Site-specific fluorescent labeling of DNA using Staudinger ligation.
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.
Wang
Charles C-Y
CC
Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Seo
Tae Seok
TS
Li
Zengmin
Z
Ruparel
Hameer
H
Ju
Jingyue
J
D001665:Binding Sites; D002850:Chromatography, Gel; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D004247:DNA