Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling |
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| Authors: | JunZhou Wu Jie Wang XinJing Tang |
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| Institution: | 1. State Key Laboratory of Natural and Biomimetic Drugs; the School of Pharmaceutical Sciences, Peking University, Beijing, 100191, China
2. State key Laboratory of Drug Research; Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China
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| Abstract: | Two new photolabile nucleotide analogues with furan-fused deoxyuridine were synthesized through Sonogashira coupling. Their enzymatic incorporation into DNA was evaluated with two DNA polymerases (Taq and Deep vent exo-) by polymerase chain reaction (PCR). Deep vent exo-recognized both nucleotides as substrates for primer extension, while Taq was much less proficient. Light irradiation of PCR products released the amino and carboxyl moieties of DNA. Further labeling with fluorescein isothiocyanate for a long DNA construct with F-dUnTP incorporation was successfully achieved. |
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| Keywords: | photolabile nucleotides enzymatic incorporation nucleotide analogues fluorescent labeling |
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