Patent ID: 7759126
Filing Date: 2010-07-20
Classification: C07H,C12Q,Y10T

Abstract:
1. 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 MB is a minor groove binder, Q is a quencher, ODN is an oligonucleotide or modified oligonucleotide, K is a bond or a linking group, Fl is a fluorophore and W is a trivalent linking group that provides sufficient spacing between the MB, F and ODN components such that: i) MB binds in the minor groove when said oligonucleotide conjugate hybridizes to said target sequence; ii) in an unhybridized form, the fluorescence of Fl in said oligonucleotide conjugate is less than 10% of unquenched fluorescence; and iii) when said oligonucleotide conjugate hybridizes to said target sequence, the fluorescence of Fl is at least 50% of its unquenched fluorescence; and wherein the conjugate forms a stable hybrid only with said target sequence that is perfectly complementary to the ODN portion of said conjugate, and the conjugate does not form a stable hybrid with any of the other polynucleotides; and (b) measuring the fluorescence produced on hybrid formation 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.