Technology Study Of PANC-1 Cell Aptamer Selection Based On CELL-SELEX

Cell aptamer selection can generalize for the following four aspects: preliminary preparation, PCR amplification, secondary ssDNA pool preparation and selection. Based on this four contents, we systematically studied the methodology of cell aptemer selection based on CELL-SELEX technology.In this aptamer selection against human pancreatic adenocarcinoma panc-1 cell which haven’t been reported in cell aptamer selection, we developed a new negative cell selection strategy, and based on the strategy, the A549 cell was selected as negative cell to carry out negative selection. The stability of cell state is very important to aptamer selection, we developed and used a high cell-passage frequency strategy to keep cell state stable throughout the the cell aptamer selection. With software analysis and PCR amplification,we investigated the sequence of primers, efficiency of PCR amplification and the ability of anti-interference, and certified the primers selected were suitable in the cell aptamer selection.PCR is widely used in CELL-SELEX to amplify secondary ssDNA pool. With random ssDNA pool as template PCR is difficult for many uncertain factors. In this paper,we adopted several PCR methods to investigate the features of random ssDNA PCR, and found that the change of annealing temperature have an effect on the efficiency of amplification, but have weak work on nonspecific amplification. And the random ssDNA PCR is extremely sensitive to the number of cycles of PCR program, the number of cycle of PCR program is much less than the conventional PCR and has a great impact on the selectivity and efficiency of PCR. In the secondary ssDNA pool PCR optimization, we geta broadly applicable PCR optimization strategy, which will has a guiding significance in secondary ssDNA pool PCR.In the secondary ssDNA pool preparation, we have developed corresponding monitoring methods for single strand isolation process, which could fed back the results of above process and confirm the recovered samples. By prolonging the incubation time of PCR product with Streptavidin-Agarose, the immobilization rate of the column to PCR product is improved. With the help of Acryl carrier, We established the ethanol precipitation ssDNA recovery method which is suitable for cell aptamer selection, the method could selectively recover target ssDNA and the recovery rate is excess 80.0%. By the method, the the problem of ssDNA desalination, pH equilibrium and ssDNA concentration were solved simultaneously. At the same time, we established the corresponding ssDNA quantity method.Selection is the core of CELL-SELEX, In this paper, we studied the method of cell aptamer selection and selection stringency regulation. 5 basic selection stringency regulation forms were confirmed(reduce cells number, increase concentration of ssDNA in incubation, shorten incubation time, raise the spreed of incubation shaker and enhance the washing intensity). The 5 forms collaboratively adjust the selection stringency, and the ssDNA concentration in incubation solution play a decisive role in the stringency regulation, and the washing time determine the whole washing stringency.Through this cell aptamer selection methodology study, we have successfully established the PANC-1 cell aptamer selection platform, and screened a candidate aptamer pool with high affinity to target cell.