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Abstract

Methods for detecting single nucleotide polymorphisms in a sample by applying an energy gradient to a mixture of probe-hybridized target wild type and mutant polynucleotides are provided. A nucleotide probe, having a nucleotide sequence complementary to at least a portion of the wild type or mutant polynucleotide(s), is exposed to a sample containing target wild type polynucleotide(s) and target mutant polynucleotide(s) under hybridizing conditions. Target wild type polynucleotide(s) form homoduplexes with the wild type probe, while target mutated polynucleotide(s) form heteroduplexes with the wild type probe. Target mutant polynucleotide(s) form homoduplexes with the mutant probes, while target wild type polynucleotide(s) form heteroduplexes with the mutant probe. An energy gradient is applied to the duplex-containing sample to induce selective denaturation of the duplexes. The gradient may be a thermal or chemical one and is preferably temporally linear. The hetero-and homo-duplexes deanneal at different times so that the detection of more than one type of polynucleotide(s) is indicative of a single nucleotide polymorphism. Capillary electropheresis, gel electropheresis, high performance liquid chromatography, or microfluidics are typically used for identification of the separated strands.

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