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  • Title: Site-specific fluorescent labeling of DNA using Staudinger ligation.
    Author: Wang CC, Seo TS, Li Z, Ruparel H, Ju J.
    Journal: Bioconjug Chem; 2003; 14(3):697-701. PubMed ID: 12757398.
    Abstract:
    We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.
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