Method for separating and purifying deoxynucleoside triphosphate

A technology of deoxynucleoside triphosphate and deoxyguanosine triphosphate, applied in chemical instruments and methods, preparation of sugar derivatives, sugar derivatives, etc., can solve problems such as pollution, time-consuming and labor-intensive use, large discharge of liquid materials, etc. , to achieve the effect of reducing environmental pollution, reducing production costs, positive social and economic effects

Inactive Publication Date: 2010-01-27
EAST CHINA UNIV OF SCI & TECH
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AI Technical Summary

Problems solved by technology

High-purity dATP (content above 98%) is very important to the success of reactions such as cDNA synthesis, sequencing and labeling, but the acquisition of high-purity products usually requires high-pressure liquid chromatography, which is time-consuming and labor-intensive. large, heavily polluted

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Separation and purification of adenine triphosphate deoxyribonucleoside dATP:

[0019] Cool 50ml of the enzyme synthesis reaction solution containing about dATP100-mM with a pH value of 7.0 to 8.0 to 4 degrees (during the synthesis reaction, HPLC is regularly performed to measure dAMP, dATP, dADP and pyruvate), and transferred to a 1L three-necked flask. Two volumes of absolute ethanol (about 200 mL) was slowly added in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no dATP peak was displayed. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value below 2.0 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully we...

Embodiment 2

[0021] Separation and purification of guanine triphosphate deoxyribonucleoside dGTP:

[0022] Cool the 50mL enzyme synthesis reaction solution containing about 20-mM dGTP at a pH of 7.0 to 8.0 to 4 degrees (during the synthesis reaction, HPLC should be performed periodically to measure dGMP, dGTP, dGDP and pyruvate), and transferred to a 1L three-necked flask , slowly added isopropanol (200 mL) in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no dGTP peak was shown. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value to about 1.5 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully weighed and dissolved in water (~...

Embodiment 3

[0024] Separation and purification of cytosine triphosphate deoxyribonucleoside dCTP:

[0025] Cool 100mL enzyme synthesis reaction solution with a pH value of 7.0 to 8.0 containing about dCTP 10-mM to 4 degrees (during the synthesis reaction, HPLC should be performed periodically to measure dAMP, dATP, dADP and pyruvate), and transferred to a 1L three-necked flask , slowly added absolute ethanol (200 mL) in an ice bath, and the resulting white precipitate was collected by centrifugation. The composition of the supernatant was analyzed by HPLC, and no peak shape of dCTP was displayed. Dissolve the precipitate with ultrapure water into 30mL aqueous solution in an ice bath, adjust the pH value to about 0.5 with hydrochloric acid, and slowly pour it into a 500mL beaker containing absolute ethanol (100mL) in an ice bath, and the resulting white precipitate is slowly After stirring, it was collected by centrifugation. A small amount of white solid (~10 mg) was carefully weighed a...

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Abstract

The invention discloses a method for separating and purifying deoxynucleoside triphosphate, which comprises the following steps: when the temperature is below 4 DEG C, depositing deoxynucleoside triphosphate biosynthesized reaction solution directly with an organic solvent to obtain a solid mixture of the deoxynucleoside triphosphate, wherein the volume ratio of the deoxynucleoside triphosphate biosynthesized reaction solution to the organic solvent is between 1:1 and 1:25; when the temperature is below the 4 DEG C, dissolving the solid mixture in ultrapure water, adjusting the pH value to between 0.1 and 2 by using acidic aqueous solution, and using the organic solvent to perform deposition again to obtain a solid matter of the deoxynucleoside triphosphate; and dissolving the solid matter in the ultrapure water, adjusting the pH value to between 5 and 11 by using sodium hydroxide alkaline aqueous solution, and performing liquid nitrogen frozen and freeze-drying to obtain a sodium salt product of the deoxynucleoside triphosphate. The method has the advantages of quick separation, good purification effect, production cost reduction, water resource pollution reduction, process energy consumption reduction, and applicability to industrial production.

Description

【Technical field】 [0001] The invention relates to the field of biochemical industry, in particular to a method for separating and purifying widely used deoxynucleoside triphosphate (dNTP) enzymatically synthesized products. 【Background technique】 [0002] Usually, the biosynthesis reaction liquid of deoxynucleoside triphosphate (dNTP) is synthesized from deoxynucleoside monophosphate (dNMP), and the dNTP generated is separated in the form of barium salt by sedimentation operation, such as using barium bromide aqueous solution to make dNTP from Sedimentation separation separated from the reaction solution, and then use ion exchange resin to elute dNTP, making it a separation and purification method for repeated sedimentation and elution of dNTP aqueous solution, or directly elute the dNTP biosynthesis reaction solution with ion exchange resin packed column , and then use high-pressure liquid chromatography to separate and purify the method. [0003] The existing method for s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H19/20C07H19/10C07H1/06
Inventor 段梅莉辛秀娟鲍杰冀亚飞英智威李慧
Owner EAST CHINA UNIV OF SCI & TECH
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