The Exploring Process Of DNA Aptamer Screening Methods For Detection

Aptamer is the oligonucleotide selected from a random sequence which is composed of a DNA or RNA molecules. Aptamer can specifically bind proteins, small organic molecules, metal ions and other ligands. Aptamer usually consist of 20 to 60 bp, screened out from the artificial random library of single-stranded oligonucleotides through systematic evolution by exponential enrichment.During the aptamer screening process, aptamer monitoring and separation are the key problems and two difficult problems. In the selection process, how to monitor aptamer effectively is an important factor which can affect selection.This article is based on the basic principle of SELEX screening technology, further improve the screening method. Through one-step screening, using double-stranded initiation, we shortened the screening time, simplified the selection process and provided a simpler and more efficient way for the selection of aptamer. In this paper, we screened different target which can specifically bind aptamer in order to explore the aptamer selection process monitoring method. The exploration of different monitoring methods provides a novel idea for aptamer monitoring and enrichs monitoring method during the aptamer selection process. The main results are as follows:1. We apply one-step method to screen P-lactamase aptamer, after one round of screening, successfully screened the corresponding aptamer, using PAGE electrophoresis to detect the affinity of aptamer, and using iodometric method to detect the specificity of aptamer.The results showed that:the screened aptamer have a good affinity with P-lactamase, but cannot inhibit the activity of P-lactamase.2. We apply one-step method to screen the aptamer of red blood cell surface membrane structure, after one round of screening, successfully screened the corresponding aptamer,using anti-A and anti-B blood grouping reagents to detect the affinity of aptamer. Blocking blood clotting Using Coagulation inhibition test to detect aptamer whether can block blood clotting or not and by this to detect the affinity and specificity of aptamer. The results showed that:The screened aptamer within the prescribed time did not block the blood clotting, and have a low affinity with red blood cell surface membrane structure.3. We apply one-step method to screen Nosema aptamer, after one round of screening, successfully screened the corresponding aptamer. Based on fluorescence detection principle, we optimize the fluorescent-5-maleimide and thiol fluorescence probe sensing system preliminarily, determining the system volume and concentration of the various components, the elution times of the aptamer which cannot bind with the target, the elution times of free thiol primer and excitation wavelength optimization. We used this system to detect the affinity and specificity of Nosema aptamer. The results showed that:this aptamer had a good affinity with Nosema. This method provide a new way to aptamer detection during the aptamer selection process,making detection faster and more convenient, and can trace detection.