Characterization of fluorescence-labeled DNA by time-resolved fluorescence spectroscopy

Claus A. M. Seidel; K. Rittinger; John A. Cortes; R. S. Goody; Malte Koellner; Juergen M. Wolfrum; Karl-Otto Greulich

doi:10.1117/12.44221

1 June 1991 Characterization of fluorescence-labeled DNA by time-resolved fluorescence spectroscopy

Claus A. M. Seidel, K. Rittinger, John A. Cortes, R. S. Goody, Malte Koellner, Juergen M. Wolfrum, Karl-Otto Greulich

Proceedings Volume 1432, Biomolecular Spectroscopy II; (1991) https://doi.org/10.1117/12.44221
Event: Optics, Electro-Optics, and Laser Applications in Science and Engineering, 1991, Los Angeles, CA, United States

Abstract

A strategy for DNA characterization was developed, which exploits the normally unwanted interactions of the dye with its environment. With a single suitable dye, acting as a sensitive probe, one can characterize the neighboring DNA-base by time-resolved fluorescence spectroscopy due to specific dynamic quenching reactions affecting the fluorescence lifetime. By specific coupling of the dye to a linker-group at the 3' terminus of DNA a one- label/one-lane concept for DNA sequencing seems to be possible and all problems with multiple dyes can be avoided. To study the feasibility of this concept, fluorescence lifetimes of various model phosphorothioate oligodeoxyribonucleotides, different in length and labelled with a specially synthesized coumarin derivative, were measured in different aqueous solutions with a DFDL-UV picosecond fluorescence spectrometer. The influence of nucleic acid-dye interactions on the lifetime and quantum yield of the coupled dye is investigated. In mononucleotides fluorescence lifetimes range between 5.3 and 1.4 ns. The order in the quenching efficiency of the bases is adenine (A) equals 0, cytosine (C) < thymine (T) < guanine (G). In labelled di- and oligonucleotides the order of quenching changes slightly: A equals 0, C < G.

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Claus A. M. Seidel, K. Rittinger, John A. Cortes, R. S. Goody, Malte Koellner, Juergen M. Wolfrum, and Karl-Otto Greulich "Characterization of fluorescence-labeled DNA by time-resolved fluorescence spectroscopy", Proc. SPIE 1432, Biomolecular Spectroscopy II, (1 June 1991); https://doi.org/10.1117/12.44221

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