A method for detecting specific DNA sequences and discriminating single
nucleotide polymorphisms (SNPs) using fluorescently labelled
oligonucleotide probes is disclosed. Oligonucleotide probes are labelled
with a reporter molecule preferentially attached to an internal
nucleotide residue. The fluorescence emission of oligonucleotide probes
varies significantly when in single-stranded and double-stranded states
despite the absence of quencher moieties, allowing reliable detection of
complementary DNA targets. The melting temperature of probe/target
duplexes permits discrimination of targets that differ by as little as a
single nucleotide residue, such that polymorphic targets may be
discriminated by fluorescence quantitation and Tm. The hybridisation
probes of this invention have been demonstrated to accurately identify
homozygous and heterozygous samples using a single fluorescent
oligonucleotide and direct investigation of saliva with hybridisation
probes permits ultra-rapid genotypic analysis within 35-40 minutes.
Target detection and SNP discrimination assays have been achieved in
homogeneous, heterogeneous, `real-time` and solid-phase formats.