Probe for detecting nucleic acid in living cells and application method thereof

A detection method and technology for living cells, applied in the nanotechnology and biological fields, can solve the problems of not suitable for in-situ detection of living cell samples, cumbersome operations, not fully applicable to living cell samples, etc., and achieve real-time in-situ detection and enhanced detection. Sensitivity, the effect of broad application prospects

Inactive Publication Date: 2011-10-19
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the Mirkin group has successfully performed real-time imaging of mRNA in living cells by using gold nanoparticle probes modified with linear oligonucleotides. However, after the probe is hybridized with the target mRNA, the fluorescent dye is released from the probe, and the fluorescent signal Cannot reflect the position of the target mRNA, and thus cannot spatially locate the target mRNA
[0005] The probes provided by existing patents CN1548551, CN101603077 and CN101812544 and the method for detecting nucleic acid have certain improvement than traditional methods, but their probes and methods are mainly suitable for detecting nucleic acids in solution and are not fully applicable to living Cell samples, especially the patent CN101603077 are mainly suitable for PCR detection technology
Although the patent CN101384730 provides probes and methods for detecting cells or tissue samples in situ, it needs to use organic reagents such as formaldehyde to fix the cells and use Triton reagents to assist, and the operation is cumbersome
The patent CN101245387 provides a probe and method for detecting nucleic acid with high sensitivity, but it is also limited to detection in solution, not suitable for in-situ detection of living cell samples, and needs to amplify the signal through chemical reactions to improve sensitivity

Method used

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  • Probe for detecting nucleic acid in living cells and application method thereof
  • Probe for detecting nucleic acid in living cells and application method thereof
  • Probe for detecting nucleic acid in living cells and application method thereof

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Experimental program
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Effect test

Embodiment 1

[0045] Signal transducer and activator of transcription 5B (STAT5B) is related to various diseases such as breast cancer, prostate cancer and leukemia, so detecting the expression of STAT5B mRNA (SEQ ID NO: 1) is helpful for early diagnosis of tumors.

[0046] Preparation of the probe: For the mRNA synthesis probe of human STAT5B, the hairpin oligonucleotide (SEQ ID NO: 2) and the target sequence (SEQ ID NO :3), the 3' end of the hairpin targeting nucleic acid was labeled with FITC, and the 5' end was modified with a sulfhydryl group. In phosphate buffered saline (PBS, 0.02mol / L, pH=8.0), fluorescent substance-labeled and sulfhydryl-modified hairpin oligonucleotides were mixed with dithiothreitol (DTT) at a molar ratio of 1:10 for 45 Minutes, and then repeated 4 times with 4 times the volume of ethyl acetate to extract the DTT in the reaction. Then add the above reaction solution dropwise into the gold nano solution at a molar ratio of 100:1, mix well, react for 24 hours, add...

Embodiment 2

[0052] Clinical studies have shown that ERCC1-negative patients have better curative effect after using cisplatin, while ERCC1-positive patients have no obvious effect, so ERCC1 gene is a drug-sensitive gene. Therefore, detecting the expression of ERCC1 can guide the clinical medication of tumor therapy.

[0053] Preparation of the probe: Synthesize the probe against ERCC1 mRNA (SEQ ID NO: 4), please use the hairpin oligonucleotide (SEQ ID NO: 5) and target Sequence (SEQ ID NO: 6), the 3' end of the hairpin targeting nucleic acid was labeled with FITC and the 5' end was modified with a sulfhydryl group. In phosphate buffered saline (PBS, 0.02mol / L, pH=8.0), fluorescent substance-labeled and sulfhydryl-modified hairpin oligonucleotides were mixed with dithiothreitol (DTT) at a molar ratio of 1:10 for 45 Minutes, and then repeated 4 times with 4 times the volume of ethyl acetate to extract the DTT in the reaction. Then add the above reaction solution dropwise into the gold nan...

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Abstract

The invention relates to the field of nanotechnology and biotechnology, in particular to a probe for detecting nucleic acid in living cells and an application method thereof. The probe is characterized in that the probe comprises three parts, namely hair clip-shaped oligonucleotide, a fluorescent labeling matter and nano-gold, wherein the hair clip-shaped oligonucleotide contains a base sequence being complimentary with target nucleic acid and has a stem-loop structure, the fluorescent labeling matter is arranged at one end of the hair clip-shaped oligonucleotide in a covalence way, and nano-gold particles are in coordination connection with hydrosulphonyl modified at the other end of the hair clip-shaped oligonucleotide. By adopting the method, the target nucleic acid in living cell samples can be simply detected in real-time, and spatial orientation can be performed on the target nucleic acid for realizing real-time in-situ detection; furthermore, transfection and a transmembrane reagent or technology are not required, thereby having important significance and extensive application prospect in the aspects of developing disease nosogenesis, tumor early diagnosis and treatment and medicament target points.

Description

technical field [0001] The invention relates to the fields of nanotechnology and biotechnology, in particular to nucleic acid hybridization and detection technology. Background technique [0002] Nucleic acid has very important biological functions, mainly storing and transmitting genetic information. Prokaryotes such as microbial pathogens are mostly bacteria and viruses, and their nuclei have no nuclear envelope, and their DNA or RNA is naked. The 16S-23S rRNA gene of bacteria is an interval sequence located between the 16S rRNA gene and the 23S rRNA gene, which has good conservation and relative variability, and is considered to be a suitable site for bacterial identification, while the genetic material of viruses is a single RNA or DNA, simple structure. These features are conducive to the detection of microbial pathogens by nucleic acid detection methods. Furthermore, in the living body, mRNA is used as a template to translate proteins, and the protein's messenger ri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 顾月清薛建鹏
Owner CHINA PHARM UNIV
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