I am working on IDH1/2 mutations and I red a lot of papers using one primer set for different mutations in the same gene IDH1 and IDH2, but I can not understand how HRM can differentiate between each different mutations in same gene using the same primer which can bind in the flanking sequences.
HRM relies on the fluorescence of a dye bound between the 2 strands of DNA. As the strands melt the melted area becomes single stranded and the fluorescence drops as the temperature rises and the strands melt and dye is released. You can get more than one result from an amplimer because of the melting characteristics of the different base changes . A change in a high GC region will melt differently from a change in a high AT region and even just the bases either side of the mutation site will make one change melt under slightly different conditions from a base change elsewhere. Both the position and the shape of the melt curve will give information about a melting change in an amplimer
HRM relies on the fluorescence of a dye bound between the 2 strands of DNA. As the strands melt the melted area becomes single stranded and the fluorescence drops as the temperature rises and the strands melt and dye is released. You can get more than one result from an amplimer because of the melting characteristics of the different base changes . A change in a high GC region will melt differently from a change in a high AT region and even just the bases either side of the mutation site will make one change melt under slightly different conditions from a base change elsewhere. Both the position and the shape of the melt curve will give information about a melting change in an amplimer
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