Amplicon melting analysis with saturation dyes

摘要

Methods are provided for nucleic acid analysis wherein a target nucleic acid that is at least partially double stranded is mixed with a dsDNA binding dye having a percent saturation of at least 50% to form a mixture. In one embodiment, the nucleic acid is amplified in the presence of the dsDNA binding dye, and in another embodiment a melting curve is generated for the target nucleic acid by measuring fluorescence from the dsDNA binding dye as the mixture is heated. Dyes for use in nucleic acid analysis and methods for making dyes are also provided.

主权项

1. A kit for amplifying and subsequently melting a plurality of target nucleic acids comprising:
(a) deoxyribonucleotide triphosphates, (b) a polymerase, and (c) a dsDNA binding dye having a percent saturation of at least 90%, wherein the dye is operable to indicate the presence of heterozygous sequence alterations in the plurality of target nucleic acids and does not inhibit amplification of the plurality of target nucleic acids at 90% saturation, (d) a protocol for amplifying each of the plurality of target nucleic acids in the presence of the dsDNA binding dye using the deoxyribonucleotide triphosphates and the polymerase, to generate a plurality of amplified nucleic acids, and (e) a protocol for generating a fluorescence melting curve from each of the plurality of amplified nucleic acids and plotting the difference between fluorescence melting curves, wherein the kit is operable to distinguish a heterozygous genotype from a homozygous genotype or for scanning for heterozygous sequence alterations in the plurality of target nucleic acids.