Solid-phase synthesis of chelate-labelled oligonucleotides: application in triple-color ligase-mediated gene analysis.
Oligonucleotides labelled with detectable groups are essential tools in gene detection. We describe here the synthesis of pyrimidine deoxynucleotide-building blocks, modified at their C-5 position with a protected form of a strongly chelating agent. These reagents can be used to introduce multiple metal ions into oligodeoxynucleotides during standard oligonucleotide synthesis. The chelating functions form strongly fluorescent complexes with europium ions, characterized by a wide separation between the excitation and emission spectra. Moreover, the long decay time of the fluorescence permits sensitive time-resolved fluorescence measurements. The chelates also have the stability required to function in triple-color assays involving europium, samarium, and terbium ions. We demonstrate the application of these reagents for ligase-based gene analysis reactions.
Kwiatkowski
M
M
Beijer Laboratory, Department of Medical Genetics, Uppsala, Sweden.
Samiotaki
M
M
Lamminmäki
U
U
Mukkala
V M
VM
Landegren
U
U
D001483:Base Sequence; D002614:Chelating Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D011088:DNA Ligases / Q000378:metabolism; D005453:Fluorescence; D006801:Humans; D008674:Metals, Rare Earth; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009705:Nucleosides; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D016133:Polymerase Chain Reaction
D002614:Chelating Agents; D008674:Metals, Rare Earth; D009705:Nucleosides; D009838:Oligodeoxyribonucleotides; D011088:DNA Ligases