Operator`s manual

22
LightCycler® 480 Gene Scanning Software
Preparing a LightCycler® 480 System Gene Scanning Experiment
Designing the Gene Scanning Assay
Preparing a LightCyclerB
®
480
System Gene Scanning
Experiment
Designing the Gene Scanning Assay1
These guidelines will help you design an effective Gene Scanning assay:
A single base variation affects the melting behavior of a 100 bp amplicon more than ►
a 500 bp amplicon; thus, short amplicons are more likely to show the effects of small
sequence changes. Therefore, it is recommended to select PCR primers that amplify a
relatively short sequence (100 - 250 bp).
Primers should anneal at temperatures around 60°C.
Nevertheless, it is possible to target longer sequences (up to 500 bp), but keep in mind
that analysis of these products will usually have lower resolution. In addition, such
products (>250 bp) are more likely to contain multiple melting domains and generate
complicated melting curves.
Secondary structures can affect the efficiency of the amplification reaction. Therefore, ►
it might be of advantage to determine the folding characteristics of both primers and
amplicon with software that can profile secondary structures.
Make sure to set the folding temperature equal to the annealing temperature that will
be used for the reaction (e.g., 60°C).
The DINAMelt Server from Rensselaer Polytechnic Institute provides appropriate soft-
ware for such secondary structure analyses; this software can make corrections for
both salt and magnesium concentration (see http://www.bioinfo.rpi.edu/applications/
hybrid/twostate.php).
Low delta-G values indicate a high level of secondary structure. Strands with high
delta-G values produce less secondary structures and so are favored in the amplifica-
tion reaction. For best results, the delta-G values should be above -1.