Patent ID: 9447458
Filing Date: 2016-09-20
CPC Classification: C12Q

Claim Text:
1. A method of distinguishing between at least two nearby neighbor variants on a locus of interest on a gene, the method comprising: (a) providing a first aliquot of said nucleic acid having the locus of interest; (b) incubating said first aliquot of said nucleic acid with a limiting primer, an excess primer, and a first probe that is designed to hybridize to said locus of interest on a target strand of said nucleic acid; (c) performing asymmetric PCR using said first aliquot to produce an excess of amplicons corresponding to the target strand to which the first probe hybridizes, thereby producing a first probe element; (d) providing a second aliquot of said nucleic acid target having the locus of interest; (e) incubating said second aliquot of said nucleic acid target with said limiting primer, said excess primer, and a second probe that is designed to hybridize to said locus of interest on the target strand, wherein said first probe differs in sequence from said second probe in length; (f) performing asymmetric PCR using said second aliquot to produce an excess of amplicons corresponding to the target strand to which the second probe hybridizes, thereby producing a second probe element, wherein said first probe and said second probe each have a sequence that is complementary to a wild-type sequence of the target strand, each of said first and second probes covering the same at least two nearby neighbor variants; (g) generating a first melting curve for the first probe element in a first mixture with a saturating binding dye by measuring fluorescence from said dye as the first mixture is heated; (h) generating a second melting curve for the second probe element in a second mixture with said saturating binding dye by measuring fluorescence from said dye as the second mixture is heated; and (i) analyzing said first melting curve and said second melting curve to distinguish between said at least two nearby neighbor variants, wherein a melting signature curve of each of said at least two nearby neighbor variants is different in said first and second melting curves.