1. www.roche-applied-science.com 1 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

2. www.roche-applied-science.com 2 Allel Specific PCR Allel 1 Allel 2

3. www.roche-applied-science.com 3 LightCycler ® 480 End-Point Genotyping Analysis of Hydrolysis Probe Assays Excel macro (“Taqxel” tool) available for analysis of LightCycler ® 480 data

4. www.roche-applied-science.com 4 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.

5. www.roche-applied-science.com 5 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)

6. www.roche-applied-science.com 6 On excel: Open FAM.xml and VIC.xml Create LightCycler ® 480 Data for “Taqxel Tool” Analysis (3)

7. www.roche-applied-science.com 7 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 of LightCycler ® 480 Data (4)

8. www.roche-applied-science.com 8 Click on Graph to visualize groups homozygote FAM homozygote VIC heterozygote “Taqxel Tool” for the Analysis of LightCycler ® 480 Data (5)

9. www.roche-applied-science.com 9 negative “Taqxel Tool” for the Analysis of LightCycler ® 480 Data (6)

10. www.roche-applied-science.com 10 unknown “Taqxel Tool” for the Analysis of LightCycler ® 480 Data (7)

11. www.roche-applied-science.com 11 Click on Analyse to visualize the listed genotypes and to adjust angle settings “Taqxel Tool” for the Analysis of LightCycler ® 480 Data (8)