Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for detecting DNA hybridization by surface cationized R-phycoerythrin

A phycoerythrin and cationization technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, biological testing, etc., can solve the problems that DNA hybridization is not involved, and achieve short preparation time, high detection sensitivity, and detection limit low effect

Active Publication Date: 2018-05-15
SHANGHAI OCEAN UNIV
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] To date, techniques using fluorescent proteins to detect DNA hybridization have not been

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
  • Method for detecting DNA hybridization by surface cationized R-phycoerythrin
  • Method for detecting DNA hybridization by surface cationized R-phycoerythrin
  • Method for detecting DNA hybridization by surface cationized R-phycoerythrin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment one: the preparation of various solutions

[0035] (1) Preparation of 50mM PBS buffer

[0036] 29.0g Na 2 HPO 4 -12H 2 O, 2.9g NaH 2 PO 4 -2H 2 O, 5.85g NaCl was dissolved in 900ml of ultra-pure water, fixed to a 1000ml volumetric flask, and then diluted 2 times, the concentration at this time was 50mM.

[0037] (2) Preparation of phycoerythrin solution

[0038] Pipette 100 μL of phycoerythrin crystals, centrifuge at 3800 rcf at 4 °C for 15 min, discard the supernatant, and dissolve the precipitated part with 500 μL of 50 mM sodium phosphate buffer (pH7.5) for 30 min; select an ultrafiltration tube with a filter membrane of 100 kD; Filter for 15 minutes for desalting, repeat ultrafiltration for 4 times, and then dilute the concentration to 1 mg / mL with PBS buffer (pH=7.5).

[0039] (3) Preparation of Cationic Polymer PDADMAC Solution

[0040] The PDADMAC (weight<100000, 35 wt.% in H2O) solution was diluted 10 times with PBS buffer, and the concentrat...

Embodiment 2

[0045] Embodiment two: the method for DNA hybridization detection

[0046] (1) After mixing R-PE and PDADMAC solution with a volume ratio of 1:10, add 5 μL of single-stranded DNA solution, then dilute to 100 μL with PBS buffer, and measure its fluorescence intensity at 575 nm two minutes later, At this time, it can be observed that the fluorescence intensity is quenched by about 88%.

[0047] (2) Mix 5 μL of single-stranded DNA solution with different concentrations of complete complementary strand solutions evenly, then add 1 μL of R-PE and 10 μL of PDADMAC solution, react for 2 minutes and dilute it to 100 μL, and measure it after two minutes The fluorescence intensity at 575nm can be observed to gradually release at this time, and finally it is basically the same as the fluorescence intensity of the protein itself.

[0048] (3) Mix 5 μL of single-stranded DNA solution with 5 μL of single-base mismatched complementary strand solution evenly, then add 1 μL of R-PE and 10 μL ...

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 belongs to the technical field of nucleic acid detection and discloses a method for detecting DNA hybridization by surface cationized R-phycoerythrin. The method includes steps: subjecting R-phycoerythrin to centrifugal ultrafiltration, desalination and dilution; adding a cationic polymer PDADMAC (polydimethyldiallyl ammonium chloride) for realizing protein surface positive charges;adding quenching group BHQ2 modified DNA single chains, wherein the fluorescence quenching rate is up to 88% approximately; adding complete complementary chains in different concentrations to enable continuous fluorescence release. The method has advantages that the natural vegetable protein R-PE and BHQ2 modified nucleic acid chains serve as donors and receptors in fluorescence resonance energy transfer, and FRET efficiency can be improved by adding of the cationic polymer; fluorescence is quenched in a free state and recovers as long as combining with target chains, high sensitivity is realized, and accordingly DNA hybridization detection is realized.

Description

Technical field: [0001] The invention belongs to the field of nucleic acid detection, in particular to a method for detecting DNA hybridization using surface cationized R-phycoerythrin. Background technique: [0002] R-phycoerythrin (R-phycoerythrin) is isolated and purified from seaweed. It is a new type of fluorescent labeling material commonly used at present. Its fluorescence intensity is 30-100 times that of fluorescein. It has good light absorption performance and very high quantum yield. The use of phycoerythrin for fluorescence analysis has incomparable advantages over traditional chemical fluorescent dyes: 1. It has a wider absorption spectrum in a wider pH range, and it is easier to choose a suitable excitation wavelength to obtain efficient fluorescence emission. And there is a specific fluorescence emission peak when excited; 2. The absorbance and fluorescence quantum yield are high, the fluorescence is strong and stable, and the sensitivity is high; 3. It has a...

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
IPC IPC(8): G01N21/64G01N33/53C12Q1/6816
CPCC12Q1/6816G01N21/64G01N21/6428G01N33/5308G01N2021/6417G01N2021/6432G01N2021/6441
Inventor 吴继魁陆云飞任宁娜张俊玲
Owner SHANGHAI OCEAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap 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