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Economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method

A technology of hydroxyphosphoric acid and nucleic acid chain, applied in the field of biochemistry, can solve problems such as high price

Inactive Publication Date: 2021-09-07
PHARMARON BEIJING +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, 5’-hydroxyphosphorylated DNA oligonucleotides are expensive in the market, while unphosphorylated DNA oligonucleotide tags are relatively cheap

Method used

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  • Economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method
  • Economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method
  • Economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1, 20 nanomoles of DNA single strand 3 before phosphorylation, with a length of 17 bases, a base sequence of CAGTGATGAGTCGGTCG, and a relative molecular mass of 5266 before phosphorylation, dissolved in nuclease-free water, and added to 20 microliters 10-fold phosphorylation buffer components, 5 units of T4 polynucleotide kinase, added nuclease-free water to a reaction volume of 50 microliters, and reacted at 37°C for 12 hours to complete the reaction. Add 5 microliters of 5 mol / liter sodium chloride aqueous solution and 125 microliters of absolute ethanol to the reaction solution, shake and mix, place in a -80°C refrigerator for 10-30 minutes, and centrifuge at a high speed (4°C, 12,000 rpm) / min, 5 minutes), a precipitate was obtained. The precipitate was dissolved in 20 microliters of water, and quantitatively detected by DNA ultraviolet spectrophotometry, the amount of the phosphorylated product was measured to be 17.5 nmoles, and the recovery rate was 88%.

Embodiment 2

[0047] Example 2, 20 nanomoles of DNA single-strand 3 before phosphorylation, 17 bases in length, base sequence CAGTGATGAGTCGGTCG, relative molecular mass before phosphorylation is 5266, dissolved in nuclease-free water, and added to 20 microliters 10-fold phosphorylation buffer composition, 5 units of T4 polynucleotide kinase, 0.2 microliter of PEG, added nuclease-free water to a reaction volume of 50 microliters, and reacted at 37°C for 2.5 hours to complete the reaction. Add 5 microliters of 5 mol / liter sodium chloride aqueous solution and 125 microliters of absolute ethanol to the reaction solution, shake and mix, place in a -80°C refrigerator for 10-30 minutes, and centrifuge at a high speed (4°C, 12,000 rpm) / min, 5 minutes), a precipitate was obtained. The precipitate was dissolved in 20 microliters of water, and quantitatively detected by DNA ultraviolet spectrophotometry, the amount of the phosphorylated product was measured to be 17.4 nmoles, and the recovery rate wa...

Embodiment 3

[0048] Example 3, 20 nanomoles of DNA single strand 3 before phosphorylation, with a length of 17 bases, a base sequence of CAGTGATGAGTCGGTCG, and a relative molecular mass of 5266 before phosphorylation, dissolved in nuclease-free water, and added to 20 microliters 10-fold phosphorylation buffer components, 5 units of T4 polynucleotide kinase, 0.5 microliter PEG, added nuclease-free water to make the reaction system 50 microliters, and reacted at 37°C for 2 hours to complete the reaction. Add 5 microliters of 5 mol / liter sodium chloride aqueous solution and 125 microliters of absolute ethanol to the reaction solution, shake and mix, place in a -80°C refrigerator for 10-30 minutes, and centrifuge at a high speed (4°C, 12,000 rpm) / min, 5 minutes), a precipitate was obtained. The precipitate was dissolved in 20 microliters of water, and quantitatively detected by DNA ultraviolet spectrophotometry, the amount of the phosphorylated product was measured to be 17.2 nmoles, and the ...

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Abstract

The invention discloses an economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method. The method comprises the following steps of taking purchased non-modified DNA (deoxyribonucleic acid) short fragments as raw materials, taking the 5'-end of the short fragments as hydroxyl, taking 10 times of a commercially purchased phosphorylation buffer solution component as a phosphoric acid source, and phosphorylating the 5'-hydroxyl of DNA under the action of T4 polynucleotide kinase. The method provided by the invention is wide in application range, mild in reaction condition, simple in post-treatment, high in recovery rate, low in cost and wide in application prospect.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to an economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method. Background technique [0002] The development of any drug is a lengthy and costly process. According to statistics on drug research and development, it takes an average of 6 to 10 years for a new drug to be put on the market from the beginning of research and development to its final approval, and the research cost is even as high as 1 billion to 3 billion US dollars. One of the important reasons why the drug development process is so long and expensive is that the discovery and optimization process of lead compounds is slow [i] . In order to solve this problem, DNA-encoded library technology (DELT for short) came into being. This concept was first proposed by Sydney Brenner (the 2002 Nobel Prize winner in Physiology and Medicine) and Richard Lerner (then director of the Scripps Research ...

Claims

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Application Information

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IPC IPC(8): C12P19/34
CPCC12P19/34
Inventor 薛丽俊张洁杨珂新胡允金
Owner PHARMARON BEIJING
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