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Aptamer selection method and method for purifying biomolecules through aptamer

A technology of biomolecules and aptamers, which is applied in the determination/inspection of microorganisms, solid separation, biological testing, etc., can solve the problems of inability to identify biomarkers, inability to achieve protein purification, labor-intensive, time-consuming, etc.

Inactive Publication Date: 2019-12-10
洪 振义 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned experimental methods all require professional technology, consume a lot of manpower, time, consumables and expensive instrument analysis operations
However, when there is no suitable monoclonal antibody with high affinity and specificity or the identity of the target biomolecule is unknown, the above methods cannot achieve the purpose of protein purification and biomarker identification

Method used

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  • Aptamer selection method and method for purifying biomolecules through aptamer
  • Aptamer selection method and method for purifying biomolecules through aptamer
  • Aptamer selection method and method for purifying biomolecules through aptamer

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preparation example Construction

[0033] Purification reagents must be prepared before purification. Regarding the preparation of purification reagents, the above-mentioned verified aptamers are used as the capture ligands of the purification reagents, and the excess biotinylated aptamers are dissolved in PBS buffer. After heating and rapid cooling to form secondary or tertiary structures as described above, they were incubated with SA-MPs and magnetically separated to remove the supernatant containing unbound aptamers and retain the magnetic particle-bound complexes. Then the magnetic particle-bound complex was dispersed in PBS buffer as a purification reagent. Once the purification reagent is prepared, it can be used to purify from a blinded sample of a positive attribute that has a high relative expression of the aptamer in a blinded analysis by q-PCR. The purification process of unknown disease-related biomolecules in the sample is shown in the figure figure 2 shown.

[0034] figure 2 It is explained ...

Embodiment

[0039] Example: Purification and identification of disease-associated biomarkers—taking non-small cell lung cancer with epidermal growth factor receptor mutation as an example

[0040] According to the biomarker discovery program (biomarker discovery) of the specific disease disclosed in the present invention. It mainly includes the screening procedure of screening aptamers with the ability to distinguish between disease patients and non-disease patients from the nucleic acid library, and using the screened aptamers as capture ligands to purify unknown biomolecules for positive attribute samples The program and other two parts are composed of figure 1 and figure 2 shown. The following will take the purification and identification of unknown disease-related biomarkers from serum samples of non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (epidermal growth factor receptor, EGFR) mutation as an example, To illustrate the specific content of th...

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Abstract

The invention provides a method for purifying specific biomolecules from a disease patient specimen through a combination of a nucleic acid aptamer and certain disease-related specific biomolecules inthe disease patient specimen and identifying the specific biomolecules to find biomarkers of specific diseases. A method for screening the aptamer, having high binding affinity with the certain disease-related specific biomolecules and not bound to other non-disease-related biomolecules, from a nucleic acid library through magnetic-assisted rapid aptamer selection (MARAS) is described. Meanwhile,the invention also provides a method for purifying the certain disease-related specific biomolecules from the disease patient specimen by using the obtained nucleic acid aptamer as a capturing carrier. Further, the biomolecules purified from a sample by using a magnetic field range applying an oscillating magnetic field as a virtual filter and having the specific binding affinity range with the aptamer serving as the capturing carrier are described.

Description

technical field [0001] The present invention generally relates to a design that utilizes positive and negative selection and structural diversity in nucleic acid libraries to find difference, and use the magnetic-assisted aptamer rapid selection (MARAS) method to screen out the aptamer (aptamer) with the ability to distinguish the difference between the two, and then use the screened aptamer as a capture ligand to extract from the positive A method for purifying certain disease-related biomolecules that bind to aptamers from samples and identifying them to discover disease-related biomarkers (biomarkers). Specifically, the present invention relates to a nucleic acid library that has the ability to distinguish the difference in biomolecules contained in disease patients and non-patient samples, and can be used as a method for purifying this difference from disease patient samples. The screening method of the aptamer of the capture ligand (capture ligand) of the disease-related...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/53G01N33/574
CPCC12N15/115G01N33/53G01N33/57423G01N33/57484C12N2310/16C12N2330/31C12N15/1048C12N2320/12C12N2320/11C12N15/1013C12Q1/6811C12Q2525/179C12Q2525/205C12Q2537/155C12Q2563/143C12Q2563/155B03C1/01B03C1/288B03C2201/18B03C2201/20C07K1/14
Inventor 洪振义洪姮娥
Owner 洪 振义