A dual-target simultaneous nucleic acid aptamer screening method based on capillary electrophoresis
A capillary electrophoresis and nucleic acid aptamer technology, which is applied in the field of protein nucleic acid separation and analysis, can solve the problems of complex screening process, high sample consumption, time-consuming and other problems.
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Embodiment 1
[0037] Raw random deoxyoligonucleotide library (ssDNA 40N ) stock solution preparation: the purchased crystal-like ssDNA 40N , centrifuged at 3000 rpm for 5 min, added pure water to make 1.0×10 -4 mol / L original ssDNA 40N The stock solution was cooled on ice in a metal bath at 94°C for 5 minutes, and stored at -20°C.
[0038] Raw ssDNA 40N The stock solution was diluted with pure water to obtain 6.0×10 -7 mol / L ssDNA 40N Sample solution; the original H-TF crystal powder was prepared with pure water to obtain 1.2×10 -6 mol / L H-TF sample solution; the original PDGF-BB protein solution was diluted with pure water to obtain 1.2×10 -6 mol / L PDGF-BB sample solution.
[0039] Take 20 μL of H-TF sample solution, PDGF-BB sample solution and ssDNA respectively40N The sample solution, after mixing, was incubated in a metal bath at 37°C for 10 minutes. In the incubation system, the two target molecules acted as reverse screening targets for each other, competing to combine with ssD...
Embodiment 2
[0047] The original ssDNA that embodiment makes 40N The stock solution was diluted with pure water to obtain 6.0×10 -7 mol / L ssDNA 40N Sample solution; the original H-TF crystal powder was prepared with pure water to obtain 1.2×10 -6 mol / L H-TF sample solution; the original PDGF-BB protein solution was diluted with pure water to obtain 1.2×10 -6 mol / L PDGF-BB sample solution.
[0048] Take 20 μL of H-TF sample solution, PDGF-BB sample solution and ssDNA respectively 40N After mixing the sample solution, incubate in a metal bath at 37°C for 10 minutes; in the incubation system, the two target molecules serve as reverse screening targets for each other, and compete to combine with ssDNA in the random oligodeoxynucleotide library to form their respective ssDNA-target molecule complexes After incubation, the mixture contains free ssDNA not bound to the two target molecules and two ssDNA-target molecule complexes bound to the two target molecules;
[0049] The capillary used d...
Embodiment 3
[0053] With the C that embodiment 1 makes H with C P The PCR amplification products were subjected to high-throughput sequencing respectively, and two sequences were selected from the sequencing results, the H-TF nucleic acid aptamer Apt H 1 with Apt H 2. PDGF-BB nucleic acid aptamer Apt p 1 with Apt p 2. In Table 1A are respectively Apt H 1. Apt H 2. Apt p 1. Apt p The sequence of 2 was characterized by affinity using CE-LIF method. Apt H 1. Apt H 2 Equilibrium dissociation constants for H-TF (K D ) were 0.059μM, 0.314μM, respectively, the equilibrium dissociation constant for PDGF-BB (K D ) were 5.890 μM and 10.560 μM, respectively, and the H-TF nucleic acid aptamer showed high affinity and specificity for H-TF. Apt P 1. Apt P 2 Equilibrium dissociation constants for PDGF-BB (K D ) are 0.380μM, 0.398μM, respectively, the equilibrium dissociation constant (K D ) were 3.650 μM and 4.996 μM, respectively, and the PDGF-BB nucleic acid aptamer showed high affini...
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