Novel technology for full cellular biosynthetic deoxyribonucleoside triphosphate

A technology of deoxynucleoside triphosphate and deoxynucleoside monophosphate, which is applied in the field of synthesizing deoxynucleoside triphosphate dNTP, can solve the problems of complex preparation, high cost, and large pollution, and achieve reduced environmental pollution, low cost, and small pollution Effect

Inactive Publication Date: 2010-07-07
NANJING UNIV OF TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a simple and efficient preparation method of deoxynucleo

Method used

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  • Novel technology for full cellular biosynthetic deoxyribonucleoside triphosphate
  • Novel technology for full cellular biosynthetic deoxyribonucleoside triphosphate
  • Novel technology for full cellular biosynthetic deoxyribonucleoside triphosphate

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0034] Example 1:

[0035] Yeast culture medium (g / L): glucose 40, urea 2.0, potassium dihydrogen phosphate 1.5, magnesium sulfate heptahydrate 0.5, zinc sulfate heptahydrate 4.0×10 -3 , Ferrous sulfate heptahydrate 3.0×10 -3 , Manganese chloride tetrahydrate 0.3×10 -3 , Anhydrous calcium chloride 1.0×10 -3 , Biotin 0.05×10 -3 . The inoculation amount of Saccharomyces cerevisiae was 10%, cultured on a shaker at 120 rpm at 30°C for 24 hours, and centrifuged at 4000 rpm for 20 minutes. Take the yeast paste and store at -7°C for later use.

[0036] The yeasts used in the following examples are all cultured in the above-mentioned culture method.

Example Embodiment

[0037] Example 2: Preparation of dATP using dAMP.

[0038] Prepared in a beaker with a capacity of 500mL from dAMP 1.49mmol, glucose 0.072mol, baker's yeast puree 100g, magnesium sulfate 14.96mmol, sodium dihydrogen phosphate 0.036mol, glycerol 3mL, cetyltrimethylamine and ammonium bromide 0.3 300 mL of the reaction solution consisting of g and water, adjusted to pH 7.0 with sodium hydroxide, stirred at 30°C for 6 hours at low speed, after the reaction, precipitated with perchloric acid, quantitatively analyzed dATP by HPLC, the conversion solution Containing 2.59g / L of dATP, the yield of dATP reaches 93.6% (in mol).

Example Embodiment

[0039] Example 3: Preparation of dATP using dAMP.

[0040] In a beaker with a capacity of 500 mL, prepare 300 mL of a reaction solution consisting of 1.66 mmol of dAMP, 0.08 mol of glucose, 110 g of air-dried baker’s yeast paste, 8.05 mmol of potassium chloride, 0.036 mol of sodium dihydrogen phosphate, 3 mL of acetaldehyde and water. Adjust the pH to 7.0 with sodium hydroxide, and stir the reaction at low speed at 30℃ for 6 hours. After the reaction, precipitate with perchloric acid and quantitatively analyze dATP by HPLC. The conversion solution contains 2.85g / L of dATP. The rate reached 92.4% (by mol).

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Abstract

The invention discloses novel technology for full cellular biosynthetic deoxyribonucleoside triphosphate, which takes deoxyribonucleoside monophosphate dNMP and phosphate anions as a substrate and saccharide as a power supplier, and uses permeable yeast cells for reaction in the presence of chemical effect substances to prepare the deoxyribonucleoside triphosphate. According to the technology, the theory of full cellular catalysis and metabolic engineering is used and the chemical effect substances are adopted to regulate and control a metabolic flow, so that a high effective power self-coupling and regeneration system is established, and high effective preparation of the deoxyribonucleoside triphosphate is finally realized. The novel technology for the full cellular biosynthetic deoxyribonucleoside triphosphate overcomes the disadvantages of the conventional production process of the deoxyribonucleoside triphosphate, and has the characteristics of simple process, high conversion, low cost and light pollution, and the like.

Description

technical field [0001] The invention relates to a method for synthesizing deoxynucleoside triphosphate dNTP from deoxynucleoside monophosphate dNMP. Background technique [0002] Deoxynucleoside triphosphate is an essential precursor material for artificially synthesized DNA, and artificially synthesized DNA fragments are widely used in genetic engineering, molecular biology, life sciences, and genetic medicine. Deoxynucleoside triphosphate is the substrate of various DNA polymerase-catalyzed reactions, and is an indispensable low molecular weight for DNA sequence analysis, gene analysis, site-directed mutagenesis, PCR (Polymerase Chain Reaction), RT-PCR, reverse transcription and DNA labeling reactions bioorganic molecules. DNA sequence analysis, site-directed mutagenesis, PCR and DNA chip technology are indispensable technologies for modern molecular biology, biochemistry and modern biomedical research. In particular, PCR technology has the characteristics of simple oper...

Claims

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Application Information

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IPC IPC(8): C12P19/40C12R1/645C12R1/85C12R1/84C12R1/78
Inventor 应汉杰姚月兰熊健陈勇柏建新苑巍张磊
Owner NANJING UNIV OF TECH
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