Operator`s manual

21
Gene Scanning with the LightCycler® 480 System
Advantages of the High Resolution Melting Technique
Advantages of the High Resolution 3
Melting Technique
Melting curve analysis is based on a robust, post-PCR physical measurement and, there-
fore, offers several advantages over mutation detection methods that derive information
from the amplification process itself:
Any amplicon can be screened for unknown sequence variants with a single high- ►
resolution dye; you do not need to target a specific variant with allele-specific probes.
You can design a genotyping assay on the basis of less sequence data. ►
Each reaction generally reveals more information. ►
In addition, the homogeneous High Resolution Melting technique can process more
samples more conveniently than traditional, non-homogeneous methods (e.g., dHPLC or
SSCP) which require amplicons to be screened for sequence variants on a separate instru-
ment after PCR.
High Resolution Melting analysis identifies heterozygous single-base changes in PCR
products with a sensitivity and specificity that is comparable or superior to non-homoge-
neous techniques. Variants can be detected regardless of their position within the frag-
ment.
Identification of homozygous sequence alterations is more difficult; most other mutation
scanning methods are unable to distinguish these alterations from wild type sequences.
By contrast, High Resolution Melting has identified homozygous sequence alterations in
several different types of amplicons.