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Light-click-reaction-based DNA marker with fluorescent responses, preparing method thereof and application thereof

A technology of light click reaction and DNA labeling

Active Publication Date: 2015-12-16
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method only realizes the modification of DNA, and there is no fluorescence response after the reaction, so it does not have various applications.

Method used

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  • Light-click-reaction-based DNA marker with fluorescent responses, preparing method thereof and application thereof
  • Light-click-reaction-based DNA marker with fluorescent responses, preparing method thereof and application thereof
  • Light-click-reaction-based DNA marker with fluorescent responses, preparing method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation of a DNA Marker Based on Optical Click Reaction and Fluorescent Response

[0068] (1) Dissolve tetrazole compound W-1 (362mg, 1.0mmol) in 6ml of tetrahydrofuran, then add 3ml of methanol to obtain a mixed solution; dissolve lithium hydroxide (840mg, 20.0mmol) in 3ml of water to obtain a lithium hydroxide solution Then drop the lithium hydroxide solution into the mixed solution, stir overnight (12h) at 50°C, and follow up the detection reaction by TLC; after the reaction is complete, adjust the pH value of the reaction solution with 1mol / L sodium bicarbonate and 1mol / L hydrochloric acid In the process of adjusting the pH value, a purple precipitate precipitated, and the pH value of the reaction solution was adjusted to 3.0 until the precipitate was completely precipitated; the precipitate was collected by filtration, and the precipitate was washed repeatedly with a large amount of water, and finally the precipitate was washed with a small amount of e...

Embodiment 2

[0076] Optical properties of embodiment 2 compound W-4

[0077] The tetrazole compound W-4 obtained in Example 1 is reacted with different olefins, acrylamide, isopropylacrylamide, dimethylaminopropylacrylamide (DMAPPA), 1-(2-hydroxyethyl)- Pyrrole-2,5-dione. The concentration of W-4 ​​is 1 μM, the concentration of the four middle olefin compounds is 40 μM, irradiate for 2 minutes under a 302nm ultraviolet lamp, measure the fluorescence of the four reaction products and calculate the fluorescence quantum yield of the reaction product, the results are shown in Table 1, Figure 4 It is the fluorescence spectrum of W-4 ​​after clicking with four different olefin compounds.

[0078] The fluorescence quantum yield of table 1 compound W-4

[0079]

Embodiment 3

[0081] Take a tube of 5' amino-labeled nucleic acid sequence and centrifuge at 12000rpm for 20min; gently open the cap of the centrifuge tube, quickly add 55 μL of 0.1M sodium borate buffer (PH8.0) and quickly cover the tube cap; shake fully on the vortex 10min; add 30μL of 50mM tetrazole compound W-3 ​​(prepared into a 50mM solution with dimethyl sulfoxide in advance); wrap the centrifuge tube with tinfoil, place in the centrifuge tube and incubate for 14 hours with shaking; add to the centrifuge tube 1ml pre-cooled absolute ethanol, shake up and down for 1min; then put it in an ultra-low temperature refrigerator for 2 hours; centrifuge, 12000rpm, 4°C, 20min; absorb the upper clear solution, add 1ml pre-cooled 1mL pre-cooled absolute ethanol , shake up and down for 1 min; then put it in an ultra-low temperature refrigerator and let it stand for 2 hours; centrifuge, 12000rpm, 4°C, 20min; absorb the upper clear solution, let stand for 30min, add 55μl sterile water, fully shake f...

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Abstract

The invention belongs to the field of nucleic acid marking, and particularly relates to a light-click-reaction-based DNA marker with fluorescent responses, a preparing method thereof and an application thereof. The DNA marker is at least one of a compound W-3 and a compound W-4. The structural formula of the W-3 and the structural formula of the W-4 are shown as the formula I and the formula II (please see the specification for the formula I and the formula II). The preparing method is simple and moderate, oxygen and water have no influences on reacting in the reacting process, operation is easy, popularization is easy, and the DNA marker, the preparing method and the application can be applied to the field of nucleic acid marking.

Description

technical field [0001] The invention belongs to the field of nucleic acid labeling, and in particular relates to a DNA marker based on a light click reaction and having a fluorescent response, a preparation method and an application. Background technique [0002] Nucleic acid is a kind of biomacromolecule polymerized by nucleotide monomers, and it is the most basic and important component of biological cells. At present, nucleic acid modification technology is a common research tool in the fields of medicine and biology, and has been widely used. The modification of nucleic acid molecules plays an important role in the basic research of molecular recognition, biomedical diagnosis and DNA nanostructure construction. [0003] The early traditional DNA modification method is to directly modify fluorescent molecules on DNA. This method requires high technology and equipment, complicated operation and complicated purification methods, which is not conducive to large-scale promoti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D405/14C07D405/04C12Q1/68
Inventor 邢达张涛吴云霞
Owner SOUTH CHINA NORMAL UNIVERSITY
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