Operator`s manual

17
Gene Scanning with the LightCycler® 480 System
Components of the LightCycler® 480 System Gene Scanning Assay
Components of the LightCycler2
®
480
System Gene Scanning Assay
To obtain meaningful gene scanning results from High Resolution Melting analysis, three
components (the DNA-binding dye, the analytical software, and the real-time PCR in-
strument itself) of the LightCycler® 480 System must work optimally. Here is a brief
look at how these three components have been engineered to work together well in a
LightCycler® 480 System Gene Scanning experiment.
LightCycler2.1
®
480 High Resolution Melting Dye
High Resolution Melting analysis relies on a new generation of dsDNA binding dyes.
LightCycler® 480 High Resolution Melting Dye is a member of this new dye family. This
unique dye can detect the presence of heteroduplexes formed during PCR (e.g., if the
sample is heterozygous for a particular mutation). This feature is not shared with other
dyes traditionally used in real-time PCR (e.g., SYBR Green I or ethidium bromide).
LightCycler® 480 High Resolution Melting Dye is not toxic to amplification enzymes.
Thus, high concentrations of the dye do not affect the PCR. These high concentrations
completely saturate the dsDNA in the sample.
dsDNA remains dye-saturated during the subsequent melting experiment. Under these
conditions, even small changes in the melting behavior result in subtle, but reproducible
changes in High Resolution Melting Dye fluorescence.
According to Wittwer et al (2003)
1)
, this occurs because the dye cannot redistribute
itself from denatured to non-denatured regions of the DNA during melting. Further,
the dyes no longer show a preference for products that melt at higher temperatures.
High fidelity correlation between fluorescence changes and DNA melting increases the
resolution of the recorded melt profiles.
1)
Carl T. Wittwer, Gudrun H. Reed, Cameron N. Gundry, Joshua G. Vandersteen, and Robert J. Pryor (2003).
High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen. Clinical Chemistry 49, 853–860