid was measured. The migration pattern is shown in FIG. The measurement was performed under the same conditions as in Example 4.

As shown in FIG. 3, in a polyacrylamide-based gel-filled capillary containing no poly (9-vinyladenine), a 12-18 mer of polydeoxyadenylic acid and a 12-18 mer of polydeoxythymidylic acid are obtained. It can be seen that separation of is impossible.

Example 5 A polyacrylamide gel-filled capillary containing 0.1% by weight of poly (9-vinyladenine) prepared in Example 2 was used to prepare 12-18 mer of polydeoxyadenylic acid and polydeoxythymidylic acid. Capillary electrophoresis of a 7: 1 mixture of 15mers was measured. The migration pattern is shown in FIG. The measurement was performed by raising the column temperature at 50 ° C. for 4 minutes and then raising the temperature to 60 ° C. Other than the column temperature,
Performed under the same conditions as in Example 5.

Comparative Example 2 A polyacrylamide gel-filled capillary containing no poly (9-vinyladenine) but otherwise prepared in the same manner as in Example 1 was used.
Capillary electrophoresis of a mixture of 18mer and 15mer of polydeoxythymidylic acid was measured. The electrophoresis pattern is shown in FIG.

The measurement was performed under the same conditions as in Example 5. As shown in FIG. 4, focusing on the separation of polydeoxyadenylic acid 12- to 18-mers and polydeoxythymidylic acid 15-mers, poly (9-vinyladenine)
In the capillary of Comparative Example 2 containing no, the 17-mer of polydeoxyadenylic acid [p (dA) ₁₇ ] → 15-mer of polydeoxythymidylic acid [p (dT) ₁₅ ] → 18-mer of polydeoxyadenylic acid This is the migration order of [p (dA) ₁₈ ]. In contrast, Example 5 containing poly (9-vinyladenine)
In the capillary (p), [p (dA) ₁₇ ] → is added by adding 0.1% by weight of poly (9-vinyladenine).
The order is [p (dA) ₁₈ ] → [p (dT) ₁₅ ], which indicates that separation of the 15-mer of polydeoxythymidylic acid is possible for the first time.

As is apparent from the above description, the separation and quantification of the target substance can be accurately performed by appropriately changing the concentration of poly (9-vinyladenine) in the capillary gel.

[Brief description of the drawings]

FIG. 1 is an electrophoretic pattern of 12 to 18 mer of polydeoxyadenylic acid using a polyacrylamide-based gel-filled capillary containing 0.03% by weight and 0.2% by weight of poly (9-vinyladenine) in Example 4. .

FIG. 2 is an electrophoretic pattern of polydeoxythymidylic acid 12-18 mer using a polyacrylamide gel-filled capillary containing 0.03% by weight and 0.2% by weight of poly (9-vinyladenine) in Example 4. .

FIG. 3 In Comparative Example 1, a polyacrylamide-based gel-filled capillary containing no poly (9-vinyladenine) was used (a) 12 to 18 mer of polydeoxyadenylic acid [p (dA) _12-18 ] , (B) 12-18 mer of polydeoxythymidylic acid [p (dT) _12-18 ], (c) 12-18 mer of polydeoxyadenylic acid [p (dA) _12-18 ] and polydeoxythymidyl 12- to 18-mer of acid [p (d
T) Electrophoresis pattern of the mixture of _12-18 ].

FIG. 4 (a) is a polyacrylamide-based gel-filled capillary containing 0.1% of poly (9-vinyladenine) of Example 5, and FIG. 4 (b) is a poly (9-vinyladenine) -free poly of Comparative Example 2. Acrylamide-based gel-filled capillaries were used, each containing 12% of polydeoxyadenylic acid.
It is an electrophoresis pattern of a mixture of 1818-mer [p (dA) _12-18 ] and polydeoxythymidylic acid 15-mer [p (dT) ₁₅ ].

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Takeo Matsumoto 2-14-9 Higashi, Tsukuba, Ibaraki Prefecture (56) References JP-A-3-163353 (JP, A) JP-A-2-6740 (JP, A) JP-A-63-118649 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 27/447 CA (STN)

Claims (1)

(57) [Claims] 1. A capillary filler comprising a polyacrylamide gel containing a 9-vinyladenine polymer.