Optimization of Polymerase Chains Reaction Amplification for ssDeoxyribonucleic Acid Library Using Capillary Electrophoresis with Laser-induced Fluorescence Detection

For random DNA libraries, the amplification of polymerase chains reaction(PCR) is very important to select aptamers by systematic evolution of ligand exponential enrichment(SELEX). The influencing factors of PCR amplification were investigated and optimized according to the changes of dsDNA product, ss-dsDNA by-products and primers using capillary electrophoresis with laser-induced fluorescence detection(CE-LIF). The results suggested that there were prominent differences between PCR amplification of homogeneous DNA templates and that of random DNA libraries. For random DNA library, the yield of products reached its maximum at the eighteenth cycle. Other influence factors including the number of initial templates, anneal temperature of PCR, concentration of DNA polymerase were also investigated. To ensure efficiency of SELEX, it is necessary to optimize the PCR amplification conditions of random DNA libraries. In this study, for the N39 DNA libraries, the optimized conditions were as follows: number of initial templates was 105 molecules, concentration of DNA polymerase was 0.05 U/μL, and annealing temperature of PCR was 70 ℃, the optimization PCR cycles was 18. This study provided important references for the selection of high efficient aptamers by SELEX method.