AMPLICON melting analysis with saturation dyes
AssigneesUniversity of Utah Research Foundation, Idaho Technology, Inc.
InventorsCarl T. Wittwer, Virginie Dujols
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Abstract
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.
Claims7
A method of PCR analysis comprising the steps of:
- providing a mixture of a dsDNA binding dye, at least one target nucleic acid, and primers configured for amplifying the target nucleic acid,
- amplifying the target nucleic acid in the presence of the dsDNA binding dye,
- monitoring fluorescence of the dsDNA binding dye,
- generating a melting curve for the target nucleic acid,
- normalizing magnitude differences of the melting curve,
- repeating the providing, amplifying, generating and normalizing steps with at least one additional target nucleic acid, and
- plotting a fluorescence difference between the magnitude-difference-normalized melting curves, wherein the magnitude-difference-normalized melting curve of one target nucleic acid is selected as a standard and is plotted as the standard across melting temperatures and the magnitude-difference-normalized melting curve for each additional target nucleic acid is plotted as a difference from the standard across the melting temperatures.
Dependencies are inferred from the claim text.