Precolumn derivatization of nucleotides based on fluorescent carbamate formation of the sugar moieties in high-performance liquid chromatography.
The fluorescence derivatization of nucleotides with 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzo++  furan in the presence of sodium azide and the separation of the derivatives by high-performance liquid chromatography are described. The reagent reacts with 5'-terminal hydroxyl groups of nucleotides to produce the corresponding fluorescent carbamates. The derivatives of mono- and oligonucleotides are separated by chromatography on a reversed phase column (TSKgel ODS-80TM) and the derivatives of octa- and deca-nucleotides on a size exclusion column (TSKgel G3000SWXL). The detection limits (signal-to-noise ratio = 3) are 0.8-6.0 pmol on column. 5'Phosphorylated nucleotide also gives a fluorescent derivative after alkaline phosphatase-mediated dephosphorylation.
Nagaoka
H
H
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Nohta
H
H
Saito
M
M
Ohkura
Y
Y
D001483:Base Sequence; D002219:Carbamates; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis*
D002219:Carbamates; D005456:Fluorescent Dyes; D009841:Oligonucleotides