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Real-time monitoring systematic evolution technology of ligands by exponential enrichment

A real-time monitoring and combination technology, applied in the field of exponential enrichment of ligand system evolution, can solve the problems of long screening process, low screening success rate, low content, etc., to achieve the effect of simple equipment and speeding up the SELEX screening process

Inactive Publication Date: 2014-07-23
HENAN ACAD OF AGRI SCI
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Problems solved by technology

[0003] However, the current SELEX method still has a long screening process, because the content of specific nucleotides in the library is low, there are disadvantages such as blind screening in the early screening, and the final screening success rate is low; equipment, which limits the wide application of this technology

Method used

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  • Real-time monitoring systematic evolution technology of ligands by exponential enrichment
  • Real-time monitoring systematic evolution technology of ligands by exponential enrichment

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example 1

[0017] Take the heavy metal lead nucleic acid aptamer screening process as an example.

[0018] (1) Coupling of lead ion and carrier protein with DTPA

[0019] This experiment mainly uses isothiocyanate method and glutaraldehyde method to couple the metal ions with the carrier protein through the bifunctional metal chelating agent DTPA to obtain the corresponding conjugate.

[0020] Isothiocyanate method: Weigh 10mg of BSA and dissolve in 0.5ml HEPES buffer (10mM Hepes, pH 9.0), 9.0 mg p-NH 2 -Bn-DTPA dissolved in 200μL of water, weighed 8mgPb(NO 3 ) 2 Dissolved in 200μL 2% HNO 2 in.

[0021] 200μl p-SCN-Bn-DTPA solution was added dropwise to 0.5 mL protein solution under stirring, and then 100 μL triethylbutylamine (1.5 M) was added, reacted at room temperature for 24 hours, and treated Centricon-30 ultrafiltration to remove unreacted Small molecule. First wash three times with HEPES buffer (10 mM, pH 9.0), and then wash twice with HBS (10 mM, pH 7.4). 200 μLPb(No) was added to the...

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Abstract

The invention discloses a real-time monitoring systematic evolution technology of ligands by exponential enrichment (SELEX). The technology comprises: fixing to-be screened targets to a solid carrier polystyrene microscale reaction plate, combining with a single-chain nucleotide library of which the downstream terminal is labeled with biotin, performing incubation reaction flushing, adding streptomycin avidin-fluorescent substance to identify the current-cycle screening effect, using a specific eluate to elute combined ssDNA, and performing amplification for next-cycle screening until an nucleic acid aptamer capable of specifically boning targets is finally screened out. The technology is capable of effectively overcoming the disadvantage that screening results cannot be detected in real time by employing conventional SELEX methods, no expensive equipment such as capillary electrophoresis equipment and the like is not needed, and the technology is widely applicable to nucleic acid aptamer rapid screening aiming at various targets.

Description

technical field [0001] The invention relates to an improved exponential enrichment ligand system evolution technology, which mainly improves the real-time monitoring of the screening process and the solid-phase method of screening targets, and is mainly used for the rapid screening of highly specific nucleic acid aptamers. Background technique [0002] Ligand System Evolution with Exponential Enrichment (SELEX) means that oligonucleotides rely on their own high affinity and high specificity for the corresponding target substances, in an oligonucleotide library containing a very large number of random sequences, through multiple The process of selective enrichment to obtain the required specific structural and functional aptamers. This technique is suitable for the enrichment of any special needs of life small molecules. The development of SELEX technology is based on monoclonal, ribozyme, and PCR technologies in the 1970s and 1980s. At present, this technology has been wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王方雨张改平胡骁飞赵东职爱民史西保邓瑞广
Owner HENAN ACAD OF AGRI SCI
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