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High-sensitivity high-flux DNA binding protein detection method

A protein-binding and detection method technology, applied in the field of DNA-binding protein detection, can solve the problems of difficult sample detection, insufficient detection sensitivity, and increased detection cost, and achieve low cost, high sensitivity, and reduced test cost.

Inactive Publication Date: 2011-11-16
XUZHOU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the previous reported methods, on the one hand, different fluorescent probes were used to detect different proteins, which led to an increase in detection costs;

Method used

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Embodiment 1

[0017] Realize the detection of NF-kappaB p50, NF-kappaB p52 and OCT-1 proteins in the crude extracts of tumor cell cultures from the same cancer pain sensitive sample: will be used to detect NF-kappaB p50, NF-kappaBp52 and OCT-1 proteins, respectively The double-stranded oligonucleotides with acrylamide modification were immobilized on a polyacrylamide gel chip (the wild-type sequence and known NF-kappaB p50 samples were used as positive controls, and the mutant sequences and known NF-kappaB p50 samples were used as positive controls. As a negative control), the tumor cell culture crude extract was incubated with the chip; then the double-stranded oligonucleotides on the chip were digested with exonuclease III. After the gel chip was subjected to low-frequency ultrasonic decontamination, the circular detection probe was hybridized with the chip, and isothermal rolling circle amplification was performed for 30 minutes. Finally, the amplification chip is hybridized with the uni...

Embodiment 2

[0019] Realize the detection of NF-kappaB p50, NF-kappaB p52 and OCT-1 proteins in the crude extracts of tumor cell cultures from the same cancer pain sensitive sample: will be used to detect NF-kappaB p50, NF-kappaBp52 and OCT-1 proteins, respectively The amino-modified oligonucleotide duplex was immobilized on the aldehyde group-modified chip (the wild-type sequence and known NF-kappaB p50 samples were used as positive controls, and the mutant sequence and known NF-kappaB p50 samples were used as negative controls. Control), the tumor cell culture crude extract was incubated with the chip; then the double-stranded oligonucleotides on the chip were digested with exonuclease III. After the gel chip was subjected to low-frequency ultrasonic decontamination, the circular detection probe was hybridized with the chip, and isothermal rolling circle amplification was performed for 30 minutes. Finally, the amplification chip is hybridized with the universal fluorescent detection prob...

Embodiment 3

[0021] Realize the detection of NF-kappaB p50, NF-kappaB p52 and OCT-1 proteins in the crude extracts of tumor cell cultures from the same cancer pain sensitive sample: will be used to detect NF-kappaB p50, NF-kappaBp52 and OCT-1 proteins, respectively The biotin-modified oligonucleotide duplexes were immobilized on streptavidin-modified magnetic microspheres or organic microspheres (the wild-type sequence and the sample of known NF-kappaB p50 were used as positive controls, and the mutant type sequence and known NF-kappaB p50 sample as a negative control), the crude extract of tumor cell culture was incubated with the chip; then the double-stranded oligonucleotides on the microspheres were digested with exonuclease III. After the microspheres were subjected to low-frequency ultrasonic decontamination, the circular detection probe was hybridized with the chip, and isothermal rolling circle amplification was performed for 30 minutes. Finally, the amplified microspheres are hybr...

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Abstract

The invention belongs to microarray chip based DNA binding protein detection technology, and in particular relates to high-sensitivity high-flux DNA binding protein detection technology by a microarray chip. Double-stranded or single-stranded oligonucleotides are fixed on the chip, and a cell crude extract to be screened and the chip are directly incubated; then exonuclease is put into the systemfor incubation; and finally, a circular probe is added into the system for rolling circle amplification. Specific lattice sequences of the chip capable of combining a target protein can be taken as an extension primer for the rolling circle amplification; and sequences not combined with the protein are digested by the exonuclease, and cannot perform the rolling circle amplification, and finally amplified products and universal fluorescent probes are hybridized. The existence of the DNA binding protein can be detected according to the existence of specific array dot signals on the chip; and the intensity of the lattice fluorescence can quantitatively detect the content of the target protein. The chip can realize the detection of the DNA binding protein with super sensitivity, low cost, high specificity and high flux, and can be widely applied to scientific research and clinical detection and diagnosis.

Description

technical field [0001] The invention belongs to a DNA binding protein detection technology based on a microarray chip, in particular to a DNA binding protein detection method with high sensitivity and high throughput. Background technique [0002] For various life activities, the specific binding of proteins to DNA is a key step for many cells to express various biological activities, including gene transcription regulation, translation, and DNA replication, recombination and repair. Therefore, the detection of DNA-binding proteins and the identification of specific binding sites are very important for exploring gene expression mechanisms and analyzing cellular functions. In turn, probing this information can help to pinpoint the causes of developmental aberrations in biological organisms. Recently, DNA-binding proteins and binding site information have become potential tags and targets for many disease diagnosis and drug discovery. Early diagnosis of related transcription...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 潘志强张励才邵翠杰张成标
Owner XUZHOU MEDICAL COLLEGE