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Method for template-free synthesis of non-natural base-containing oligonucleotide chain by using terminal deoxyribonucleotide transferase and application of non-natural base-containing oligonucleotide chain

A deoxyribonucleotide and base oligonucleotide technology, applied in biochemical equipment and methods, genetic engineering, DNA preparation, etc., can solve the problem of unresearched oligonucleotide chains containing non-natural bases, etc. problem, to achieve the effect of high coupling efficiency, low cost and convenient operation

Pending Publication Date: 2022-03-18
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regarding all the above-mentioned in vivo and in vitro applications, it is necessary to synthesize oligonucleotide chains containing unnatural bases first. Currently, the synthesis of oligonucleotide chains containing unnatural bases faces the same challenges as the synthesis of natural oligonucleotide chains. However, it has not been studied whether TdT enzyme can be used to synthesize oligonucleotide chains containing unnatural bases

Method used

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  • Method for template-free synthesis of non-natural base-containing oligonucleotide chain by using terminal deoxyribonucleotide transferase and application of non-natural base-containing oligonucleotide chain
  • Method for template-free synthesis of non-natural base-containing oligonucleotide chain by using terminal deoxyribonucleotide transferase and application of non-natural base-containing oligonucleotide chain
  • Method for template-free synthesis of non-natural base-containing oligonucleotide chain by using terminal deoxyribonucleotide transferase and application of non-natural base-containing oligonucleotide chain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Preparation of oligonucleotide chains with unnatural base as dATP

[0053] (1) Mix 500nM priming chain single-chain FAM-T36-G, 1×TdT reaction buffer, the pH of TdT reaction buffer is 7.4, and 10mM magnesium acetate solution to obtain mixed solution 1;

[0054] (2) Add 50 μM dATP to the mixed solution 1 in step (1) to obtain the mixed solution 2;

[0055] (3) Add 1U / μL enzyme TdT to the mixed solution 2 described in step (2) to obtain the mixed solution 3;

[0056] (4) Incubate the mixed solution 3 in step (3) at 37° C. for 15 minutes, then heat-treat at 75° C. for 20 minutes to inactivate the enzyme TdT, and obtain the mixed solution 4;

[0057] (5) Add 2×TBE-Urea loading buffer to the mixed solution 4 in step (4), heat treatment at 95°C for 10 minutes to completely denature the oligonucleotide chain; analyze the enzyme TdT by denaturing gel The effect of adding an unnatural base to the 3' end of an oligonucleotide strand.

Embodiment 2

[0059] To prepare an oligonucleotide chain whose unnatural base is dTTP, the steps are the same as in Example 1, wherein dTTP is added in step (2), and the incubation time in step (3) is 30 minutes.

Embodiment 3

[0061] To prepare an oligonucleotide chain whose unnatural base is dGTP, the steps are the same as in Example 1, wherein dGTP is added in step (2).

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Abstract

The invention relates to a method for template-free synthesis of a non-natural base-containing oligonucleotide chain by using terminal deoxyribonucleotide transferase and application of the non-natural base-containing oligonucleotide chain. The method comprises the following steps: mixing initiation chain single-stranded DNA, a TdT reaction buffer solution and a magnesium acetate solution to obtain a mixed solution 1; adding one of natural deoxyribonucleotide, dTPT3TP, a dTPT3TP derivative or dNaMTP into the mixed solution 1 to obtain a mixed solution 2; adding TdT into the mixed solution 2 to obtain a mixed solution 3; incubating the mixed solution 3, and carrying out heat treatment to inactivate TdT to obtain a mixed solution 4; adding a loading buffer solution into the mixed solution 4, heating to completely denature the oligonucleotide chain, and analyzing the effect of adding the non-natural basic group to the 3'terminal of the oligonucleotide chain by TdT through denatured gel. The method is high in coupling efficiency, low in cost, convenient to operate, few in by-product and free of pollution waste. The oligonucleotide chain has potential application value in DNA end labeling and six-base information storage.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a template-free method for synthesizing oligonucleotide chains containing unnatural bases by terminal deoxyribonucleotide transferase and its application. Background technique [0002] The synthesis of oligonucleotide chains is one of the important basic technologies of molecular biology, which is crucial to modern biological research and has very important applications in the fields of genetic engineering, clinical diagnosis and treatment, such as polymerase chain amplification (PCR), genome synthesis, sequencing, aptamer enriched by exponential enrichment ligand system evolution technology (SELEX) nucleic acid aptamer library construction provides DNA fragments, etc., and is expected to realize new "digital biology" applications, such as DNA-based data storage and computing. [0003] At present, the synthesis of oligonucleotide chains mainly uses the solid-phase phosphoramidite me...

Claims

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Application Information

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IPC IPC(8): C12P19/34C12N15/115C12N15/10
CPCC12P19/34C12N15/115C12N15/111C12N2310/16C12N2330/30
Inventor 陈庭坚王光远何传平刘家韵
Owner SOUTH CHINA UNIV OF TECH
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