Kit for aptamer screening and use and detection method thereof

A nucleic acid aptamer and kit technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA preparation, etc., can solve the problems of many steps, low success rate, long screening period, etc.

Pending Publication Date: 2019-07-19
安徽省昂普拓迈生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many steps in the whole process, and the screening cycle is very long. It takes several months to screen out nucleic acid aptamers with high specificity for the target.
With the rapid development of nucleic acid aptamers, more and more people have paid attention to this field in recent years. However, due to the many steps, there is no conventional kit for screening at present. Many experimental conditions make the screening time-consuming and labor-intensive, and the success rate is very low

Method used

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  • Kit for aptamer screening and use and detection method thereof
  • Kit for aptamer screening and use and detection method thereof
  • Kit for aptamer screening and use and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] A kit for nucleic acid aptamer screening, the raw materials of which include: 1 magnet of 1 cm × 2 cm × 3 cm, 10 7 2mL streptavidin-modified magnetic beads per μL, 3 tubes of 1.3nmol / tube (dry powder) nucleic acid library, 1 tube of 5nmo / tube (dry powder) short sequence, 30 parts of 1mL PCR master mix, 150mL ePCR microbeads 1 part of droplet forming oil, 20 parts of 300 μL fluorescent quantitative PCR master mix, 1 part of 100 μL molecular weight standard sample;

[0073] Wherein, the sequence of the nucleic acid library is 5'-ATTGGCACTCCACGCATAGG-40N-CCTATGCGTGCTACCGTGAA-3', wherein 40N represents a sequence formed by connecting 40 arbitrary nucleotide bases;

[0074] The sequence of the short sequence is: 5'-CCTATGCGTGGAGTGCCAAT-biotin-3';

[0075] The raw materials of the PCR master mix include: 100 μM FAM modified nucleic acid library forward primer 5 μL, 100 μM polyA modified nucleic acid library reverse primer 5 μL, 10 mM dNTP 20 μL, 5 U / μL DNA polymerase 4 μL, 1...

Embodiment 2

[0081] The nucleic acid aptamer screening of Example 2 was carried out with the reagents in Example 1.

[0082] A method for using a kit for nucleic acid aptamer screening, comprising the steps of:

[0083] S1. Immobilizing the nucleic acid library on magnetic beads:

[0084] Take the nucleic acid library and short sequences and use DPBS buffer as a solvent to prepare solution A. Solution A contains a nucleic acid library of Mnmol. Take solution A and pair it with a PCR machine to obtain solution B;

[0085]Wherein, during the first round of screening, the concentration of the nucleic acid library in solution A was 5 μM, and the concentration of the short sequence was 10 μM; from the second round of screening, the concentration of the nucleic acid library in solution A was 350 nM, and the concentration of the short sequence was 700 nM; The instrument pairing procedure is to keep at 95°C for 10 minutes, cool down to 60°C at a rate of 0.1°C / s, hold for 1 minute, and then cool d...

Embodiment 3

[0110] The detection in Example 3 was carried out with the fluorescent quantitative PCR master mix in Example 1.

[0111] Fluorescent quantitative PCR detection method described in embodiment 2 is:

[0112] Draw a standard curve: take the nucleic acid library described in Example 1 and add it to the fluorescent quantitative PCR premix described in Example 1 to prepare standard curve solutions with different nucleic acid library concentrations, detect the Cq values ​​of each standard curve solution by fluorescent quantitative PCR, and use each standard The lg value of the nucleic acid library concentration of the curve solution is the ordinate, and the Cq value of each standard curve solution is the abscissa to draw a standard curve to obtain a regression equation.

[0113] The values ​​of the standard curve are shown in the table below:

[0114] X axis (Cq value) Y axis (log value of nucleic acid library concentration) Nucleic acid library concentration (pM) ...

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Abstract

The invention discloses a kit for aptamer screening. The kit comprises the following raw materials: a magnet, streptavidin-modified magnetic beads, a nucleic acid library, a short sequence, a PCR liquid premix, ePCR micro-droplet generation oil, a fluorescence quantitative PCR liquid premix and a molecular weight standard sample. The invention also discloses a using method of the kit for aptamer screening. The method comprises the following steps: S1, immobilizing the nucleic acid library on the magnetic beads; S2, performing screening; S3, performing PCR amplification; S4, preparing ssDNA; and S5, performing multi-round screening. The invention also discloses a method for detecting the binding rate of aptamer screened by a magnetic bead method and a target. According to the invention, thescreening period is shortened, the screening time is saved, the experimental flow is simplified, the repeatability is good, and the screening efficiency is high.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for nucleic acid aptamer screening and its use and detection methods. Background technique [0002] Nucleic acid aptamer (aptamer) refers to the DNA or RNA molecule screened and isolated through the exponential enrichment of ligands evolution technology (Systematicevolution of ligands by exponential enrichment, referred to as SELEX), also known as chemical antibody. It can bind with high affinity and specificity to targets such as proteins, metal ions, small molecules, peptides and even whole cells. Compared with traditional antibodies, nucleic acid aptamers have the advantages of small molecular weight, better stability, easy modification, no immunogenicity, short production cycle, and can be artificially synthesized, eliminating the need for animal immunization, feeding, and protein extraction And a series of processes such as purification. These advantages make it have bro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/6851
CPCC12N15/115C12N15/111C12Q1/6851C12N2310/16C12N2330/31C12Q2531/113C12Q2563/107C12Q2525/205
Inventor 罗昭锋杨伟丽方晓娜王进军
Owner 安徽省昂普拓迈生物科技有限责任公司
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