期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2003
卷号：100
期号：2
页码：409-413
DOI：10.1073/pnas.242729099
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Three single-stranded DNA molecules of different lengths were synthesized and characterized, each containing a fluorescent dye (6-carboxyfluorescein) connected to the 5' end via a photocleavable 2-nitrobenzyl linker and a biotin moiety at the 3' end. UV irradiation ({lambda} {approx} 340 nm) of solutions containing these fluorescent DNA molecules caused the complete cleavage of the nitrobenzyl linker, separating the fluorophore from the DNA. The photocleavage products were characterized by HPLC and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. Our experimental results indicated that the proximity of the chromophore 6-carboxyfluorescein to the 2-nitrobenzyl linker did not hinder the quantitative photocleavage of the linker in the DNA molecules. The biotin moiety allowed immobilization of the fluorescent DNA on streptavidin-coated glass chips. The photocleavage of the immobilized DNA was investigated directly by fluorescence spectroscopy. The results demonstrated that close to 80% of the fluorophore was removed from the immobilized DNA after UV irradiation at 340 nm. These results strongly support the application of the 2-nitrobenzyl moiety as an efficient photocleavable linker, connecting fluorescent probes to DNA molecules for a variety of biological analyses such as DNA sequencing by synthesis.
关键词：oligonucleotides‖fluorescence‖DNA sequencing by synthesis
