High-resolution melting analysis

Abstract

The present invention relates to methods and systems for the analysis of the dissociation behavior of nucleic acids and the identification of nucleic acids. Methods and systems are disclosed for identifying a nucleic acid in a sample including an unknown nucleic acid and for detecting a single nucleotide polymorphism in a nucleic acid in a sample. Methods and systems are disclosed for identification of a nucleic acid in a biological sample including at least one unknown nucleic acid by fitting denaturation data including measurements of a quantifiable physical change of the sample at a plurality of independent sample property points to a function to determine an intrinsic physical value and to obtain an estimated physical change function, and identifying the nucleic acid in the biological sample by comparing the intrinsic physical value for at least one unknown nucleic acid to an intrinsic physical value for a known nucleic acid.

1 . (canceled) 
     
     
         2 . A method for identifying one or more nucleic acids in at least one sample, the method comprising:
 providing a microfluidic device comprising at least one microfluidic channel;   filling at least one of the microfluidic channels with at least one sample;   amplifying the at least one sample in at least one of the microfluidic channels;   increasing the temperature of the at least one sample while illuminating the sample and obtaining images of the sample depicting a signal emanating from the sample during the temperature increase;   processing the images to obtain thermal dissociation data for the one or more nucleic acids;   providing a mathematical representation to model the thermal dissociation data, the mathematical representation being a sum including one or more terms, wherein each of the one or more terms is associated with one of the nucleic acids in the sample, wherein each of the one or more terms depends on the temperature of the sample and parameters defining the one of the nucleic acids in the sample;   fitting the measured thermal dissociation data to the mathematical representation to calculate the parameters defining each of the one or more nucleic acids in the sample; and   identifying each of the one or more nucleic acids in the sample based on the calculated parameters.   
     
     
         3 . The method of  claim 2 , further comprising:
 determining a number of peaks in the thermal dissociation data presented as a thermal dissociation curve; and   modeling the thermal dissociation data based on the determined number of peaks, wherein a number of the terms representing nucleic acids in the mathematical representation equals the number of peaks in the thermal dissociation curve.   
     
     
         4 . The method of  claim 2 , wherein the step of identifying each of the one or more nucleic acids comprises comparing the calculated parameters for each nucleic acid in the sample to parameters defining known nucleic acids. 
     
     
         5 . The method of  claim 2 , further comprising modelling a homozygous wild type by one term in the mathematical representation. 
     
     
         6 . The method of  claim 2 , further comprising modelling a heterozygous mutant by two terms in the mathematical representation. 
     
     
         7 . The method of  claim 2 , wherein the mathematical representation includes a baseline signal measured in absence of the nucleic acids. 
     
     
         8 . The method of  claim 2 , wherein the parameters defining the one or more nucleic acids are used as inputs to a classifier trained to predict a genotype of the sample. 
     
     
         9 . The method of  claim 2 , wherein the sample further includes a fluorescent dye, and wherein the signal emanating from the sample and indicative of a nucleic acid denaturation is selected from fluorescence intensity and ultraviolet absorbance of the dye. 
     
     
         10 . A system for identifying one or more nucleic acids in a sample, the method comprising:
 a microfluidic device comprising at least one microfluidic channel;   a heating unit to increase and decrease temperature of the at least one microfluidic channel;   a measuring unit to obtain thermal melt data for the one or more nucleic acids by measuring a signal emanating from the sample as the temperature of the sample is ramped, the signal being indicative of a nucleic acid denaturation;   a modelling unit to provide a mathematical representation for the thermal melt data, the mathematical model being a sum including one or more terms, wherein each of the one or more terms is associated with one of the nucleic acids in the sample and depends from the temperature of the sample and parameters defining the one of the nucleic acids in the sample;   a fitting unit to fit the measured melting data to the mathematical representation to calculate the parameters defining the one or more nucleic acids in the sample; and   an identification module to identify each of the one or more nucleic acids based on the calculated parameters.