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Proceedings Paper

Hole-burning and picosecond time-resolved spectroscopy of isolated molecular clusters
Author(s): Stacey A. Wittmeyer; Andrew J. Kaziska; Maria I. Shchuka; Michael R. Topp
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Paper Abstract

A precolumn derivatization method has been developed for high performance liquid chromatographic (HPLC) analysis of DNA damage using fluorescence detection. The modified nucleotide, having excised enzymatically from the exposed DNA, is enriched from the normal nucleotides and labeled with a fluorescent reagent. The labeling procedure involves phosphoramidation of the nucleotide with ethylenediamine (EDA) followed by conjugation of the free amino end of the phosphoramidate with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride. The dansylated nucleotide can be analyzed with a sub-picomole limit of detection (LOD) by conventional HPLC using a conventional fluorescence detector. By combining microbore HPLC with laser-induced fluorescence (LIF) detection, we present the development of an analytical system that has sub-femtomole LOD for real-time analysis of the dansylated nucleotide. The application of the developed system in fluorescence postlabeling assay of a small alkyl-modified nucleotide (5-methyldCMP) in calf-thymus DNA is discussed.

Paper Details

Date Published: 1 July 1991
PDF: 12 pages
Proc. SPIE 1435, Optical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications, (1 July 1991); doi: 10.1117/12.44250
Show Author Affiliations
Stacey A. Wittmeyer, Univ. of Pennsylvania (United States)
Andrew J. Kaziska, Univ. of Pennsylvania (United States)
Maria I. Shchuka, Univ. of Pennsylvania (United States)
Michael R. Topp, Univ. of Pennsylvania (United States)

Published in SPIE Proceedings Vol. 1435:
Optical Methods for Ultrasensitive Detection and Analysis: Techniques and Applications
Bryan L. Fearey, Editor(s)

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