Operator`s manual

19
Gene Scanning with the LightCycler® 480 System
Components of the LightCycler® 480 System Gene Scanning Assay
Temperature shifting:3. The next step is to shift the temperature axis of the normal ized
melting curves at the point where the entire double-stranded DNA is completely de-
natured. For this, the software automatically applies a default Temp Shift Threshold of
5% to all data. (If you wish, you can set this threshold manually to a different value.)
Now, samples with heterozygous SNPs can easily be distinguished from the wild type
by the different shapes of their melting curves.
normalized melting curves normalized, temp-shifted melting curves
Difference Plot:4. The final step is to further analyze the differences in melting curve
shape by subtracting the curves from a reference curve (also called “base curve”), thus
generating a Difference Plot, which helps cluster samples automatically into groups
that have similar melting curves (e.g., those with the same genotype).
The way melting curves for homozygotes and heterozygotes are plotted depends on
the base curve you selected for the Difference Plot. In the example above, a homozy-
gote sample was selected as base curve, resulting in negative melting curves for the
heterozygotes. (This is because heterozygotes melt at a lower temperature than ho-
mozygotes.) If you selected a heterozygote sample as base curve, homozygotes would
appear as positive melting curves in the example shown.