A multi -price adaptation DNA nano ladder and its preparation methods and applications
A DNA molecule and aptamer technology, applied in the field of biomedicine, can solve problems such as multidrug resistance and drug resistance, and achieve the effects of strong biocompatibility, high affinity and selectivity, and easy functional modification.
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Embodiment 1
[0052] Example 1: Preparation method of rolling circle amplification (RCA) product
[0053] Rolling circle amplification (RCA) reactions were performed with circular DNA templates A and B, respectively, and 2 μL of primers (10 μM), 2 μL of circular DNA template (10 μM), and 2 μL of 10×Phi29 DNA polymerase buffer were added to each reaction system in turn. and 2μL of dNTPs (7mM), supplemented with ddH 2 O to the total volume of 20 μL, add 0.8 μL Phi29 DNA polymerase (10 U / μL) to initiate the reaction, mix well, centrifuge at appropriate low speed, place it in a constant temperature shaker and incubate at 30 °C for 10 min, and then incubate at 65 °C for 10 min to inactivate Phi29 DNA polymerase to produce rolling circle amplification (RCA) product chain A and chain B. Rolling circle amplification (RCA) products were characterized by agarose gel electrophoresis about 8000 nt in length.
[0054] Table 1: Oligonucleotide sequences involved in Example 1
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Embodiment 2
[0057] Example 2: Preparation method of DNA nano-ladder
[0058] The rolling circle amplification (RCA) products prepared in Example 1 were 1 μL each of chain A and chain B, and 1 μL each of short chains 1-6 (concentration 100 μM), were added to a solution containing 42 μL of 2×TAE-Mg 2+ (24mM) in a centrifuge tube, supplemented with ddH 2 0 to 100 μL, mix well, centrifuge at low speed appropriately, heat treatment at 95°C for 5 min, and anneal to 25°C naturally to prepare DNA nano-ladder.
[0059]Transmission electron microscopy was performed at an accelerating voltage of 200KV. Sample preparation for transmission electron microscopy: 5 μL of DNA nano-ladder sample was dropped onto the copper grid, allowed to stand for 5 minutes, stained with 1% uranyl acetate for 1 minute, and tested after the sample was completely dried.
[0060] The self-assembled products were characterized by transmission electron microscopy, such as image 3 As shown in (a), it can be seen that the s...
Embodiment 3
[0067] Example 3: Drug Loading Experiment
[0068] The concentration of immobilized Dox was 200nM, and the concentration of DNA nanoladder was 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, respectively, mixed well, centrifuged, and incubated at 30°C for 30min.
[0069] The detection method of the microplate reader is fluorescence intensity detection, the detection type is end-point scanning mode, the excitation wavelength is set to 485 nm, the emission wavelength is set to 590 nm, and the detection temperature is 30 °C.
[0070] Preparation method of DNA nanoladder-Dox complex: 200nM antitumor drug adriamycin hydrochloride (Dox) was mixed with 1nM, 0.4nM, 0.2nM, 0.1nM, 0.04nM, 0.02nM, 0.01nM, 0.005nM, 0.0025nM, respectively. DNA nanoladders were incubated at 30°C for 30min.
[0071] The antitumor drug doxorubicin hydrochloride (Dox) is inserted between the G-C bases of the DNA double-strand by physical binding to form a DNA nano-ladder-Dox complex, and ...
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