Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping...
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![Page 1: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/1.jpg)
1 www.roche-applied-science.com
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
![Page 2: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/2.jpg)
2 www.roche-applied-science.com
Allel Specific PCR
Allel 1 Allel 2
![Page 3: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/3.jpg)
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3
LightCycler® 480 End-Point Genotyping Analysis of Hydrolysis Probe Assays
Excel macro (“Taqxel” tool) available for analysis of LightCycler® 480 data
![Page 4: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/4.jpg)
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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.
![Page 5: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/5.jpg)
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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)
![Page 6: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/6.jpg)
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On excel: Open FAM.xml and VIC.xml
Create LightCycler® 480 Data for “Taqxel Tool” Analysis (3)
![Page 7: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/7.jpg)
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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)
![Page 8: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/8.jpg)
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Click on Graph to visualize groups
homozygote FAM
homozygote VIC
heterozygote
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (5)
![Page 9: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/9.jpg)
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negative
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (6)
![Page 10: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/10.jpg)
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unknown
“Taqxel Tool” for the Analysis ofLightCycler® 480 Data (7)
![Page 11: Www.roche-applied-science.com 1 Content Principles and advantages of melting-curve based genotyping LightCycler ® 480 Genotyping Software - examples Discrimination.](https://reader035.fdocuments.in/reader035/viewer/2022062804/56649ebf5503460f94bc9ea9/html5/thumbnails/11.jpg)
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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)