Operator`s manual

15
Gene Scanning with the LightCycler® 480 System
High Resolution Melting
High Resolution Melting1
The key technique in gene scanning, High Resolution Melting, is a refinement of earlier,
well-established DNA dissociation (or “melting”) techniques (e.g., to determine the T
m
of a
DNA hybrid). Like all melting analyses, the technique subjects DNA samples to increasing
temperatures and records the details of their dissociation from double-stranded (dsDNA)
to single-stranded form (ssDNA).
Before a High Resolution Melting analysis can be performed, the target sequence must be
available in high copy number. The easiest way to accomplish this is to perform a DNA
amplification reaction (PCR) before the High Resolution Melt.
Both procedures are performed in the presence of a fluorescent dye that binds only dsDNA.
The dye does not interact with ssDNA, but fluoresces strongly in the presence of dsDNA.
This change in fluorescence can be used both to measure the increase in DNA concentra-
tion during PCR and then to directly measure thermally-induced DNA dissociation dur-
ing High Resolution Melting.
For detection of sequence variations, differences in the melting curves of the amplicons
are analyzed. Heterozygote DNA forms heteroduplices that begin to separate into single
strands at a lower temperature and with a different curve shape than homozygote DNA.
Depending on the individual sequence, most of the different homozygotes give distin-
guishable melting curves, too.
In a melting experiment, fluorescence is initially high because the sample starts as dsDNA,
but fluorescence diminishes as the temperature is raised and DNA dissociates into single
strands. The observed “melting” behavior is characteristic of a particular DNA sample.
Mutations in PCR products are detectable because they change the shape of the melt-
ing curve. When the mutant sample is compared to a reference “wild type” sample, these
changes are visible. Below is an example of an experiment with the LightCycler® 480
Instrument and the LightCycler® 480 High Resolution Melting Dye that identifies a single
nucleotide polymorphism (SNP).