MUTATION DETECTION IN HIGHLY HOMOLOGOUS GENOMIC REGIONS

摘要

PROBLEM TO BE SOLVED: To provide methods for distinguishing highly homologous genomic regions and detecting variants at a locus of interest.SOLUTION: In one embodiment, the method comprises the followings: (a) providing an aliquot of a nucleic acid having a locus of interest; (b) incubating the aliquot of the nucleic acid with a limiting primer, an excess primer, and a probe that is designed to hybridize to the locus of interest on a target strand of the nucleic acid; (c) performing asymmetric PCR using the aliquot to produce an excess of amplicons corresponding to the target strand to which the probe hybridizes, thereby producing a probe-amplicon element; (d) generating a melting curve for the probe-amplicon element in a mixture with a saturating binding dye by measuring fluorescence from the dye as the mixture is heated; and (e) analyzing the melting curve to determine whether a variant is present or not at the locus of interest.SELECTED DRAWING: Figure 8