JPH0637510B2 - Nucleic acid separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for separating a nucleic acid, more specifically, an average pore size of 150.
The present invention relates to a method for separating nucleic acids by liquid chromatography, which comprises using a packing material for reverse phase chromatography obtained by reacting silica gel having a large pore size of angstrom or more with a silane compound having an alkyl group having 20 or more carbon atoms. It is a thing.

(Prior Art and Problems) A packing material obtained by reacting a silane compound having an alkyl group having 1 to 18 carbon atoms with silica gel is widely used in reverse phase chromatography, and a nucleic acid having a small molecular weight or its constituent substance is used. Is also very useful for. However, when these usual packing materials are used, the width of the chromatographic peak becomes wider as the molecular weight of the nucleic acid becomes larger, and the separation becomes difficult.

(Problems to be Solved) As a result of intensive studies on the physical properties of silica gel and the relationship between the type of alkylsilane compound and the separability with respect to nucleic acids, the inventors have found that the average pore size is 150 angstroms or more. The packing material obtained by reacting silica gel with a silane compound having an alkyl group having 20 or more carbon atoms provides a method for separating nucleic acids which gives an extremely sharp chromatographic peak even for nucleic acids having 10 or more bases.

(Means for Solving Problems) Hereinafter, the present invention will be described in detail.

The silica gel used in the present invention has a particle size of 1 to 1
00 micron, preferably 3 to 30 micron spherical or crushed porous silica gel with an average pore size of 150
It is more than Angstrom. When the pore size is less than 150 Å, the sharpness of the peak is lost as the number of bases increases with respect to the nucleic acid having 10 or more bases, which is not preferable. That is, it is considered that when the pore size is small, the pores tend to be blocked in the surface modification with the long-chain alkylsilane compound, and the larger the nucleic acid, the more the molecules cannot move smoothly in the pores. The optimum pore size varies depending on the size of the target nucleic acid, but in the present invention, the target number of bases is 5
150 to 1.00 in the oligomer region below 0
A range of 0 Angstroms is suitable.

The long-chain alkylsilane used in the present invention has 2 carbon atoms.
Having 0 or more alkyl groups, for example, eicosyltrichlorosilane, eicosyltrimethoxysilane, eicosyltriethoxysilane, docosylmethyldichlorosilane, docosylmethyldimethoxylane, docosylmethyldiethoxysilane, docosyltriethoxysilane Chlorosilane, docosyltrimethoxysilane, docosyltriethoxysilane, triacontyldimethylchlorosilane, triacontyldimethylmethoxysilane, triacontyldimethylethoxysilane, triacontyltrichlorosilane, triacontyltrimethoxysilane, triacontyltriethoxysilane An alkylsilane compound having 1 to 3 halogen groups or alkoxy groups such as.

In the present invention, the reaction between silica gel and the alkylsilane compound is carried out by a usual method. That is, the reaction can be carried out by heating the dried silica gel together with the alkylsilane compound in the presence of a suitable solvent.

The necessary amount of the alkylsilane compound in the reaction is usually equivalent to or less than the silanol group of silica gel.
It is preferable to form a monomolecular weight that is as uniform as possible.
The desirable introduction amount of the alkylsilane compound depends on the physical properties of silica gel and the type of the alkylsilane compound. Usually, an elemental analysis value of carbon of about 10 to 20% is suitable.

It is necessary that the solvent used in the reaction dissolves the alkylsilane compound well and is inert to the alkylsilane compound. Preferred solvents include saturated hydrocarbons such as n-hexane, n-heptane, n-octane, n-decane, isooctane and cyclohexane, halogenated carbons such as chloroform, carbon tetrachloride, dichloroethane, trichloroethane, tetrachloroethane and dichloropropane. Aromatic hydrocarbons such as hydrogen, benzene, toluene, xylene, ethylbenzene, and chlorobenzene,
Examples thereof include aromatic halides such as dichlorobenzene and trichlorobenzene. The reaction temperature may be usually selected from room temperature to the boiling point of the solvent, and the reaction is generally carried out at 50 to 100 ° C. for several hours to 20 hours.

The filler thus obtained has a few micromoles / m ² of residual silanol groups. Since the alkylsilane compound used is very large, the steric hindrance limits the silanol groups that can participate in the reaction among the silanol groups that silica gel has, and when using a polyhalogenated alkylsilane compound, a new silanol group is added. By generating. Therefore, when there is a possibility that the residual silanol group may be involved in the irreversible adsorption of the cationic substance present in the sample, it is desirable to eliminate it as much as possible. In order to eliminate the residual silanol group, a method of conducting a condensation reaction with an alkylsilane compound having a small number of carbon atoms is usually performed. Examples of the lower alkylsilane compound include trimethylchlorosilane, trimethylpromosilane, trimethylmethoxysilane, trimethylethoxysilane, trimethylsilylacetamide, bis (trimethylsilyl) acetamide, and ethyldimethylchlorosilane.

For the reaction for eliminating the silanol group, the same method as that for reacting the above-mentioned silica gel with an alkylsilane compound can be applied. Usually, in order to complete the reaction, the lower alkylsilane compound is used in an amount of at least equivalent to the silanol group, and the reaction is carried out at a temperature not higher than the boiling point of the lower alkylsilane compound for 5 to 20 hours.

In the present invention, as the conditions for liquid chromatography, known conditions for ordinary reverse phase chromatography can be applied. That is, the packing material obtained by the method described above is packed in a column for proper chromatography, and the separation is performed by a gradient elution method in which the concentration of the organic solvent is gradually increased, using a suitable buffer solution and a suitable organic solvent. To do. As the organic solvent, a wide range of organic solvents that are water-soluble and inert to nucleic acids can be applied, but normally, lower alcohols such as methanol, ethanol, n-propanol, iso-propanol, acetonitrile, tetrahydrofuran, etc. are used. To be

In liquid chromatography, one of the indexes representing the resolution of a column is the height equivalent to a theoretical plate, which is defined by the following formula.

Height equivalent to theoretical plate = L / N = (L / 16) x (W / Ve) where L is column length (mm) N is theoretical plate number W is peak width (ml) Ve is elution volume (ml) theoretical plate The smaller the numerical value of the equivalent height, the smaller the spread of the peak (the peak is sharper), indicating that the column performance is excellent. However, since the height equivalent to a theoretical plate depends on the size of the particles (the smaller the particle size, the larger), it is necessary to compare with the same particle size as much as possible.

The height equivalent to a theoretical plate measured with a synthetic nucleic acid having 26 bases was in the range of 0.01 mm to 0.1 mm for ordinary commercially available 5 micron octadecyl silylated silica gel (ODS silica). For agents, from 0.001 mm to 0.005 mm
The value was extremely small and excellent.

Hereinafter, the present invention will be specifically described with reference to examples.

Example 1 30 equipped with a reflux condenser, a stirrer and a nitrogen inlet tube
Dry in a 0 ml three-necked flask at 80 ° C. under reduced pressure for 16 hours, average pore size 250 Å, specific surface area 28
0 m ² / g, 10 g of silica gel with an average particle size of 10 microns
Then, 90 ml of dry toluene is added thereto, and the mixture is degassed in an ultrasonic bath for about 5 minutes. To this, 6.4 g of triacontyldimethylchlorosilane was added and reacted at 80 ° C. for 10 hours. Hydrogen chloride generated during the reaction is introduced by introducing nitrogen into a gas absorption tube containing a sodium hydroxide solution to be absorbed. After the reaction, the gel is filtered with a glass filter, and then unreacted triacontyldimethylsilane is removed,
The gel was rinsed with 300 ml of 80 ° C toluene in 3 to 5 portions. Then, toluene is removed with 300 ml of acetone, and then hydrogen chloride is removed with 300 ml of water. further,
After washing with 300 ml of acetone, it was dried under reduced pressure at 60 ° C. for 16 hours. The alkylsilylated silica gel thus obtained was transferred to the above-mentioned apparatus, 90 ml of dry toluene was added thereto, deaerated in an ultrasonic bath for about 5 minutes, 10 g of trimethylchlorosilane was added and reacted at 40 ° C. for 10 hours. .
After the reaction, the gel was filtered with a glass filter, toluene,
Wash with acetone, water, and acetone in that order, and then use 1 at 60 ° C under reduced pressure.
It was dried for 6 hours. The elemental analysis value of the silica gel incorporating triacontyl group obtained by such a surface modification reaction is carbon 17.
It was 0% and hydrogen 3.1%.

To evaluate the ability of this gel to separate nucleic acids in liquid chromatography, an inner diameter of 4.6 mm and a length of 15 cm
The column number was 7, 9, 23 by the elution method of a linear gradient (0-25% linear gradient) of acetonitrile from a 0.1 M aqueous solution of ammonium acetate using a UV absorption spectrophotometer as the detector. The chromatogram obtained by separating the synthetic nucleic acid mixture of No. 26 and No. 26 is shown in FIG.

The height corresponding to a theoretical plate calculated from the peak of a nucleic acid having 26 bases is 0.0016 mm, which is an extremely sharp peak.

Example 2 Silica gel having an average pore size of 150 Å,
Evaluation was carried out by the surface modification reaction and liquid chromatography in the same manner as in Example 1 except that silica gel having a specific surface area of 350 m ² / g and an average particle size of 5 microns was used and the amount of triacontyldimethylchlorosilane used was 8.0 g. went.
Elemental analysis values of the obtained gel are carbon 19.3%, hydrogen 3.5%
Met. Further, the height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.0019 mm.

Example 3 Silica gel having an average pore size of 500 Å,
Evaluation was carried out by the surface modification reaction and liquid chromatography in the same manner as in Example 1 except that silica gel having a specific surface area of 220 m ² / g and an average particle size of 10 microns was used and the amount of triacontyldimethylchlorosilane used was 5.0 g. went. The elemental analysis value of the obtained gel is 9.8% carbon, 2.1 hydrogen.
%Met. The height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.0015 mm.

Example 4 Evaluations were carried out by the surface modification reaction and liquid chromatography in the same manner as in Example 1 except that eicosyltrichlorosilane was used as the alkylsilane compound. The elemental analysis values of the obtained gel were carbon 15.8% and hydrogen 3.0%. Further, the height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.0020 mm.

Example 5 The evaluation by surface modification reaction and liquid chromatography was performed in the same manner as in Example 1 except that docosylmethyldimethoxysilane was used as the alkylsilane compound. The elemental analysis value of the obtained gel is carbon 16.0%, hydrogen 3.
It was 1%. The height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.0018 mm.

Example 6 The evaluation by surface modification reaction and liquid chromatography was performed in the same manner as in Example 1 except that the reaction for removing the residual silanol group using trimethylchlorosilane was omitted. The elemental analysis values of the obtained gel were carbon 16.5% and hydrogen 3.0%. Further, the height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.0020 mm.

Comparative Example 1 Evaluations were carried out by the surface modification reaction and liquid chromatography in the same manner as in Example 2 except that octadecyltrichlorosilane was used as the alkylsilane compound.
Elemental analysis values of the obtained gel are carbon 19.1%, hydrogen 3.5%
Met. The height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.035 mm.

Comparative Example 2 Silica gel having an average pore size of 100 Å,
Evaluation was carried out by the surface modification reaction and liquid chromatography in the same manner as in Example 1 except that silica gel having a specific surface area of 400 m ² / g and an average particle diameter of 5 μm was used. The elemental analysis values of the obtained gel were carbon 19.7% and hydrogen 3.6%. The height corresponding to the theoretical plate of the peak of the nucleic acid having 26 bases was 0.083 mm.

(Effect) As described above, the packing material obtained in the present invention is a packing material for so-called reverse-phase chromatography, and has a nucleic acid, particularly about 10 to 100 bases important in future genetic engineering, as compared with the conventional packing material. It is a very useful packing material that exhibits excellent separation ability for nucleic acids of size, and can be used for analysis and separation / purification.

[Brief description of drawings]

FIG. 1 shows a chromatogram obtained by separating a nucleic acid mixture. 1: nucleic acid having 7 bases, 3: nucleic acid having 23 bases, 2: nucleic acid having 9 bases, 4: nucleic acid having 26 bases

Claims (1)

[Claims] 1. A packing material for reverse phase chromatography, which is obtained by reacting a silica gel having a large pore diameter of 150 angstroms or more with a silane compound having an alkyl group having 20 or more carbon atoms. Method for separating nucleic acid by liquid chromatography.