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
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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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