JPS6281400A - Method of separating nucleic acid - Google Patents

Abstract

PURPOSE:To obtain a nucleic acid having large number of bases important in genetic engineering in extremely improved separation ability and readily, by using a reaction product of silica gel having large pore diameter and a long-chain alkylsilane as a filler for reversed phase chromatography. CONSTITUTION:Firstly, silica gel having >=150Angstrom average pore diameter is reacted with a silane compound (e.g., eicosyltrichlorosilane, etc.,) containing >=20C alkyl group usually by a method wherein dried silica gel and the alkylsilane compound are heated in a solvent such as n-hexane, etc., at 50-100 deg.C. Then, the prepared reaction is used as a filler for reversed phase chromatography to separate a nucleic acid by liquid chromatography. In a case where there is fear that a silanol group remaining in the filler causes irreversible adsorption of cationic substances in a test specimen, preferably the silanol group is eliminated by condensation with an alkylsilane compound with a few carbon atoms.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for separating nucleic acids, and more particularly, to
Relating to a method for separating nucleic acids by liquid chromatography, which uses a reversed phase chromatography packing material obtained by reacting a silane compound having an alkyl group with a carbon number of 20 or more with silica gel having a large pore diameter of angstrom or more. It is something.

(Prior art i and problems) A packing material obtained by reacting silica gel with a silane compound having an alkyl group having 1 to 18 carbon atoms is widely used in reversed phase chromatography, and it It is also known to be extremely useful for constituent substances. However, when these ordinary packing materials are used, the molecule size of the nucleic acid becomes thicker (the wider the chromatographic peak becomes, making separation difficult).

(Problems to be Solved) As a result of extensive research into the relationship between the physical properties of silica gel, the type of alkylsilane compound, and the separation ability for nucleic acids, the present inventors found that large pores with an average pore diameter of 150 angstroms or more The packing material obtained by reacting silica gel with a silica/compound having an alkyl group having 20 or more carbon atoms provides a method for separating nucleic acids that provides extremely sharp chromatography even for nucleic acids having 10 or more bases. be.

(Means for solving problems) The present invention will be explained in detail below.

The silica gel used in the present invention has a particle size of 1 to 1.
00 microns, preferably 3 to 30 microns, spherical or crushed porous silica gel with an average pore size of 150 microns.
It is more than Angstrom. If the pore diameter is less than 150 angstroms, the sharpness of the peak will be lost as the number of bases increases for nucleic acids having 10 or more bases, which is not preferable. That is, it is thought that if the pore diameter is small, the pore tends to be blocked during surface modification with a long-chain alkyl 7-rane compound, and that as the nucleic acid becomes larger, molecules cannot move smoothly within the pore. The optimal pore diameter varies depending on the size of the target nucleic acid, but in the present invention, the number of target bases is 5.
In the oligomer region below 0, from 150 to i, o
A range of o 0 angstroms is suitable.

The long chain alkylsilane used in the present invention has 2 carbon atoms.
Those having 0 or more alkyl groups, such as eicosyltrichlorosilane, eicosyltrimethoxysilane, and eicosyltriethoxysilane.

Tocosylmethyldichlorosilane, tocosylmethyldimethoxylane, tocosylmethyljethoxy7ran, tocodyltrichlorosilane, tocodyltrimethoxysilane, tocodyltriethoxysilane, triacontyldimethylchlorosilane, triacontyldimethylmethoxysilane, triaco/tyldimethylethoxysilane, triacontyltrichlorosilane, triacontyltrimethoxysilane.

It is an alkylsilane compound having 1 to 3 groups or alkoxy groups, such as triacontyltriethoxysilane 1.

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

The required amount of the alkylsilane compound in the reaction is usually an equivalent amount or less based on the silanol group of the silica gel.

(It is preferable to form a uniform monomolecular layer.)

The preferred introduction of the argylsilane compound depends on the physical properties of the silica gel and the type of the alkylsilane compound. Usually, an elemental analysis value of carbon of about 10 to 20 is appropriate. The solvent used for the reaction needs to dissolve the alkylsilane compound well and be inert to the alkylsilane compound. Preferred solvents include saturated hydrocarbons such as n-hexane, n-hebutane, n-octane, n-decano, isooctane, and cyclohexane, chloroform, and carbon tetrachloride.

Examples include halogenated hydrocarbons such as dichloroethane, trichloroethane, tetrachloroethane, and dichloropropane, aromatic hydrocarbons such as benzene, toluene, xylene, and ethylbenzene, and aromatic halides such as chlorobenzene, dichlorobenzene, and trichlorobenzene. The reaction temperature is usually selected between room temperature and the boiling point of the solvent (generally, the reaction is carried out at about 50 to 100°C for several hours to 20 hours.

The filler thus obtained has a few micromol/- of residual silanol groups. Since the alkylsilane compound used is very large, the number of silanol groups that can participate in the reaction among the silanol groups in silica gel is limited due to steric hindrance, and when a polyhalogenated alkylsilane compound is used, new silanol groups are created. By generating.

Therefore, if the remaining silanol groups are likely to be involved in irreversible adsorption of cationic substances present in the sample, it is desirable to eliminate them as much as possible. K, which eliminates remaining silanol groups, is usually subjected to a condensation reaction with an alkylsilane compound having a small number of carbon atoms. Examples of lower alkylsilane compounds include trimethylchlorosilane, trimethylbromosila/trimethylmethoxysilane.

Examples include trimethylethoxysilane, trimethylsilylacetamide, bis(trimethylnoryl)acetamide, and ethyldimethylchlorosilane.

For the reaction of eliminating silanol groups, a method similar to that for reacting the alkylsilane compound with silica gel described above can be applied. Usually, in order to complete the reaction, the lower alkylsilane compound is used in an amount equivalent to or more than the silanol group, and the reaction is carried out at a temperature below the boiling point of the lower alkylsilane compound for 5 to 20 hours.

In the present invention, known conditions for ordinary reverse phase chromatography can be applied as conditions for liquid chromatography. That is, the packing material obtained by the above method is packed into an appropriate chromatography column, and separation is performed using an appropriate buffer and an appropriate organic solvent by gradient elution in which the concentration of the organic solvent is gradually increased. conduct. As the organic solvent, a wide range of organic solvents that are water-soluble and inert to nucleic acids can be used, but methanol, ether/
Lower alcohols such as -ol, n-7' lobanol, and 1so-chlorohanol, acetonitrile, and tetrahydrofuran are used.

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

Theoretical plate equivalent height - L/N - (L/16) x (W/Ve)
Here, L is the column length (radius), N is the number of theoretical plates, W is the peak width (rnt), Vθ is the elution volume (, t), and the smaller the value, the smaller the peak spread (the sharper the peak), and the smaller the height of the column. Indicates superior performance. However, since the height equivalent to a theoretical plate depends on the size of the particles (the smaller the particle size, the larger the particle size), it is necessary to compare 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 α01 to 11 tin for ordinary commercially available 5 micron octadecyl silylated silica gel (ODS silica), but for the packing material of the present invention, α001as to α005
It showed an excellent value, which is considered to be a small amount.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Practical Example 1 5 equipped with reflux condenser, stirrer and nitrogen inlet tube
00IILt in a three-necked flask at 80°C under reduced pressure.
Dry for 6 hours, average pore diameter 250 angstroms, specific surface area 280? F? /9, silica gel 109 with an average particle size of 10 microns is added, 90-g dry toluene is added thereto, and degassed in an ultrasonic bath for about 5 minutes. Add 6.4 g of triacontyldimethylchlorosilane to this, and
The reaction was allowed to proceed for 0 hours.

Hydrogen chloride generated during the reaction is hydroxylated by introducing nitrogen into the gas absorption tube containing the IJium solution and absorbed. After the reaction, the gel was passed through a glass filter, and then in order to remove unreacted triacontyldimethylsilane,
The gel was rinsed with 300 ml of toluene at 80° C. in 3 to 5 portions. Thereafter, toluene was removed with acetone saomt, and then hydrogen chloride was removed with 300 ILt of water.

Furthermore, after washing with acetone 300°C, 60°C under reduced pressure was used.
It was dried for 16 hours. The alkylsilylated silica gel obtained by pressing in this manner was transferred to the above-mentioned apparatus, 90% of dry toluene was added, and after degassing in an ultrasonic bath for about 5 minutes, 109%
of trimethylchlorosilane and reacted at 40°C for 10 hours. After the reaction, filter the gel through a glass filter and add toluene, acetone, and water.

It was washed successively with acetone and dried under reduced pressure at 60°C for 16 hours. The elemental analysis values of the triacontyl group-introduced silica gel obtained by such surface modification reaction were 17.0% carbon and 51% hydrogen.

In order to evaluate the ability of this gel to separate nucleic acids in liquid chromatography, the inner diameter was 4.61+5.

Packed into a stainless steel column with a length of 15 crn, and using an ultraviolet absorption spectrophotometer for detection, a 0-25% linear gradient elution method of acetonitrile from a 11M ammonium acetate aqueous solution was used, and the number of bases was 7.9. Chromatograms separating synthetic nucleic acid mixtures of .25 and 26 are shown in the first FyJK.

The height equivalent to a theoretical plate calculated from the peak of a nucleic acid with 26 bases is α0016. It can be seen that this is an extremely sharp peak.

Example 2 Silica gel with an average pore diameter of 150 angstroms,
The surface modification reaction and liquid chromatography were carried out in the same manner as in Example 1, except that silica gel with a specific surface area of 35 (ly//l; Evaluation was performed.The elemental analysis values of the gel obtained were: carbon 1'13%;
The hydrogen content was 55%. In addition, the peak height equivalent to a theoretical plate for a nucleic acid with 26 bases is α0019. Met.

Example 5 Silica gel with an average pore diameter of 500 angstroms,
Evaluation by surface modification reaction and liquid chromatography was carried out in the same manner as in Example 1, except that silica gel with a specific surface area of 220 d/9 and an average particle size of 10 microns was used, and the amount of triacontyldimethylchlorosilane used was 5.09. Ta. The elemental analysis values of the obtained gel were 9.8% carbon and λ hydrogen.
It was 1%. In addition, the height equivalent to a theoretical plate of a peak of a nucleic acid having 26 bases is α0015. Met.

Example 4 Surface modification reaction and liquid chromatography evaluation were carried out in the same manner as in Example 1 except that eicosyltrichlorosilane was used as the alkyl sila/compound. The elemental analysis of the gel obtained was 15.8% carbon and 50% hydrogen.
Met. Further, the height equivalent to a theoretical plate of the peak of a nucleic acid having 26 bases was [LO0201Ij.

Example 5 Evaluation by surface modification reaction and liquid chromatography was carried out in the same manner as in Example 1 except that tocosylmethyldimethoxysilane was used as the alkylsilane compound. The elemental analysis values of the obtained gel were 160% carbon and 51% hydrogen.
%Met. Further, the height equivalent to a theoretical plate of the peak of a nucleic acid having 26 bases was αOO18su.

Example 6 Evaluation by surface modification reaction and liquid chromatography was carried out in the same manner as in Example 1 except that the reaction for removing residual silanol groups using trimethylchlorosilane was omitted. The elemental analysis values of the obtained gel were 145% carbon and 50% hydrogen.

In addition, the height equivalent to a theoretical plate of the peak of a nucleic acid with 26 bases is α
It was 0020°.

Comparative Example 1 Evaluation by surface modification reaction and liquid chromatography was carried out in the same manner as in Example 2 except that octadecyltrichlorosilane was used as the alkylsilane compound.

The elemental analysis of the gel obtained was 191% carbon and 55% hydrogen.
Met. Further, the height equivalent to a theoretical plate of a peak of a nucleic acid having 26 bases was 1035.

Comparative Example 2 Silica gel with an average pore diameter of 100 Å/gström,
Evaluation by surface modification reaction and liquid chromatography was carried out in the same manner as in Example 1, except that silica gel with a specific surface area of 40 OFF//9 and an average particle diameter of 5 microns was used. The elemental analysis values of the obtained gel were 19.7% carbon and 56% hydrogen.
Met. In addition, the height equivalent to a theoretical plate of a peak of a nucleic acid having 26 bases is α083. Met.

(Effects) As described above, the packing material obtained by the present invention can be used as a packing material for so-called reversed phase chromatography, and has a higher number of bases than conventional packing materials, which is important for nucleic acids, especially in future genetic engineering. It is an extremely useful packing material that exhibits excellent separation ability for nucleic acids of a certain size, and can be used for analysis and separation and purification.

[Brief explanation of drawings]

FIG. 1 shows a chromatogram separating a nucleic acid mixture.

Claims (1)

[Claims] 1) A liquid chromatograph characterized by using a packing material for reversed phase chromatography obtained by reacting a silane compound having an alkyl group with a carbon number of 20 or more with a silica gel having a large pore diameter of 150 angstroms or more. A method for separating nucleic acids using graphics.