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Content
Principles and advantages of melting-curve based genotyping
LightCycler® 480 Genotyping Software - examples
Discrimination by allel specific PCR, end-point
genotyping on the LightCycler® 480 System
High-Resolution Melting for mutation scanning, genotyping and
GC-tail Tm shift
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Allel Specific PCR
Allel 1 Allel 2
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LightCycler® 480 End-Point Genotyping Analysis of Hydrolysis Probe Assays
Excel macro (“Taqxel” tool) available for analysis of LightCycler® 480 data
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Create LightCycler® 480 Data for “Taqxel Tool” Analysis (1)
After PCR with Hydrolysis probes (run on LC480 or another instrument)
perform an endpoint measurement by programming a short "Melting Curve"
from 60-61°C.
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After run open analysis "Tm Calling" and export raw data with a right mouse click into the Melting Curves graph, save as .xml format: set filter combination: 533; color comp: on, for FAM data
set filter combination: 568; color comp: on, for VIC/Hex data
Create LightCycler® 480 Data for “Taqxel Tool” Analysis (2)
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On excel: Open FAM.xml and VIC.xml
Create LightCycler® 480 Data for “Taqxel Tool” Analysis (3)
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Open “Taqxel tool“ and copy/paste the FAM and VIC excel sheets into the respective FAM/VIC folder of the analysis macro
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (4)
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Click on Graph to visualize groups
homozygote FAM
homozygote VIC
heterozygote
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (5)
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negative
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (6)
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unknown
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (7)
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Click on Analyse to visualize the listed genotypes and to adjust angle settings
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (8)