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

Method capable of detecting activity of multiple DNA glycosylases

A technology of glycosylase and DNA probe, applied in the field of biological analysis, can solve the problems of unsuitable for sample detection, low sensitivity, unsuitable for quantitative analysis, etc.

Pending Publication Date: 2020-07-14
EPITAS BIOSCIENCES (SHANGHAI) CO LTD
View PDF5 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing traditional methods such as gel electrophoresis using fluorescently labeled substrates have low sensitivity, require a large amount of sample, and have low throughput, which is not suitable for quantitative analysis; replacing fluorescently labeled substrates with radioactive isotope-labeled substrates can improve sensitivity and reduce However, isotope labeling will cause radioactive contamination; high-performance liquid chromatography has strong qualitative and quantitative capabilities, and requires a small amount of sample, but the sample pretreatment is complicated and the throughput is not high; enzyme-linked immunosorbent assay and Western blotting requires specific antibodies that bind to proteins, and the operation steps are cumbersome, which is not suitable for the detection of a large number of samples; fluorescent methods rely on external labeling with fluorophores and quenchers for homogeneous assays, and are suitable for high-throughput, but Its signal window is relatively small, and substrate probe design is complex and expensive

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 capable of detecting activity of multiple DNA glycosylases
  • Method capable of detecting activity of multiple DNA glycosylases
  • Method capable of detecting activity of multiple DNA glycosylases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] Example 1: Pure UDG activity detection

[0167] First design a double-stranded DNA probe T1-T2 containing a uracil base, wherein the nucleotide sequence of the T1 strand is: 5'-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3' (SEQ ID NO: 1) The nucleotide sequence of the T2 chain is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), and the T1 and T2 chains are complementary to form a double-stranded DNA probe T1-T2.

[0168] Double-stranded DNA probes T1-T2 and UDG of different concentrations (0nM, 15nM, 30nM, 60nM, 120nM) were added to the reaction buffer, the composition of the reaction buffer was: 20mM Tris-Cl pH8.0, 1mM EDTA , 1mM DTT; the final volume is 100μL, the concentration of double-stranded DNA probes T1-T2 in the system is 30nM, the system is incubated at 25°C for 30 minutes to allow the base excision reaction to occur, and then 1μL of streptavidin magnetic beads Add the reaction solution to fully combine with the biotin-labeled DNA for 1 hour, add NaOH to the re...

Embodiment 2

[0170] Example 2: Pure TDG activity detection

[0171] First design a double-stranded DNA probe T1-T2 containing a uracil base, wherein the nucleotide sequence of the T1 strand is: 5'-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3' (SEQ ID NO: 1) The nucleotide sequence of the T2 chain is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), and the T1 and T2 chains are complementary to form a double-stranded DNA probe T1-T2.

[0172] Double-stranded DNA probes T1-T2 and different concentrations (0nM, 1.85nM, 5.56nM, 16.67nM, 50nM) of TDG were added to the reaction buffer, the composition of the reaction buffer was: 20mM HEPES pH7.5, 100mM NaCl, 0.2mM EDTA, 2.5mM MgCl 2 ; The final volume is 100 μL, the concentration of double-stranded DNA probes T1-T2 in the system is 30 nM, the system is incubated at 25°C for 30 minutes to allow the base excision reaction to occur, and then 1 μL streptavidin magnetic beads are added to the reaction solution Fully combine with biotin-labeled DNA for ...

Embodiment 3

[0174] Example 3: Pure SMUG1 activity detection

[0175] First design a double-stranded DNA probe T1-T2 containing a uracil base, wherein the nucleotide sequence of the T1 strand is: 5'-FAM-TAA UGT GAA TGG AGC TGA AAT-biotin-3' (SEQ ID NO: 1); the nucleotide sequence of the T2 chain is 5'-ATT TCA GCT CCA TTC ACG TTA-3' (SEQ ID NO: 2), and the T1 chain and the T2 chain are complementary to form a double-stranded DNA probe T1-T2;

[0176] Double-stranded DNA probes T1-T2 and SMUG1 at different concentrations (0nM, 12.5nM, 25nM, 50nM, 100nM) were added to the reaction buffer, the composition of the reaction buffer was: 10mM Tris-Cl pH7.0, 10mM MgCl 2 , 1mM DTT; the final volume is 100μL, the concentration of double-stranded DNA probes T1-T2 in the system is 30nM, the system is incubated at 25°C for 30 minutes to allow the base excision reaction to occur, and then 1μL of streptavidin magnetic beads Add the reaction solution to fully combine with the biotin-labeled DNA for 1 hour...

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 detection system for detecting DNA glycosylases. The detection system comprises (a) a double-stranded DNA probe, wherein the double-stranded DNA probe comprises two strandsT1 and T2; the two strands T1 and T2 can form a double-stranded DNA structure; the strand T1 comprises at least one base capable of being recognized by to-be-detected DNA glycosylases, a fluorophore and a separate label; and the fluorophore and the separate label are located at two ends of the at least one base capable of being recognized by the DNA glycosylases separately; (b) a component capableof breaking a glucoside-phosphate bond at a nucleic acid abasic site; and (c) a solid-phase carrier with a separate bound label. The detection system provided by the invention is high in flux, smallin required sample volume, large in signal window, simple and convenient to operate and low in cost, and can detect multiple DNA glycosylases.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a method capable of detecting the activities of various DNA glycosylases and an application thereof. Background technique [0002] BER (base excision repair) pathway is the main way to repair endogenous DNA base damage caused by oxidation, alkylation and deamination. Among them, DNA glycosylase is an important link in the BER pathway, including: UDG (uracil DNA glycosylase), TDG (thymine DNA glycosylase) and OOG1 (8-hydroxyguanine DNA glycosylase) )Wait. DNA glycosylase can specifically excise N-β-glycosidic bonds on damaged or mismatched nucleotides, forming abasic sites (AP sites) on the DNA chain. Then the AP endonuclease will cut the glycoside-phosphate bond of the damaged nucleotide, and remove the small fragment of DNA including the AP site nucleotide, and synthesize a new fragment by DNA polymerase I, and finally DNA ligase connects the two into ...

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 Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6883G01N33/574G01N33/573
CPCC12Q1/6886C12Q1/6883G01N33/57423G01N33/573C12Q2600/156G01N33/574C12Q1/34C12Q1/44G01N21/64
Inventor 傅新元闫娟毛卓郭剑南杨盛莲
Owner EPITAS BIOSCIENCES (SHANGHAI) CO LTD
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