Xu F.-L.,Chongqing Cancer Institute and Chongqing Cancer Hospital |
Zhou Q.,Chongqing Cancer Institute and Chongqing Cancer Hospital |
Kuang Y.,Chongqing Cancer Institute and Chongqing Cancer Hospital |
Chen X.-L.,Chongqing Cancer Institute and Chongqing Cancer Hospital |
And 2 more authors.
Journal of Shanghai Jiaotong University (Medical Science) | Year: 2013
Objective: To establish commonly used technical platform of cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) for screening DNA aptamers specifically binding to tumor cells. Methods: A random double-stranded DNA (dsDNA) pool was established by PCR, and single-stranded DNA (ssDNA) library was then separated from dsDNA pool by using avidin coated Sepharose. In order to get DNA aptamers for cancer cells, cell-SELEX and PCR were performed by using cultured gastric cancer cell line as positive screening target while normal gastric mucosal cell line as negative. Flow cytometry (FCM) was used to monitor the enrichment efficiency and affinity of aptamers. Cloning, sequencing, and sequence analysis of aptamers by conventional molecular biology techniques were also carried out. Results: A random ssDNA pool was successfully established and specific aptamers of tumor cells were got after 12 runs' cell-SELEX. Of 120 positive sequenced clones, 21 aptamers were identified. Accurate alignment of Blastn implied that not any similar sequence to 21 submitted aptamers was found in NCBI data base. Conclusion: The article provided a technical platform for obtaining DNA aptamers specifically binding to tumor cells in a relatively short period of time. Source