SU1655534A1 - Method for obtaining affinic sorbent for fractionating nucleic acids - Google Patents

Abstract

The invention relates to sorbents for affinity chromatography and makes it possible to increase the selectivity of the separation of nucleic acids. The microgranulated invention relates to sorbents for affinity chromatography and can be used to fractionate DNA and RNA. The purpose of the invention is to increase the selectivity of the sorbent to nucleic acids. EXAMPLE 1. 100 cm3 of microgranulated cellulose is mixed with 100 cm of distilled water, 10 cm of 40% sodium hydroxide and 10 cm3 of epichlorohydrin are added. The ratio of reagents, wt.%: Cellulose 45; caustic soda 1,8; epichlorohydrin 4,5. The suspension is stirred for 2 hours at 40 ° C, cellulose (100 cm is mixed with 100 ml of distilled water, 10 ml of 40% sodium hydroxide and epichlorohydrin are added in an amount of 4.5-15% by weight of the reaction mixture. The suspension is stirred 2 h at 40 ° C, washed with water and suspended with an aqueous solution of diaminodecane (0.06-0.35% by weight of the mixture). Stir for 20 h at 35 ° C and washed with water and dimethylformamide, add 0 , 8-1.9 wt.% Of azide of M- (2-nitro-3-hydroxy-4-chlorobenzoyl) -5-amino-caproic acid in ethyl acetate with triethylamine and stirred at 4 ° C for 18 hours. dimethylformamide, methanol and 7-12 wt.% sodium dithionite are added, stirred for 3 hours at 20 ° C. Washed with water, 50% CH 2 COOH and ethyl alcohol. Para-quinone (1.3-3, 4 wt.%) And SchZ3-dimethylaminopropyl} hydrochloride - 2-amino-3-hydroxy-4-methylbenzamide (0.27-0.51 wt.%) In ethyl alcohol. Stir for 24 hours at 22 ° C and wash alcohol and water. A sorbent is used to separate nucleic acids. 1 tab. then the epoxy cellulose is washed with distilled water (3000 cm3) and 150 mg of diaminodecane (0.06% diaminodecan concentration) are suspended in an aqueous solution for 35 hours at 35 ° C. After that, aminodecyl cellulose is washed with distilled water (4000 cm) and dimethylformamide (500. To 10 cm of aminodecyl cellulose, add 10 cm3 of dimethylformamide, 0.5 cm of triethylamine and a solution of 0.3 g of M- (2-nitro-3-hydroxy) azide -4-chlorobenzoyl) -5-amino-caproic acid in 20 cm3 of ethyl acetate. The content of M- (2-nitro-3-hydroxy-4-chloroben (L c ate ate)

Description

zoyl) -5-aminocaproic acid in the reaction mixture, 0.8 wt.%. The suspension is stirred at 4 ° C for 18 hours and then washed with dimethylformamide (1000 CMJ the resulting intermediate.

In order to reduce the nitro groups in it, the intermediate is washed with methanol (50 cm3), 100 cm3 of 10% aqueous solution of sodium dithionite is added and stirred for 3 hours at 20 ° C. The content of sodium dithionite is 7%. Then the sorbent is washed with water (500 cm3), 50% acetic acid (200 cm3), distilled water (300 cm3, ethyl alcohol (200 cm).

The formation of immobilized derivatives of actinocin is carried out directly on cellulose by stirring a suspension of the sorbent in 30 cm of ethanol containing 0.1 g of N- (3-dimethylaminopropyl) -2-amino-3-hydroxy-4-methylbenzamide hydrochloride and Oi5 g of paraquinone for 24 h at 22 ° C. The content of N- (3-dimethylaminopropyl) -2-amino-3-hydroxy-4-methylbenzamide and para-quinone hydrochloride is 0.27% and 1.3%, respectively.

The resulting sorbent is washed with ethyl alcohol (300 cm and distilled water (500 cm3).

During the preparation of the sorbent, M- (2-nitro-3-hydroxy-4-chlorobenzoyl) -5-aminocaproic acid azide is used, which is obtained by the following procedure.

Synthesis of M- (2-nitro-3-hydroxy-4-chlorobenzoyl) -5-aminocaproic acid azide. 0.5 g of M- (5-ethoxycarbonylpentyl) -2-nitro-3-oxo-4-chlorobenzamide is dissolved in 15 cm3 of methyl alcohol, 3 cm3 of hydrazine hydrate are added, evaporated in two steps, and 3 more are added cm3 hydrazine hydrate. It is kept for 12 hours at 18-20 ° C, protected from direct light, and evaporated to dryness in vacuo. The evaporation residue is dissolved in 7 cm of water, and then acidified with acetic acid to pH 6.0 and cooled to 5-7 ° C. After 10 hours, the precipitate formed is filtered off, washed with water and dried. The yield is chromatographically homogeneous in butanol-2: 3% ammonia (5: 2) systems on Silufol UV-254 M- (5-hydrazocarbonyl) -2-nitro-Z-oxy-4-chlorobenzamide plates with mp. 180- 182 ° С, 0.46 g (77%), so pl. 183.5-185, 0 ° С (after crystallization from methanol). Found,%: N 15.80; 16.17. CisHnCIN / iOs. Calculated,%: N 16,25. To a solution of 0.69 g (2 mmol) of M- (5-hydrazocarbonyl-pentylU2-nitro-3-oxy-4-chlorobenzamide in 40 cm of a mixture of acetic and 15% hydrochloric acid at 0-5 ° C and stirring A solution of 0.27 g (4 mmol) of sodium nitrite in 1 cm of water is mixed. The mixture is stirred for 15 minutes, 40 cm of water are added and the mixture is extracted with ethyl acetate in two portions of 20 cm 3.

for the derivatization of aminodecyl cellulose. Example 2-5. Conducted according to the method of example 1. The ratios of the reagents used are presented in the table.

PRI me R 6. DNA chromatography, On

A 10 x 30 mm column containing 4 cm of affinity sorbent prepared according to Example 1 was coated with 0.2 cm3 of DNA solution from salmon milk (Sigma, United States) in 0.1 M sodium phosphate buffer pH 7.8 (the content of native DNA is 7.7 OE or 380 μg of DNA). Unbound material is washed off with the same buffer (50 cm3). DNA is eluted with a buffer solution containing. 0.5% sodium dodecyl sulfate,

0.5% sodium lauryl sarcosinate (150 cm). Collect fractions of 4 cm3 each. The measurement of the UVU is the absorption of each fraction at 260 nm. the fractions containing the UV absorbing material are combined and precipitated

adding 2.5 volumes of ethanol in the cold. The precipitate is separated by centrifugation, dissolved and the absorption spectrum is taken in the region of 220-310 nm.

Wash fractions contain a small amount of UV absorbing material precipitated with ethanol. The absorption spectrum of the precipitate is characteristic of polysaccharides and is a curve that smoothly decreases from 220 to 310 nm. Eluate

contains pure DNA with a characteristic spectrum (Amaks 258 nm, E263 / j; 230 2.5; E260 / E280 2.2). The DNA yield was-7.5 O.E. or 98.7%.

Example. DNA chromatography On

a 10 x 30 mm column containing 4.0 cm3 of sorbent prepared in accordance with Example 2 is deposited 7.6 O.E. native DNA from salmon milt. DNA application, washing, UV absorbing, collecting and

analysis of the fractions was carried out under the conditions of Example 6. DNA elution was performed with a linear gradient of 0.0-0.5% sodium dodecyl sulfate in a 0.1 M cation phosphate buffer, pH 7.8. Two fractions of DNA are clearly visible on the chromatogram. Fraction I (eluted from 30 to 40% of the gradient, contains 6.2 OE DNA or 81% of the applied material) corresponds to the bulk of the chromosomal DNA from salmon milk. Fraction II (eluted from 50% to 70% gradient, contains 1.3 OE DNA or 17%) corresponds to GC-rich satellite DNA.

PRI me R 8, DNA chromatography. Under the conditions of Example 7, the separation is carried out

drug D N To (4,0 O. E. 2bo) from salmon milk, denatured by heating. The DNA yield in the eluent (sodium dodecyl sulfide and sodium sercosilate) is 3.8 O.E., or 95%.

PRI me R 9. Chromatography of RNA. A column of 10 x 30 mm, containing 4.0 cm3 of sorbent prepared in accordance with Example 5, was deposited with 2.6 O.E. yeast RNA solution (Fluka. Switzerland). The application of RNA, washing, recording of UV absorption, collection and analysis of fractions are performed under the conditions of Example 6. The elution of the RNA with the Y-linear gradient 0-i M sodium perchlorate in 0.1 M sodium phosphate buffer pH 7.8. RNA is eluted in a 30-40% gradient (0.3-0.4 M sodium perchlorate). The yield of RNA is 2.4 O.E., or 92%.

PRI me R 10. DNA chromatography. Under the conditions of Example 9, the separation of the preparation of native DNA (8.0 OE) from salmon milk was carried out. No significant amounts of DNA are found in any of the eluate fractions. At the same time, DNA is quantitatively eluted (yield: 96%) under conditions of regeneration of the column: upon elution with 0.5% sodium dodecyl sulfate; 0.5% sodium sarcosylate in 0.1 M sodium phosphate buffer pH 7.8.

PRI me R 11. Chromatography of DNA and RNA. An artificial mixture of RNA (4.0 O. E.) and DNA (7.0 O.E.) was applied to a column of 10 x 30 mm containing 4.0 cm3 of the sorbent obtained in accordance with Example 1. The application of the sample, washing, recording of the UV absorption, collection and analysis of the fractions are performed under the conditions of Example 6. The elution of RNA and DNA is carried out in a stepwise gradient. At the first stage - 0.5 M sodium perchlorate in 0.1 M sodium phosphate buffer pH 7.8 - RGC quantitatively eluted (yield 95%), at the second stage 0.5% sodium dodecyl sulfate, 0.5% sodium sarcosylate in 0.1 M sodium phosphate buffer pH 7.8 - DNA is eluted quantitatively (yield 97%).

PRI me R 12. Chromatography of DNA. Under the conditions of example B, the preparation of native DNA preparation (8.0 O.E) from salmon milk was carried out on a sorbent prepared in accordance with Example 3. No significant amounts of DNA were detected in any of the eluate fractions. Regeneration of the column upon elution with 0.5% sodium sarcosylate, 0.5%

Sodium dodecyl sulfate in 0.1 M sodium phosphate buffer pH 7.8 does not allow desorption of the bound DNA.

PRI me R 13. Chromatography of DNA. Under the conditions of Example 6, the preparation of a preparation of native DNA (8.0 OE) from salmon milk was carried out on a sorbent prepared in accordance with Example 4. DNA was quantitated (95%, or 7.6 OE) found in slip through with equilibrating buffer. That is, there is no binding to the sorbent.

Under the conditions of examples 6-11, 20 cycles of fractionation of nucleic acid preparations were carried out on a 10 x 30 mm column with a sorbent prepared according to examples 1,2,5. After each cycle, the column is regenerated by washing successively with 0.5% sodium dodecyl sulfate, 0.5% sodium sarcosylate (20 cm and then equilibrated with 0.1 M sodium phosphate buffer solution pH 7.8.

The spherical form of microgranulated cellulose allows 20 fractionation cycles to be performed without compromising hydrodynamic properties. The analog of actinocin used as a ligand allows selectively sorbing nucleic acids and separating DNA from RNA without decreasing the capacity of the sorbent, which is not achieved on the sorbent prepared in accordance with the prototype.

Claims (1)

Claims of Invention A method for producing an affinity sorbent for fractionating nucleic acids, including immobilizing a DNA complexon on the surface of a polymeric carrier, characterized in that, in order to increase the selectivity of the sorbent for nucleic acids, microgranulated cellulose is used as the polymeric carrier. which is successively treated with epichlorohydrin at a concentration of 4 5-15% by weight of the reaction mixture, diaminodecane 0.06-0.035 wt.%, M- (5-azidocarbonylpentyl) -2-nitro-3-hydroxy-4-chlorobenzamide 0.8-1.9% by weight, sodium dithionite 7-12% by weight and para-quinone 1.3-3.4% by weight in the presence of H- (3-dimethylaminopropyl) -2-amino-3-hydroxy - -4-methylbenzamide hydrochloride in a concentration of 0.27-0.51% by weight of the reaction mixture.