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
Editing Samples
Use the ► Subset Editor if necessary, to define subsets of samples subdividing the multiwell plate into several areas which can be ana-
lyzed separately. In all cases the full plate (all samples) will be measured.
Use the ► Sample Editor to record information about the samples in the experiment. You can enter sample information manually
before, during or even after an experiment is completed.
Step 1: Select the workflow Endpt Geno.
Step 2: Select the samples you want to edit.
Step 3: Configure the properties: Replicate of, Sample Names and Sample Types.
Start Run
Check if the multiwell plate is loaded into the LightCycler® 480 Instrument. ►
Click ► Start Run and name the experiment.
Preparing the Reaction Mix and Setting up the Multiwell Plate
Prepare a 20x conc. solution that contains all specific PCR primers (18µM each) and both hydrolysis probes, FAM-labeled specific ►
for allele X, HEX (VIC)-labeled specific for allele Y (4µM each) or use comercially available SNP assay mix.
In a 1.5 ml reaction tube on ice, prepare the PCR mix without target nucleic acid according to your sample amount: ►
5µl reaction volume per well for 384 plate or 20µl reaction volume per well for 96 plate. Use LightCycler® 480 Multiwell Plates,
only white for this application. Add the following components in the order listed below:
PCR Mix (without target nucleic acid)
5µl for 384 Well Plate 20µl for 96 Well Plate
Reagent 1 Reaction 1 Reaction
LightCycler
®
480 Probes Master, 2x conc. 2.5 µl 10 µl
Assay Mix, 20x conc. 0.25 µl 1 µl
Water, PCR-grade - 4 µl
Volume 2.75 µl 15 µl
Pipet 2.75 µl (384 wells) or 15 µl (96 wells) PCR Mix into the respective wells of the LightCycler® 480 Multiwell Plate.
Target Nucleic Acid
5µl for 384 Well Plate 20µl for 96 Well Plate
DNA sample or PCR-grade water for NTC 2.25 µl 5 µl
Add 2.25 µl (5-20ng) respectively 5 µl (5-40ng) of the DNA template of all unknown samples. Always include a
No-Template-Control (NTC) in your experiment; use PCR-grade water for the NTC.
Cover the multiwell plate with a sealing foil; use an adhesive seal applicator for correct sticking. ►
Centrifuge the multiwell plate at 1,500 × g for two minutes. ►
Load the multiwell plate into the LightCycler® 480 Instrument. ►
Selecting the Protocol
Change to the ► Overview screen by clicking and choose the plate type White Plates.
Click ► New Experiment from Template.
Select ► Endpoint Genotyping (PCR Read) for detection of FAM and HEX and set the respective reaction volume.
Procedure Overview
Alternatively you can perform the amplification reaction in a non real-time block cycler instrument. The resulting endpoint
fluorescence is determined by using a short measuring program (Post Read) after PCR for analysis. Additionally this short pro-
gram can also be run prior to PCR (Pre Read), allowing a background correction.
For more precise results use the optional Background Correction ►
1. Change to the Overview screen by clicking
and choose the plate type White Plates.
2. Click New Experiment from Template.
3. Select the Roche Run Template Endpoint Genotyping (Pre-Post Read) for the pre-PCR experiment.
4. Check if the multiwell plate is loaded into the LightCycler® 480 Instrument and start the run.
6. After the run load the multiwell plate into the non real-time block cycler instrument and perform the amplification.
Load the multiwell plate into the LightCycler® 480 Instrument. ►
On the ► Overview screen of the LightCycler® 480 Software click New Experiment from Template.
Select the Roche Run Template ► Endpoint Genotyping (Pre-Post Read).
Start the run to generate Post PCR data.. ►
Please note: If you have performed the optional Pre Read, use the option Use external experiment for background correction for the
analysis of the Post Read run. Browse in the Navigator to import the corresponding Pre Read experiment.
time
15-60‘
5-15‘
75-95‘
5‘
Prepare reaction mix and set-up multiwell plate
Select protocol and edit samples
Perform PCR using LightCycler
®
480 Instrument
Data acquisition Pre Read
Perform block cycler PCR
Data acquisition Post Read
Analyze data (call allele types)
Generate report
2Endpoint Genotyping 3Endpoint Genotyping 4Endpoint Genotyping
Editing Samples
Use the ► Subset Editor if necessary, to define subsets of samples subdividing the multiwell plate into several areas which can be ana-
lyzed separately. In all cases the full plate (all samples) will be measured.
Use the ► Sample Editor to record information about the samples in the experiment. You can enter sample information manually
before, during or even after an experiment is completed.
Step 1: Select the workflow Endpt Geno.
Step 2: Select the samples you want to edit.
Step 3: Configure the properties: Replicate of, Sample Names and Sample Types.
Start Run
Check if the multiwell plate is loaded into the LightCycler® 480 Instrument. ►
Click ► Start Run and name the experiment.
Preparing the Reaction Mix and Setting up the Multiwell Plate
Prepare a 20x conc. solution that contains all specific PCR primers (18µM each) and both hydrolysis probes, FAM-labeled specific ►
for allele X, HEX (VIC)-labeled specific for allele Y (4µM each) or use comercially available SNP assay mix.
In a 1.5 ml reaction tube on ice, prepare the PCR mix without target nucleic acid according to your sample amount: ►
5µl reaction volume per well for 384 plate or 20µl reaction volume per well for 96 plate. Use LightCycler® 480 Multiwell Plates,
only white for this application. Add the following components in the order listed below:
PCR Mix (without target nucleic acid)
5µl for 384 Well Plate 20µl for 96 Well Plate
Reagent 1 Reaction 1 Reaction
LightCycler
®
480 Probes Master, 2x conc. 2.5 µl 10 µl
Assay Mix, 20x conc. 0.25 µl 1 µl
Water, PCR-grade - 4 µl
Volume 2.75 µl 15 µl
Pipet 2.75 µl (384 wells) or 15 µl (96 wells) PCR Mix into the respective wells of the LightCycler® 480 Multiwell Plate.
Target Nucleic Acid
5µl for 384 Well Plate 20µl for 96 Well Plate
DNA sample or PCR-grade water for NTC 2.25 µl 5 µl
Add 2.25 µl (5-20ng) respectively 5 µl (5-40ng) of the DNA template of all unknown samples. Always include a
No-Template-Control (NTC) in your experiment; use PCR-grade water for the NTC.
Cover the multiwell plate with a sealing foil; use an adhesive seal applicator for correct sticking. ►
Centrifuge the multiwell plate at 1,500 × g for two minutes. ►
Load the multiwell plate into the LightCycler® 480 Instrument. ►
Selecting the Protocol
Change to the ► Overview screen by clicking and choose the plate type White Plates.
Click ► New Experiment from Template.
Select ► Endpoint Genotyping (PCR Read) for detection of FAM and HEX and set the respective reaction volume.
Procedure Overview
Alternatively you can perform the amplification reaction in a non real-time block cycler instrument. The resulting endpoint
fluorescence is determined by using a short measuring program (Post Read) after PCR for analysis. Additionally this short pro-
gram can also be run prior to PCR (Pre Read), allowing a background correction.
For more precise results use the optional Background Correction ►
1. Change to the Overview screen by clicking
and choose the plate type White Plates.
2. Click New Experiment from Template.
3. Select the Roche Run Template Endpoint Genotyping (Pre-Post Read) for the pre-PCR experiment.
4. Check if the multiwell plate is loaded into the LightCycler® 480 Instrument and start the run.
6. After the run load the multiwell plate into the non real-time block cycler instrument and perform the amplification.
Load the multiwell plate into the LightCycler® 480 Instrument. ►
On the ► Overview screen of the LightCycler® 480 Software click New Experiment from Template.
Select the Roche Run Template ► Endpoint Genotyping (Pre-Post Read).
Start the run to generate Post PCR data.. ►
Please note: If you have performed the optional Pre Read, use the option Use external experiment for background correction for the
analysis of the Post Read run. Browse in the Navigator to import the corresponding Pre Read experiment.
time
15-60‘
5-15‘
75-95‘
5‘
Prepare reaction mix and set-up multiwell plate
Select protocol and edit samples
Perform PCR using LightCycler
®
480 Instrument
Data acquisition Pre Read
Perform block cycler PCR
Data acquisition Post Read
Analyze data (call allele types)
Generate report
2Endpoint Genotyping 3Endpoint Genotyping 4Endpoint Genotyping
Editing Samples
Use the ► Subset Editor if necessary, to define subsets of samples subdividing the multiwell plate into several areas which can be ana-
lyzed separately. In all cases the full plate (all samples) will be measured.
Use the ► Sample Editor to record information about the samples in the experiment. You can enter sample information manually
before, during or even after an experiment is completed.
Step 1: Select the workflow Endpt Geno.
Step 2: Select the samples you want to edit.
Step 3: Configure the properties: Replicate of, Sample Names and Sample Types.
Start Run
Check if the multiwell plate is loaded into the LightCycler® 480 Instrument. ►
Click ► Start Run and name the experiment.
Preparing the Reaction Mix and Setting up the Multiwell Plate
Prepare a 20x conc. solution that contains all specific PCR primers (18µM each) and both hydrolysis probes, FAM-labeled specific ►
for allele X, HEX (VIC)-labeled specific for allele Y (4µM each) or use comercially available SNP assay mix.
In a 1.5 ml reaction tube on ice, prepare the PCR mix without target nucleic acid according to your sample amount: ►
5µl reaction volume per well for 384 plate or 20µl reaction volume per well for 96 plate. Use LightCycler® 480 Multiwell Plates,
only white for this application. Add the following components in the order listed below:
PCR Mix (without target nucleic acid)
5µl for 384 Well Plate 20µl for 96 Well Plate
Reagent 1 Reaction 1 Reaction
LightCycler
®
480 Probes Master, 2x conc. 2.5 µl 10 µl
Assay Mix, 20x conc. 0.25 µl 1 µl
Water, PCR-grade - 4 µl
Volume 2.75 µl 15 µl
Pipet 2.75 µl (384 wells) or 15 µl (96 wells) PCR Mix into the respective wells of the LightCycler® 480 Multiwell Plate.
Target Nucleic Acid
5µl for 384 Well Plate 20µl for 96 Well Plate
DNA sample or PCR-grade water for NTC 2.25 µl 5 µl
Add 2.25 µl (5-20ng) respectively 5 µl (5-40ng) of the DNA template of all unknown samples. Always include a
No-Template-Control (NTC) in your experiment; use PCR-grade water for the NTC.
Cover the multiwell plate with a sealing foil; use an adhesive seal applicator for correct sticking. ►
Centrifuge the multiwell plate at 1,500 × g for two minutes. ►
Load the multiwell plate into the LightCycler® 480 Instrument. ►
Selecting the Protocol
Change to the ► Overview screen by clicking and choose the plate type White Plates.
Click ► New Experiment from Template.
Select ► Endpoint Genotyping (PCR Read) for detection of FAM and HEX and set the respective reaction volume.
Procedure Overview
Alternatively you can perform the amplification reaction in a non real-time block cycler instrument. The resulting endpoint
fluorescence is determined by using a short measuring program (Post Read) after PCR for analysis. Additionally this short pro-
gram can also be run prior to PCR (Pre Read), allowing a background correction.
For more precise results use the optional Background Correction ►
1. Change to the Overview screen by clicking
and choose the plate type White Plates.
2. Click New Experiment from Template.
3. Select the Roche Run Template Endpoint Genotyping (Pre-Post Read) for the pre-PCR experiment.
4. Check if the multiwell plate is loaded into the LightCycler® 480 Instrument and start the run.
6. After the run load the multiwell plate into the non real-time block cycler instrument and perform the amplification.
Load the multiwell plate into the LightCycler® 480 Instrument. ►
On the ► Overview screen of the LightCycler® 480 Software click New Experiment from Template.
Select the Roche Run Template ► Endpoint Genotyping (Pre-Post Read).
Start the run to generate Post PCR data.. ►
Please note: If you have performed the optional Pre Read, use the option Use external experiment for background correction for the
analysis of the Post Read run. Browse in the Navigator to import the corresponding Pre Read experiment.
time
15-60‘
5-15‘
75-95‘
5‘
Prepare reaction mix and set-up multiwell plate
Select protocol and edit samples
Perform PCR using LightCycler
®
480 Instrument
Data acquisition Pre Read
Perform block cycler PCR
Data acquisition Post Read
Analyze data (call allele types)
Generate report
2Endpoint Genotyping 3Endpoint Genotyping 4Endpoint Genotyping
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
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
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