Real-time linear detection probes: sensitive 5′-minor groove binder-containing probes for PCR analysis

A method for distinguishing between wild-type, mutant and heterozygous target polynucleotides, said method comprising:

• (a) contacting a sample containing a target polynucleotide with two probes wherein a first probe is specific for said wild-type target polynucleotide and a second probe is specific for said mutant target polynucleotide, each of said probes having a formula:
• wherein MGB is a minor groove binder, Q is a quencher, W is a trivalent linking group, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group and Fl is a fluorophore;
• wherein said first and second probes have different fluorophores and each of said probes forms a stable hybrid only with the target sequence that is perfectly complementary to the ODN portion of said probes;
• and
• (b) measuring the fluorescence produced on hybrid formation for each fluorophore, wherein said measuring is carried out at two wavelengths and is measured as a function of temperature, and using melting curve analysis to indicate the presence or absence of each of said wild-type, mutant and heterozygous target polynucleotides.