Analysis
Click ► Analysis in the Module bar, select Endpoint Genotyping and your corresponding analysis subset in the Create New Analysis
dialog box.
Assign allele X and allele Y to the corresponding filter combinations ( ► e.g., FAM for allele X and HEX for allele Y).
The ► Genotyping Analysis screen opens - click Calculate to view the Scatter Plot or the Fluorescence data, result table and multiwell
plate image for automatic genotype calling.
The multi-select button ► Auto Group applies automated grouping, in case no sample is defined as standard in the Sample Editor.
If samples are defined as standard in the Sample Editor, the option Standards (In Run) is enabled by default, calling the genotypes
according to the standards included in the run.
Depending on chemistry and assay parameter the ► Analysis Mode 1 or Analysis Mode 2 can be selected. In most cases both settings
will result in correct grouping. If the groups are not separated properly (depending on the PCR chemistry used and the analyzed
parameters), test both settings for optimized results.
You can manually change any genotype by using the ► New Call option. To do so, select the samples in the multiwell plate image or
the result table. Alternatively use the Select button above the graph by dragging the wanted samples.
Report
Create a report by clicking the ► Report button.
Generate ► an analysis report containing general and detailed experiment information and analysis results. You can customize the
report to include several result items.
Print the report, or save the report as pdf file. ►
The LightCycler
®
480 System
Short Guide
Topic: Endpoint Genotyping
Purpose: Describes how to set up and perform Endpoint Genotyping assays using Hydrolysis probes for the
detection of known SNPs
Assay Principle: A signal is generated when dyes, bound to allele-specific probes, are cleaved. Dependent on the dif-
ferent hybridization intensity between probe and target caused by mismatch or perfect match, the
corresponding fluorescent signal can be measured.
Detection Format: Hydrolysis Probe assays, dual color, FAM / HEX (VIC)-labeled
Result: Automatic sample grouping according to genotype.
An optional control concept can be applied when standards and negative control are defined.
Results can be displayed as scatter plot and table view.
Requirements
System LightCycler
®
480 System (96 or 384)
LightCycler
®
480 Software, Version 1.5
LightCycler
®
480 Multiwell Plate 96 or 384, white
Reagents LightCycler
®
480 Probes Master
Unknown samples (5-40ng purified genomic DNA in 5µl or 20µl reaction volume) and optionally stan-
dards and negative controls
SNP assay mix containing specific primers,
one Hydrolysis Probe labeled with FAM dye to detect the allele X sequence, and one Hydrolysis Probe
labeled with HEX (VIC) dye to detect the allele Y sequence
For detailed information on Endpoint Genotyping please refer to the LightCycler® 480 Operator’s Manual,
section Endpoint Genotyping Analysis.
Ordering Information
Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related
products, please visit our special interest sites for the LightCycler® 480 Real-Time PCR System: http://www.lightcycler480.com
Software
LightCycler
®
480 Software, Version 1.5 1 software package 04 994 884 001
LightCycler
®
480 LIMS Interface Module 1 software package 05 066 310 001
LightCycler
®
480 Gene Scanning Software 1 software package 05 103 908 001
LightCycler
®
480 Multiple Plate Analysis Software 1 software package 05 075 122 001
Disposables
LightCycler
®
480 Multiwell Plate 96, white 50 plates with 50 sealing foils 04 729 692 001
LightCycler
®
480 Multiwell Plate 384, white 50 plates with 50 sealing foils 04 729 749 001
LightCycler
®
480 Multiwell Plate 96, clear 50 plates with 50 sealing foils 05 102 413 001
LightCycler
®
480 Multiwell Plate 384, clear 50 plates with 50 sealing foils 05 102 430 001
PCR Reagents
LightCycler
®
480 High Resolution Melting Master 5 × 100 µl (500 reactions, 20 µl each) 04 909 631 001
LightCycler
®
480 SYBR Green I Master 1 kit (5 × 100 reactions, 20 µl each)
1 kit (10 × 500 reactions, 20 µl each)
04 707 516 001
04 887 352 001
LightCycler
®
480 Probes Master 1 kit (5 × 100 reactions, 20 µl each)
1 kit (10 × 500 reactions, 20 µl each)
1 kit (1 × 5000 reactions, 20 µl each)
04 707 494 001
04 887 301 001
04 902 343 001
LightCycler
®
480 Genotyping Master 1 kit (4 × 96 reactions, 20 µl each) 04 707 524 001
LightCycler
®
480 RNA Master Hydrolysis Probe 1 kit (5 × 100 reactions) 04 991 885 001
Isolation of Nucleic Acids
High Pure PCR Template Preparation Kit 100 purifications 11 796 828 001
For technical support please contact:
www.roche-applied-science.com
For life science research only.
The product is covered in-part by US 5,871,908, co-exclusively licensed from Evotec OAI AG.Parts of the
Software used for the LightCycler
®
480 System are licensed from Idaho Technology Inc., Salt Lake City,
UT, USA. This product is covered by one or more of U.S. 6,197,520, 6,303,305, 6,387,621, 6,503,720, 6,730,501
and corresponding claims in their non-U.S. counterparts, owned by Roche Diagnostics GmbH and/or
licensed from Idaho Technology, Inc. This LightCycler
®
480 Real-Time PCR System is licensed under
U.S. Patent 6,814,934 and corresponding claims in its non-U.S. counterparts and under one or more of U.S.
Patents Nos. 5,038,852, 5,656,493, 5,333,675, 5,475,610, 5,602,756, 6,703,236, 7,238,517 or corresponding
claims in their non-U.S. counterparts, for use in life science research, in vitro diagnostics and other applied
fields. No rights are conveyed expressly, by implication, or by estoppel under any patent claims or for any
other application.
LIGHTCYCLER and HIGH PURE are trademarks of Roche. Other brands or product names are trademarks of
their respective holders..
Published by
Roche Diagnostics GmbH
Roche Applied Science
68298 Mannheim
Germany
© 2009 Roche Diagnostics
All rights reserved.
05465052001 0209
5Endpoint Genotyping