Amplicon melting analysis with saturation dyes

摘要

The invention provides:
A kit for amplifying and subsequently melting a plurality of target nucleic acids comprising:
(a) amplification reagents;
(b) a dsDNA binding dye having a percent saturation of at least about 90%;
(c) information on amplifying each of the plurality of target nucleic acids in the presence of the dsDNA binding dye using the amplification reagents, to generate a plurality of amplified nucleic acids; and
(d) information on generating a fluorescence melting curve from each of the plurality of amplified nucleic acids and plotting the difference between each of the fluorescence melting curves. The invention also provides a method of PCR analysis comprising the steps of:
providing replicates of a mixture of a dsDNA binding dye, a target nucleic acid having a specific genotype, and primers configured for amplifying the target nucleic acid,
amplifying each of the replicates of the target nucleic acid in the presence of the dsDNA binding dye,
monitoring fluorescence of the dsDNA binding dye, generating a melting curve for each of the replicates of the target nucleic acid,
repeating the providing, amplifying and generating steps with at least one additional target nucleic acid,
establishing the target nucleic acid melting curve as a standard across temperatures using the replicates of the target nucleic acid having the specific genotype, and
plotting a fluorescence difference between the standard and the melting curve of the at least one additional target nucleic acid.