Nucleic acid melting analysis with saturation dyes

摘要

The invention provides a method for nucleic acid analysis comprising the steps of
mixing a target nucleic acid with a saturating dsDNA binding dye and an unlabeled probe configured to hybridize to a portion of the target nucleic acid, to form a mixture,
amplifying the target nucleic acid to generate an amplicon,
allowing the unlabeled probe to hybridize to the amplicon to form a probe/amplicon duplex,
generating a melting curve for the probe/amplicon duplex by measuring fluorescence from the dsDNA binding dye as the mixture is heated, and
analyzing the shape of the melting curve. Also provided are kits for analyzing a target nucleic acid comprising:
oligonucleotide primers configured for amplifying the target nucleic,
a saturating dsDNA binding dye.
a thermostable polymerase, and
acid an unlabeled probe configured to hybridize at least partially to the target nucleic acid.