Melting curve (dissociation) analysis

/Melting curve (dissociation) analysis

Melting curve (dissociation) analysis

2017-12-05T09:46:47+00:00 December 5th, 2017|

Every piece of dsDNA has a melting point (Tm) at which temperature 50% of the DNA is single stranded. The temperature depends on the length of the DNA, sequence order, G:C content and Watson-Crick pairing. When DNA-binding dyes are used, as the fragment is heated, a sudden decrease in fluorescence is detected when Tm is reached (due to dissociation of DNA strands and release of the dye). This point is determined from the inflection point of the melting curve or the melting peak of the derivative plot (what is meant by derivative plot is the negative first-derivative of the melting curve). The same analysis can be performed when hybridization probes are used as they are still intact after PCR. As hydrolysis probes (e.g., TaqMan®) are cleaved during the PCR reaction, no melting curve analysis possible if they are used (because of their specificity, there is no need either). Mismatch between a hybridization probe and the target results in a lower Tm. Melting curve analysis can be used in known and unknown (new) mutation analysis as a new mutation will create an additional peak or change the peak area. See Ririe, 1997 & an overview by PremierBiosoft for details of melting curve analysis. High-resolution melting curve analysis can be achieved on most real-time PCR instruments (for example CFX96 or Corbett’s Rotor-Gene 6000) or on dedicated instruments like Idaho Technology’s LightScanner®. See Human Mutation Special Issue (June 2009) on HRM.

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