Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor

A technology of molecular beacon probes and nucleic acid aptamers, which is applied in material excitation analysis, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve the problems of serious non-specific reactions, many operation steps, and high price, and achieve high sensitivity High, good selectivity, simple operation effect

Inactive Publication Date: 2012-10-17
HUNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this method requires antibodies and enzyme-labeled proteins, so it is...

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  • Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor
  • Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor
  • Nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor

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Embodiment 1

[0023] Embodiment 1: Preparation of nucleic acid aptamer molecular beacon probe

[0024] a kind of like figure 1 The nucleic acid aptamer molecular probe of the present invention detecting PDGF-BB, this nucleic acid aptamer probe comprises nucleic acid aptamer molecular probe and the fluorophore and the quenching group that connect 5' end and 3' end respectively group. The nucleic acid aptamer molecular probe is PDGF-BB recognition sequence 5' - AAG GCTACG GCA CGTAGA GCATCA CCATGATCCTG-3' (SEQ ID NO. 2). The group is FITC, and the quenching group is Dabcyl. In this example, the nucleic acid aptamer molecular probe was designed and then synthesized in ABI3400 DNA / RNA synthesizer. Synthetic starting materials were purchased from Glen Research (Sterling, VA). DNA purification was achieved by ProStar HPLC (Varian). DNA concentration was determined by Cary Bio-300 UV-visible spectrometer (Varian).

Embodiment 2

[0025] Example 2: Specific investigation of nucleic acid aptamer molecular beacon probes

[0026] Dilute the aptamer beacon probe in a physiological buffer solution (20 mM Tris-HCl, pH 7.1, 140 mM sodium chloride, 5 mM potassium chloride, 1 mM calcium chloride, and 1 mM magnesium chloride), Its final concentration is 20 nM. Then take a clean 96 microwell plate, add 100 μl 20 nM nucleic acid aptamer molecular beacon probe to 11 microwells, and add 50 μl physiological buffer solution and bovine serum at a final concentration of 100 nM Albumin (BSA), hemoglobin (HEM), lactate dehydrogenase (LDH), lysozyme (LYZ), myoglobin (MYO), thrombin (THR), epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), insulin-like growth factor 1 (iGF1), and the target protein platelet growth factor (PDGF-BB). The fluorescent signal value of the nucleic acid aptamer molecular probe at 520 nm was recorded by a Fluorolog spectrophotometer (Jobin Yvon Horiba) after the protein...

Embodiment 3

[0027] Embodiment 3: Nucleic acid aptamer molecular beacon probe is used for the detection of sample in bovine serum

[0028] The simulated biological sample of PDGF-BB was prepared by adding 100 nmol of PDGF-BB to DMEM medium containing 0.5% fetal bovine serum (FBS), and the final concentration of DGF-BB was 100 nM. Proteins in 100 microliters of the control sample Sb (DMEM medium containing 0.5% FBS) and the biological sample Sa were enriched by filtration with Sephadex G-10, respectively. Then the protein was dispersed in 100 microliters of physiological buffer solution, and the final concentration of 50nM nucleic acid aptamer molecular probe was added. After 60 minutes of reaction, Fluorolog spectrophotometer (Jobin Yvon Horiba) recorded the fluorescence change value of the nucleic acid aptamer molecular probe at 520 nm before and after the addition of the protein. Experimental results such as image 3 As shown, the samples dispersed in DMEM-containing medium (containi...

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Abstract

The invention discloses a nucleic acid aptamer molecular beacon probe for the detection of platelet-derived growth factor. The nucleic acid aptamer molecular beacon probe comprises a PDGF-BB nucleic acid aptamer, a fluorescent group which connects to the 5' end of the PDGF-BB nucleic acid aptamer and a quenching group which connects to the 3' end of the PDGF-BB nucleic acid aptamer. According to the invention, high specificity and high affinity of the nucleic acid aptamer probe to the target protein and high sensitivity type conversion mechanism are subtly utilized, and fluorescence quenching mechanism is also utilized. The nucleic acid aptamer molecular beacon probe provided by the invention has a high sensitivity and a good selectivity. The detection process by the use of the nucleic acid aptamer molecular beacon probe provided by the invention is simple to operate with only one step, and the operation cost can be reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid aptamer molecular beacon probe for detection of platelet-derived growth factor. [0002] Background technique [0003] Protein is the most important material basis of life activities and plays an important role in life functions. PDGF-BB is a human platelet growth factor protein that is involved in the growth of fibroblasts, glial cells, and smooth muscle cells. PDGF-BB is the coding product of the proto-oncogene c-sis. The abnormal increase of its autocrine activity can promote tumor cell proliferation and inhibit its apoptosis. It is a good tumor marker. At present, the method for detecting PDGF-BB is mainly enzyme-linked immunosorbent assay. The method is capable of detecting picograms per milliliter of sample. However, this method requires antibodies and enzyme-labeled proteins, so it is expensive; at the same time, there are many steps and non-spec...

Claims

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Application Information

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IPC IPC(8): G01N21/64C12N15/115
Inventor 谭蔚泓方晓红赵子龙胡蓉
Owner HUNAN UNIV
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