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A kind of dna pulldown method and kit

A magnetic bead and pretreatment technology, applied in the biological field, can solve the problems of low binding specificity between DNA probes and proteins and the influence of nuclease contamination, so as to save experimental costs, prevent contamination, and improve specificity.

Active Publication Date: 2018-03-20
GUANGZHOU BIOSENSE BIOSCI
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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.

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  • A kind of dna pulldown method and kit

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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 ...

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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

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Application Information

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Patent Type & Authority Patents(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
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