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Chip for non-label detecting DNA bindin, preparation and use method thereof

A protein-binding and labeling detection technology, which is applied in the direction of biological testing, material inspection products, etc., can solve the problems of low-abundance protein loss, improve detection efficiency and sensitivity, disadvantages, etc., achieve comprehensive biological significance, save detection costs, and avoid pollution effect

Inactive Publication Date: 2003-08-13
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods must perform fluorescent molecular labeling on proteins before detection, which may lead to the loss of low-abundance proteins during the labeling process, which is not conducive to improving detection efficiency and sensitivity

Method used

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  • Chip for non-label detecting DNA bindin, preparation and use method thereof
  • Chip for non-label detecting DNA bindin, preparation and use method thereof
  • Chip for non-label detecting DNA bindin, preparation and use method thereof

Examples

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Embodiment 1

[0025] Refer to attached figure 1 , 2 3. A chip for non-labeled detection of DNA-binding proteins, the structure of which has a double-stranded oligonucleotide probe, and the sequence of the double-stranded oligonucleotide probe is specific to the DNA recognition sequence of the detected DNA-binding protein. Designed to be divided into two half-site fragments I and II with cohesive ends between the recognition site containing the DNA-binding protein to be tested, i.e. probe 1 and probe 2 (such as figure 1 ), one of the double-stranded oligonucleotide fragments, probe 1, is immobilized on the substrate, and the other double-stranded oligonucleotide fragment, probe 2, introduces a label (-FAM) (such as figure 2 ). Or probe 2 does not label any fluorescent groups, but it is designed as a protruding sticky end away from the 5' end of the half site, and the base sequence of the protruding end is 3'-ANNA-5' (wherein N represents any base )(Such as image 3 ). Example 1 Prepara...

Embodiment 2

[0038] Example 2 Preparation and application method of a chip for non-labeled detection of DNA-binding proteins

[0039]1. Preparation of probes

[0040] The design and preparation of the probe are basically the same as in Example 1, except that the probe 2 does not label any fluorescent groups, but its distal end is designed as a protruding sticky end, and the base sequence of the protruding end is 3'-ANNA- 5' (where N represents any base) (such as image 3 ).

[0041] 2. The preparation and hybridization of the microarray are the same as in Example 1.

[0042] 3. Incorporation of fluorescent molecules

[0043] After hybridization, the chip was exposed to DNA polymerase 1 large fragment (Klenow enzyme) extension system at 37°C for 1 hour, and fluorescently labeled deoxyuridine adenosine triphosphate (such as Cy3-dUTP) was introduced. Wash free Cy3-dUTP (such as Figure 5 ).

[0044] 4. Fluorescent signal detection and result analysis

[0045] Also utilize the incorpora...

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Abstract

A chip for the non-label detection of DNA bindin has the dual-chain oligonucleotide probes designed according to the specific DNA recognizing sequence for the DNA bindin to be detected. The probe A is fixed to the substrate, and a label is introduced to the probe B. The probe B and the solution of the DNA bindin are inculated together and then hybridized with chip. If DNA bindin exists, the probes A and B have very high binding efficiency and the label of probe B can be detected at the position of probe A.

Description

technical field [0001] The invention relates to a chip for non-marker detection of DNA binding protein and its preparation and application methods. The DNA-binding protein here mainly refers to regulatory factors with specific DNA-binding sequences. The chip can simultaneously detect multiple DNA-binding proteins, and has the advantages of high throughput, high sensitivity, direct view, simple preparation, low cost, and no need to label proteins. Background technique [0002] The sequence-specific recognition and interaction of DNA and proteins play an important role in the regulation of biological gene expression, and the occurrence and development of almost all diseases are related to the abnormality of DNA-binding proteins. The study of DNA-binding proteins has attracted more and more attention, and has become one of the important contents of genome and proteome research. It is very important to detect the amount of one or more proteins in cells or diseased tissues. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/68
Inventor 白云飞葛芹玉陆祖宏李同祥王进科
Owner SOUTHEAST UNIV
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