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Fluorochrome for marking oligonucleotide and protein, method for preparing same and use

A fluorescent dye and protein labeling technology, applied in the field of oligonucleotide and protein labeled fluorescent dyes and their preparation, can solve the problems of ineffective application, affecting the effect of electrophoretic separation, difference in energy absorption and emission capabilities, etc.

Active Publication Date: 2008-11-19
AGCU SCIENTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are many technical difficulties in achieving effective multiplex real-time quantitative fluorescence detection, especially when a single laser source is required, such as in electrophoretic analysis or automated gene sequencing systems for separating nucleic acids
First of all, it is difficult to find a group of more than five fluorescent dyes that are well resolved in the 200nm spectral band, because generally the half-beam width of a single fluorescence is 40-80nm; second, even if such a group is found that is well resolved in the spectral band Fluorescent dyes are often not effectively applied due to the difference in the energy absorption a

Method used

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  • Fluorochrome for marking oligonucleotide and protein, method for preparing same and use
  • Fluorochrome for marking oligonucleotide and protein, method for preparing same and use
  • Fluorochrome for marking oligonucleotide and protein, method for preparing same and use

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preparation example Construction

[0025] The preparation reaction principle of fluorescent dye of the present invention is as follows:

[0026] Synthesis principle of infrared wavelength fluorescent dyes synthesized by energy conversion method

[0027]

[0028] Infrared wavelength fluorescent molecular synthesis mechanism:

[0029]

[0030] The long-wavelength fluorescent dye DY-630 succinate and aminated short-wavelength fluorescein are dissolved in dimethylformamide, and under the catalysis of triethylamine, they are connected to form a new one that can efficiently absorb energy at a wavelength of 480-520nm And release fluorescent molecules with strong fluorescence at 650nm wavelength

Embodiment 1

[0032] Aminated short-wavelength fluorescein with a wavelength of 460-520nm, which is a single isomer, and 0.1 mmole of long-wavelength fluorescent dye DY-630 succinate were dissolved in 100 μl of dimethylformamide at a molar ratio of 1:1. 10 μl of triethylamine was added and under its catalysis, the reaction was carried out at room temperature. After 1 hour of reaction, 200 μl of weakly alkaline sodium carbonate solution with pH 7-10 was added and mixed. The reaction was further carried out at 60° C. for 1 hour. After the reaction finishes, remove the precipitate by high-speed centrifugation, after the supernatant is concentrated under reduced pressure (heatable, but should be lower than 60°C), dissolve the methanol in 10ml 5% solution in dichloromethane, and use 1000ml5-30% ( The concentration gradient solution of methanol in dichloromethane (volume ratio concentration) is separated by silica gel column chromatography to remove reaction by-products and unreacted compounds, c...

Embodiment 2

[0034] Aminated short-wavelength fluorescein, which is a mixture of isomers, and 0.1 mmole of long-wavelength fluorescent dye DY-630 succinate were dissolved in 200 μl of dimethylformamide at a molar ratio of 20:1. Add 20 μl of triethylamine under its catalysis, react at below 60° C., after 12 hours of reaction, add 500 μl of pH 7-10 weak alkaline sodium carbonate aqueous solution and mix. Then react at 60°C for 24 hours. After the reaction finishes, high-speed centrifugation removes the precipitate, and the supernatant is concentrated under reduced pressure (heatable, but should be lower than 60°C), and then dissolved in 10ml of 5% methanol in dichloromethane, and mixed with 1000ml of 5-30% The concentration gradient solution of methanol in dichloromethane (volume specific concentration) is separated by silica gel column chromatography, removes reaction by-products and unreacted compounds, collects the last colored fluorescent component, and concentrates under reduced pressur...

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Abstract

The invention discloses a flourescent dye used for labeling oligonucleotide and protein and a method for preparing the same as well as an application. The method mainly comprises the following steps that: succinate in the flourescent dye with amination and a short absorption wavelength and succinate in the activated flourescent dye with a long absorption wavelength and an emission wavelength react according to the mol ratio of 1-100 to 1 to produce reaction products; or succinate in the flourescent dye with amination and long absorption wavelength and succinate in the activated flourescent dye with short longest absorption wavelength and emission wavelength react according to the mol ratio of 1-100 to 1 to produce the reaction products; the reaction side products and a compound which does not react in the reaction products are removed by methods of dialysis, column flow, hyperfiltration or high pressure liquid phase and the novel and effective flourescent dye which can absorb laser energy at a shorter wavelength and emit strong fluorescence at a longer wavelength. The flourescent dye which is adopted can fulfill the aims of detecting and analyzing the fluorescence in real time, quantificationally and multiplexly, thereby greatly improving the specificity, reliability, uniformity and sensitivity of the detection.

Description

technical field [0001] The invention relates to a fluorescent dye and its preparation method and application, in particular to a fluorescent dye for oligonucleotide and protein labeling, its preparation method and application. Background technique [0002] As a tool for modern biological and clinical detection, non-radioactive fluorescent labeling methods, especially new fluorescent reagents, are widely used in life sciences, biological It plays an increasingly important role in the application of technology and clinical medicine, and gradually replaces traditional technologies such as isotope-labeled radiochemical reagents and becomes an indispensable and effective tool. At present, it has been widely used in gene automatic sequencing, multiple real-time quantitative fluorescent detection PCR (DNA polymerase chain reaction), nucleic acid hybridization and molecular immunology analysis. [0003] The simultaneous detection of one or more analytic targets using resolvable mul...

Claims

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Application Information

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IPC IPC(8): C09K11/06G01N21/64C12Q1/68
Inventor 郑卫国
Owner AGCU SCIENTECH
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