DNA pulldown method and kit

A technology of reagents and magnetic beads, applied in the biological field, can solve the problems of the influence of nuclease contamination and the low specificity of DNA probe and protein binding, and achieve the effects of preventing contamination, saving experimental costs and improving specificity.

Active Publication Date: 2016-11-09
GUANGZHOU BIOSENSE BIOSCI
View PDF4 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, there are still many shortcomings in DNA pulldown experiments, such as the low specificity of DNA probes binding to proteins, and the experimental process is also affected by nuclease contamination.

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
  • DNA pulldown method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] In this example, specific DNA probes were designed for the P53 protein in the target region, and the probes were labeled with desthiobiotin 8-oxoG. The design and labeling methods of probes are well known to those skilled in the art and will not be repeated here. Streptavidin coupled to magnetic beads (BeaverBeads TM Streptavidin, Beaver Biotechnology Co., Ltd.) and affinity binding with desthiobiotin; nuclei extracts were incubated with magnetic beads-DNA probes, and the protein molecules could specifically bind to the probes; after washing, the non-specific binding proteins could be Molecule removal; finally, elution is performed with an eluent (for streptavidin) to obtain the target probe-protein complex, and then the protein type is identified by Western Blot. Specific steps are as follows:

[0039] S1. Magnetic bead pretreatment

[0040] ①Put 80 μL of streptavidin-labeled magnetic beads into an EP tube, add 1 mL of 1×TES to wash the magnetic beads;

[0041] ② ...

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 DNA pulldown method and a kit. The method includes the following steps of pretreating magnetic beads, wherein the magnetic beads marked by BSA(bovine serum albumin) and oligonucleotide random primer closed streptavidin are used; preparing a probe-magnetic bead compound; extracting and pretreating cell nucleus protein, wherein deoxyribonuclease is added into an extracting nucleoprotein sample for incubation, the magnetic beads are added for incubation and then removed, and supernatant is collected; carrying out pulldown; collecting the protein sample. The DNA pulldown method has the following advantages that BSA and an oligonucleotide random primer are used for closing the magnetic beads in advance, and nonspecific binding between the magnetic beads, protein and genomic DNA is reduced. Nuclease contamination is prevented through systemic nuclease protection, stability and reliability of experiments are improved, repeatability is high, and the experiment process is simplified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA pulldown method and kit for studying the interaction between DNA and protein. Background technique [0002] With the rise of functional genomics, the study of protein-DNA interactions has become increasingly important. Protein-DNA interactions are involved in many biological functions, such as gene regulation, DNA repair and DNA recombination (ROHS R, JIN X, WEST SM, et al. 2010. Origins of specificity in protein-DNA recognition. Annu RevBiochem[J], 79:233-269). There are many technical solutions for studying DNA-protein interaction, such as yeast one-hybrid (Y1H) method, ChIP-seq method, gel shift assay (EMSA) and DNA pulldown method. [0003] DNA pulldown method is a very important method for the study of protein-DNA interaction. This method allows the isolation of DNA-protein complexes within the same sample. Usually, the DNA is labeled with a high-affinity tag (such as ...

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): G01N33/68
CPCG01N33/68
Inventor 王晓香董先辉张娟曾健周剑
Owner GUANGZHOU BIOSENSE BIOSCI
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
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap