Method for screening aptamer specifically bound with alpha-fetoprotein

A nucleic acid aptamer and alpha-fetoprotein technology, applied in the field of analytical chemistry, can solve the problems of immunogenicity and high cost of antibodies

Inactive Publication Date: 2014-11-05
ZHONGSHAN HOSPITAL FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the qualitative and quantitative detection of AFP in clinical and scientific research mostly relies on antibodies. Although the sensitivity and specificity are high, antibodies have disadvantages such as high cost and immunogenicity.

Method used

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  • Method for screening aptamer specifically bound with alpha-fetoprotein
  • Method for screening aptamer specifically bound with alpha-fetoprotein
  • Method for screening aptamer specifically bound with alpha-fetoprotein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Screening of nucleic acid aptamers

[0026] 1. Instruments and materials: P / ACE MDQ capillary electrophoresis instrument (Beckman-Coulter, USA), equipped with diode array (PDA) and laser-induced fluorescence (LIF) detectors; PVA-coated elastic quartz capillary column with a total length of 40 cm, effective Length 30 cm, inner diameter 50 μm, outer diameter 360 μm.

[0027] 2. Electrophoresis buffer preparation: use ultrapure water to prepare 30 mM NaH 2 PO 3 , adjusted the pH value to 7.5 with 1 M NaOH solution, and filtered through a 0.45 μm membrane filter.

[0028] 3. Design and synthesize a random oligonucleotide library with a full length of 75 bases. The sequence is shown in SEQ ID NO.1, 5'-GTGACGCTCCTAACGCTGAC-35N-CCTGTCCGTCCGAACCAATC-3', with 20 bases at both ends The fixed sequence of , the middle 35 bases are random sequences.

[0029] 4. Synthesize upstream primers and downstream primers for PCR amplification, the sequence is:

[0030] Upstre...

Embodiment 2

[0038] Example 2 Cloning and sequencing of nucleic acid aptamers

[0039] Prepare the following solutions in a microcentrifuge tube, the total volume is 5 μl: pMD19-T Vector 1 μl, oligonucleotide PCR product 0.2 pmol, ddH 2O supplemented to 5 μl. Add 5 μl of Solution I. React at 16°C for 30 min. The whole amount (10 μl) was added to 100 μl Trans5α competent cells, and placed in ice for 30 min. After heating at 42°C for 45 s, place in ice for 1 min. Add 890 μl LB medium, shake and culture at 37°C for 60 min. Cultured on LB agar plate medium containing X-Gal, IPTG, and Amp at 37°C to form a single colony. White monoclonal colonies were selected and placed in 2 ml LB (Amp 80 μg / ml) liquid medium, shaken at 200 rpm and 37 °C for 16 h, and the bacterial liquid was submitted to Shanghai Sangon for sequencing.

Embodiment 3

[0040] Example 3 Verification of nucleic acid aptamers

[0041] 83 oligonucleotide sequences were obtained by sequencing. After sorting out the sequencing results and predicting the structure, 14 sequences with representative structures were selected for capillary electrophoresis analysis, and one sequence with high specificity, aptamer-273, was selected.

[0042] The sequence of Aptamer-273 is shown in SEQ ID NO.4.

[0043] Investigation 1: Capillary electrophoresis detection of binding specificity between fluorescently labeled aptamer-273 and AFP protein in this example.

[0044] Electrophoresis buffer is 30 mM NaH 2 PO 3 , pH 7.5, AFP concentration 0.1 mg / ml, aptamer concentration 10 μM. The inner diameter of the PVA-coated capillary column is 50 μm, the total length is 40 cm, and the effective length is 30 cm; the separation voltage is -8 kV, the pressurization is 34 mbar; the temperature is 25 ℃; the pressure is 34 mbar, and the fluorescent (FAM)-labeled aptamer-273...

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Abstract

The invention relates to a method for screening an aptamer specifically bound with an alpha-fetoprotein by using a capillary electrophoresis technology. The method comprises the following steps of designing and establishing a random oligonucleotide library; enriching oligonucleotides which can be bound with alpha-fetoprotein by combining a capillary electrophoresis technology and an SELEX (Systematic Evolution of Ligands by Exponential Enrichment) screening method; and cloning, sequencing, and carrying out capillary electrophoresis analysis and immunofluorescence verification to finally obtain oligonucleotides which can be specifically bound with the alpha-fetoprotein, namely the aptamer. The invention provides an efficient and specific aptamer screening method. The method has the advantages that the screening time is shortened by means of the advantage of the capillary electrophoresis technology, and the screening operation can be finished through four rounds of circulation; and meanwhile, the non-specific screening caused by a matrix effect is reduced.

Description

technical field [0001] The invention relates to the technical field of analytical chemistry, in particular to a screening method for nucleic acid aptamers specifically binding to alpha-fetoprotein. Background technique [0002] Aptamer (aptamer) is a single-stranded oligonucleotide (ssDNA or RNA) that recognizes target molecules through structure specificity. Its principle of action is similar to that of antibodies, and at the same time, it has the advantages of non-immunogenicity, chemical synthesis, easy modification, low cost, and good stability. It has broad application prospects in clinical diagnosis, new drug development, and basic research. [0003] The Aptamer screening technology is named as the systematic evolution of ligands by exponential enrichment, referred to as SELEX (Systematic evolution of ligand by exponential enrichment), which is a new combinatorial chemistry technology developed in the early 1990s. The basic idea is: construct an oligonucleotide librar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12N15/1048C12Q2565/125
Inventor 吴伟忠胡洪卫郭玮冬黎黎谈绮文
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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