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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)