CELL-SELEX-based Selection Of DNA Aptamers For PANC-1

Pancreatic cancer is a highly malignant digestive tumor.It develops rapidly,having a occ?Lt clinical manifestation and a very poor prognosis.It has been a major challenge in oncology.At present,the clinical discovery and diagnosis of pancreatic cancer is basically dependent on histological imaging and cell detection,and it is diffic?Lt to achieve early diagnosis.In recent years,cell aptamers have attracted much attention because of their various advantages such as high affinity and high selectivity.CELL-SELEX technology has also been rapidly developed due to its unique advantages,providing a new type of solution to these problems.In this work,we adopted CELL-SELEX strategy to obtain nucleic acid aptamers that can specifically recognize human pancreatic cancer cell line PANC-1,and human lung adenocarcinoma cell line A549 was used as a control.The main research contents include:1.We made a detailed discussion of the PCR involved in selection.In this study,highly efficient and specific PCR res?Lts were obtained mainly by adjusting the number of PCR cycles.A large number of templates were put into the front part of selection for PCR to reduce the loss of effective sequences,and the effects of different PCR conditions on the selection were discussed.2.We made a detailed study of the preparation and quantification of the evolutionary library.In the preparation of the evolution library,fluorescence monitoring was monitored for the process of double-stranded dissociation.The advantages and disadvantages of the ethanol precipitation method and desalting drying method were compared in the desalting treatment of the evolutionary library.In the quantification of the evolution library,we used 0.2M Na OH and 1×BB as background against the sensitivity of FAM to p H.3.We conducted a innovative selection to improve the application of the obtained aptamer pop?Lation in plasma.By controlling the number of the cell and library,combined with incubation time,washing intensity,the pressure of selection was gradually enhanced.After 14 rounds of selection,a high affinity and evolution pool of PANC-1 was initially screened.On this basis,two rounds of HBS selection were introduced in the last two rounds and aptamer pop?Lations of PANC-1 that were resistant to HBS interference were successf?Lly obtained.