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Site-specific fluorescent labeling of DNA using Staudinger ligaton

Bioconjugate Chemistry, ISSN: 1043-1802, Vol: 14, Issue: 3, Page: 697-701
2003
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Article Description

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 (∼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.

Bibliographic Details

Wang, Charles C-Y; Seo, Tae Seok; Li, Zengmin; Ruparel, Hameer; Ju, Jingyue

American Chemical Society (ACS)

Biochemistry, Genetics and Molecular Biology; Chemical Engineering; Engineering; Pharmacology, Toxicology and Pharmaceutics; Chemistry

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