Capillary Electrophoresis Method for On-line Reaction Separation of Protein-oligonucleic Acid Complex

A capillary electrophoresis and protein technology, which is applied in the direction of material analysis, material analysis, and material excitation analysis through electromagnetic means, can solve the problems of tedious optimization of reaction conditions, time-consuming and labor-intensive, and large amount of target molecules, so as to save the need for incubation Step, inhibition of dissociation, effect of increased complex amount

Active Publication Date: 2017-06-27
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the traditional CE-SELEX technology for screening aptamers, the amount of target molecules used is large, the optimization process of reaction conditions is cumbersome, and time-consuming and labor-intensive. Method, described method is based on capillary electrophoresis on-line reaction and separation method, is used for the efficient screening of target molecular nucleic acid aptamer

Method used

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  • Capillary Electrophoresis Method for On-line Reaction Separation of Protein-oligonucleic Acid Complex
  • Capillary Electrophoresis Method for On-line Reaction Separation of Protein-oligonucleic Acid Complex
  • Capillary Electrophoresis Method for On-line Reaction Separation of Protein-oligonucleic Acid Complex

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Embodiment 1

[0029] In Example 1 below, the 15-base H-Thr adapter (Apt15) sequence: 5'-GGT TGG TGT GGT TGG-3', the 29-base H-Thr adapter (Apt29) sequence: 5'-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3', the above aptamers were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; in Examples 2-7, fluorescently labeled 15-base H-Thr aptamer (Apt15- FAM) sequence: 5'-FAM-GGT TGG TGT GGT TGG-3', fluorescently labeled 29 bases H-Thr adapter (Apt29-FAM) sequence: 5'-FAM-AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-3', the aptamers were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; human thrombin (H-Thr) was purchased from Enzo Life Science Company; 80 fluorescent library: 5'-AGCAGCACAGAGGTCAGATG-(40N)- CCTATGCGTGCTACCGTGAA-3' was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0030] The capillary used for the online reaction of the reactant in the capillary zone electrophoresis is a coated capillary, which was purchased from Aoteike Biotechnology Co., Ltd., Handan Develo...

Embodiment 2

[0040] Preparation of original Apt29-FAM stock solution: Centrifuge the purchased Apt29-FAM crystals at 3000 rpm for 5 min, add pure water to make 1.0×10 -4 mol / L of the original Apt29-FAM stock solution; the original Apt29-FAM stock solution was diluted with pure water to obtain 1.0×10 -6 The Apt29-FAM sample solution of mol / L; The original H-Thr stock solution that embodiment 1 makes is diluted with pure water to obtain 1.0 * 10 -6 mol / L H-Thr sample solution.

[0041] (1) Perform capillary zone electrophoresis in a Beckman P / ACE MDQ capillary electrophoresis instrument, take H-Thr sample solution, electrophoresis buffer and Apt29-FAM sample solution respectively, and inject H-Thr sample with slow migration rate first during operation Solution, then inject electrophoresis buffer, and finally inject Apt29-FAM sample solution with fast migration rate, the injection conditions are as follows: H-Thr injection: 0.5psi, 20s, electrophoresis buffer injection: 0.5psi, 10s, Apt29 -...

Embodiment 3

[0045] Determination of complex collection time: According to the formula: The peak elution time adopts the time before and after the peak elution of the compound in Example 2 (the time before the peak elution: 9 minutes, the time after the peak elution: 13 min), and the calculated complex collection time is: 11.3min~16.3min.

[0046] Collection of complexes: Carry out capillary zone electrophoresis according to the method of Example 2. When the electrophoresis runs to 11 minutes, transfer the outlet end of the capillary to a complex collection bottle containing 20 μL of pure water. To ensure sufficient collection, continue Run the electrophoresis for 10 minutes. After one collection, use the same method to collect four more H-Thr and Apt29-FAM complexes in the same collection bottle. A total of 5 H-Thr and Apt29-FAM capillary online reaction complexes were collected. .

[0047] Detection of the complex: the collected 5 complexes in 20 μL of pure water were injected into the...

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Abstract

The invention relates to a method applied to on-line reaction and separation of a protein-oligonucleotide compound by capillary electrophoresis, belonging to the technical field of biological separation and analysis. The method comprises the following steps: carrying out capillary zone electrophoresis on reactants; sequentially feeding samples, namely reactants with low migration rate in the capillary, an electrophoresis buffer solution and reactants with high migration rate in the capillary; reacting and mixing the reactants with different migration rates in the capillary during the electrophoresis process online and then carrying out electrophoresis, separating and collecting electrophoresis products, and carrying out capillary electrophoresis-laser-induced fluorescence detection on the products. The products comprise protein and ssDNA. According to the method, the two reactants are mixed and reacted online in the capillary electrophoresis process in the absence of incubation; the compound is quickly, simply and conveniently separated online; the method is high in efficiency and high in speed, few in samples and high in utilization rate of samples, and is especially suitable for protein which is inadequate in source and high in cost.

Description

technical field [0001] The invention relates to a method for capillary electrophoresis on-line reaction separation of protein-oligonucleic acid complexes, which belongs to the technical field of biological separation and analysis. Background technique [0002] Single-stranded oligonucleotide (single-stranded DNA, ssDNA) or RNA has a specific secondary structure, which can form high-affinity and specific complexes with protein molecules. The oligonucleotide library is a class containing 10 13 ~10 15 A mixture of single-stranded DNA molecules (ssDNA) with different bases, which may contain one or several ssDNAs that can bind to target molecules with high specificity and high affinity, that is, nucleic acid aptamers (Aptamers). Nucleic acid aptamers are screened from random oligonucleotide libraries by exponential enrichment ligand system evolution (SELEX) technology, and have oligonucleotide sequence ligands with high affinity and high specificity for target molecules , whi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447G01N21/64
Inventor 屈锋胡猷浩吴景刚赵新颖
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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