Melt Curve Analysis

摘要

A method of high resolution melting curve analysis for characterizing nucleic acid molecules such as PCR products having a distinct Tm using fluorescence is provided. The technique comprises modeling the raw melting curve data as a sum of at least two signal components, the first signal component representing the light intensity emitted by unbound/free fluorophores and the one or more second signal components representing the combined light intensity emitted by fluorophores bound to double stranded DNA. Numerical analysis is used to determine the values of the different components contributing to the total signal such that the model matches the raw fluorescence data as closely as possible. The method enables an improved resolution of mixtures of target nucleic acids even at non-saturating dye concentrations because it takes into account the effect of redistribution of intercalating dye from low-temperature duplexes to duplexes that melt at higher temperatures.

主权项

1. A method for analyzing a melt curve characterizing the melt of a solution comprising one or more populations of nucleic acid molecules and a constant number of fluorophores of at least first type, the method comprising obtaining a fluorescence signal descriptive of melt curve data over a temperature range, the fluorescence signal representing the intensity of the light emitted by said fluorophores as a function of temperature,
modeling the fluorescence signal at a plurality of temperatures within the temperature range as a sum of a first signal component representing the combined light intensity emitted by unbound fluorophores of said first type in the solution at a given temperature and a set of one or more second signal components, each representing the combined light intensity emitted by said fluorophores bound to the respective nucleic acid molecule population at the given temperature,
wherein the first signal component is provided as a product of a first term representing the relative number of unbound fluorophores of said first type at the given temperature and a second term representing the emission efficiency of an unbound fluorophore of said first type at said given temperature, andwherein each second signal component is provided as a product of a respective third term representing the relative number of said fluorophores bound to the respective nucleic acid molecule population at the given temperature and a respective fourth term representing the emission efficiency of said fluorophore bound to the respective nucleic acid molecule population at said given temperature, and utilizing numerical analysis to determine the values of said first, second, third and fourth terms at said plurality of temperatures such that the difference between the fluorescence signal and the modeled fluorescence signal meets a predefined criterion.