Synthesis and studies of modified nucleotides and oligonucleotides

Abstract

Large-scale genotyping programmes employ fluorescently-labelled nucleoside triphosphates as a means of facilitate detection. A novel protocol for the solid-phase synthesis of these compounds is described.

A series of nucleoside-5'-triphosphate analogues with structurally dissimilar linker arms is outlined. A variety of dyes were introduced to the terminus of the linker arms whilst attached to the solid support.

A series of PCR labelling experiments were performed to evaluate the incorporation of the labelled nucleoside triphosphate analogues by DNA polymerase enzymes. Good yields were obtained when replacing 40-90% of the unmodified dTTP with the modified dUTP analogues.

A simple, novel real-time PCR based assay, which is amenable to high throughput genotyping programmes is described. The assay requires a probe with a single fluorescent dye, two primers and triphosphate analogues labelled with a quencher molecule. Initially the system is fluorescent, however during PCR a decrease in fluorescence will be observed as the probe hybridises to the target and the fluorophore is quenched by the modified triphosphates incorporated into the nascent strand. The synthesis of a Peptide Nucleic Acid monomer and a Carbocyclic nucleoside is outlined.

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