MUTATION DETECTION IN HIGHLY HOMOLOGOUS GENOMIC REGIONS

摘要

The present invention relates to methods, kits, primers and probes for use in distinguishing highly homologous genomic regions and detecting variants in a locus of interest. In one aspect, the present invention is useful for distinguishing Exon 9 of the CFTR gene from a large homologous region of chromosome 20 in order to determine the presence of the A455E variant.

主权项

1. A method of identifying a variant on a target strand of a nucleic acid molecule having a locus of interest that is substantially homologous to a second genomic region, comprising:
(a) incubating an aliquot of said nucleic acid with a limiting primer, an excess primer, and a probe that is designed to hybridize to said locus of interest on a target strand of said nucleic acid; (b) performing asymmetric PCR using said aliquot to produce an excess of amplicons corresponding to the target strand to which the probe hybridizes, thereby producing a probe-amplicon element; (c) 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; (d) analyzing the melting curve to determine whether a variant is present.