Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Pyrene-marked single-chain DNA (deoxyribonucleic acid) fluorescent probe and preparation method thereof

A technology of labeling and pyrene, which is applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc. It can solve the problems of complex synthesis operation, and achieve the effect of not losing sensitivity, good reference value and high accuracy.

Inactive Publication Date: 2015-04-01
UNIV OF SCI & TECH BEIJING
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, all the labeled fluorescent probes described above connect the fluorescent group to the nucleic acid molecular chain through a covalent bond, and the synthesis operation is relatively complicated.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Pyrene-marked single-chain DNA (deoxyribonucleic acid) fluorescent probe and preparation method thereof
  • Pyrene-marked single-chain DNA (deoxyribonucleic acid) fluorescent probe and preparation method thereof
  • Pyrene-marked single-chain DNA (deoxyribonucleic acid) fluorescent probe and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044]Dissolve 6 mmol of pyrenemethanol in 150 mL of chloroform at freezing point temperature, add 3 mmol of phosphorus tribromide to the solution, stir in an ice bath for 12 hours, then add saturated aqueous sodium bicarbonate to neutrality, separate layers, and take the organic layer for Distillation, recrystallization, and drying give pyrene bromide. Dissolve 12mmol of poly-4-vinylpyridine in 10mL of chloroform, then add 1.2mmol of pyrenemethyl bromide, stir and reflux at 68°C for 24 hours, then slowly add the reaction solution dropwise into tetrahydrofuran to obtain a precipitate, filter, wash, and dry Then intermediate products are obtained. Dissolve 5 mmol of the intermediate product in 8 mL of ethanol, add 14 mol of n-bromobutane, stir and reflux at 85°C for 24 hours, then slowly add the reaction solution dropwise into tetrahydrofuran to obtain a precipitate, filter, wash, and dry to obtain the product cationic poly Electrolyte (P4VP-Py-Bu).

Embodiment 2

[0046] (1) Preparation of samples: Use PBS (10 mM, pH = 7.4) buffer to prepare each sample solution, as follows: image 3 1) P4VP-Py-Bu, 2) P4VP-Py-Bu / ssDNA1, 3) P4VP-Py-Bu / ssDNA2, 4) P4VP-Py-Bu / ssDNA1+ssDNA2, 5) P4VP-Py- Bu / ssDNA1+ssDNAt, 6) P4VP-Py-Bu / ssDNA2+ssDNAt, 3') P4VP-Py-Bu / ssDNA3, 4') P4VP-Py-Bu / ssDNA2+ssDNA3, 6') P4VP-Py-Bu / ssDNA3+ssDNAt total 9 samples, the final concentration of polyelectrolyte in each sample solution was 2.0×10 -7 M, each DNA concentration is 1.0×10 -6 M.

[0047] (2) Heat treatment sample: Put the sample prepared in (1) into a 90°C oven for heat treatment for 15 minutes, and then quickly move to a 40°C oven for heat treatment for 30 minutes.

[0048] (3) Fluorescence detection of the sample: under the condition of excitation wavelength of 344nm, pass image 3 It can be clearly seen from the graph and histogram of fluorescence intensity at 377nm that when DNA1 or DNA3 interacts with polyelectrolyte to form a fluorescent probe, the fluore...

Embodiment 3

[0050] (1) Preparation of samples: Use PBS (10 mM, pH = 7.4) buffer to prepare each sample solution, as follows: Figure 5 1) DNA1 / DNA2, 2) P4VP-Py-Bu + DNA1 / DNA2, 1’) DNA1 / DNA3, 2) P4VP-Py-Bu + DNA1 / DNA3 marked in a total of 4 samples. The final concentration of polyelectrolyte in each sample solution was 2.0 × 10 -5 M, each DNA concentration is 1.0×10 -4 M.

[0051] (2) Heat treatment sample: Put the sample prepared in (1) into a 90°C oven for heat treatment for 15 minutes, and then quickly move to a 40°C oven for heat treatment for 30 minutes.

[0052] (3) Circular dichroism test on the sample: The circular dichroism test is done to confirm that the pyrene molecule is indeed intercalated into the DNA double helix structure. Such as Figure 5 In the spectrum shown, the circular dichroism intensity after adding polyelectrolyte P4VP-Py-Bu all decreased significantly, and the positive band appeared blue shifted, and the negative band appeared red shifted. The only possibili...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for synthesizing a poly-4-vinyl pyridine cationic polyelectrolyte grafted with fluorophore pyrene and preparing a single-chain DNA (deoxyribonucleic acid) composite fluorescent probe therefrom. The synthesis process of the pyrene-grafted poly-4-vinyl pyridine cationic polyelectrolyte comprises the following steps: (1) preparing bromomethyl pyrene from pyrene methanol and phosphorus tribromide; (2) grafting the bromomethyl pyrene onto poly-4-vinyl pyridine according to a certain monomer mol ratio; and (3) performing quaternary ammonification on the product in the step (2) with n-bromobutane to obtain the water-soluble cationic polyelectrolyte. The polyelectrolyte and single-chain nucleic acid are subjected to mutual adsorption in a buffer solution under the electrostatic and hydrophobic effects so as to form the composite probe, and whether basic groups of target single-chain DNA and the DNA of the probe are complementary is detected according to the variation of fluorescence in experiments. The invention has the advantages of simple synthetic route, easy operation process and low cost; and the probe can quickly and sensitively perform specific recognition on DNA.

Description

technical field [0001] The invention relates to a method for synthesizing a water-soluble fluorescent polyelectrolyte and a method for using the polyelectrolyte to prepare a fluorescent probe for detecting oligonucleotide sequences, belonging to the fields of functional polymer technology, analytical chemistry and bioanalytical chemistry. technical background [0002] Functional polymers are polymers with specific functions formed by combining specific functional groups in their macromolecular chains, or compounding macromolecules with other materials with specific functions, or both. Materials, and its application in the field of bioanalytical chemistry, especially in the detection of biomacromolecules, has attracted close attention from scholars at home and abroad. The specific recognition of nucleic acid base sequences is a current international research hotspot, and the use of functional polymers with fluorescent properties as fluorescent probes for specific recognition ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10C08F126/06C08F8/44
Inventor 王国杰张瑞辰杨领叶赵敏董杰
Owner UNIV OF SCI & TECH BEIJING
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com