C M LightCycler ® 480 Gene Scanning Software Version 1.5 www.roche-applied-science.
Table of Contents Prologue 5 I Revision History.........................................................................................................................5 II Contact Addresses....................................................................................................................5 III Trademarks..................................................................................................................................6 IV Intended Use.................................
Table of Contents 4 E Supplementary Functions 43 1 Gene Scanning Template............................................................................................................................ 43 2 Result Control ..................................................................................................................................................44 3 Unlicensed Features..................................................................................................................
Prologue Revision History Prologue I Revision History Version Revision Date 1.0 February 2007 2.0 February 2008 © Copyright 2008, Roche Diagnostics GmbH. All rights reserved. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express written permission of Roche Diagnostics GmbH. Questions or comments regarding the contents of this manual can be directed to the address below or to your Roche representative.
Prologue Trademarks III Trademarks LIGHTCYCLER, LC, MAGNA PURE and HIGH PURE are trademarks of Roche. Other brands or product names are trademarks of their respective holders. IV Intended Use With the LightCycler® 480 Gene Scanning Software, the user can detect heteroduplex structures in DNA samples on the LightCycler® 480 Instrument.
Prologue Software License Agreement VI Software License Agreement Read the following terms and conditions of this Software License Agreement (“Agreement”) carefully before installing the LightCycler® 480 Software, hereinafter referred to as (“Software”). Proceeding with the installation of the Software will constitute acceptance of the terms and conditions of this Agreement.
Prologue Software License Agreement Licensee may not: a. Use the Software, in whole or in part, except as expressly provided in this Agreement. b. Use the Software on more than five workstations at a time. c. Copy, sell, or otherwise transfer the Software or assign its rights under this Agreement, in whole or in part, to another party, except as expressly provided in this Agreement. d. Rent, distribute, license or sublicense the Software. e. Create derivative works based on Software. f.
Prologue Software License Agreement 4 Disclaimer of Warranties THE WARRANTY SET FORTH IN THE PREVIOUS PARAGRAPH, IS IN LIEU OF ALL OTHER WARRANTIES, EXPRESS OR IMPLIED, ARISING BY LAW, FROM A COURSE OF PERFORMANCE, A COURSE OF DEALING, TRADE USAGE, OR OTHERWISE.
Prologue Software License Agreement 6 General Information Licensee may not sublicense, assign or transfer the license or the Software, in whole or in part, except as expressly provided in this Agreement. Any attempt otherwise to sublicense, assign or transfer any of the rights, duties or obligations hereunder is void. 7 Intellectual Property Rights Licensee shall only hold those rights to the Software that are expressly described in Section 2 of this Agreement.
Prologue Software License Agreement 8 Duration and Termination The Agreement is effective until terminated. Licensee may terminate this Agreement at any time by destroying the Software and documentation relating to the Software in any form. The Agreement will terminate automatically and without notice from Supplier, if Licensee fails to comply with any term or condition of this Agreement. Licensee agrees to destroy the Software upon termination of this Agreement by Supplier.
Prologue Conventions Used in this Manual VII Conventions Used in this Manual Text Conventions To impart information that is consistent and memorable, the following text conventions are used in this Operator‘s Manual: Numbered listing Steps in a procedure that you must perform in the order listed. Italic type, blue Points to a different chapter in this Operator’s Manual, which you should consult. Italic type Points to a software function or element.
Prologue Warnings and Precautions VIII Warnings and Precautions The LightCycler® 480 System is equipped with software, enabling the user of the Product to connect it with a network. Roche draws the attention of the user to the fact that such connection may have an adverse effect on the Product’s integrity, e.g., due to an infection of the Product with malicious code (viruses, Trojan horses, etc.) or access by unauthorized third parties (e.g., intrusion by attackers).
Gene Scanning with the LightCycler® 480 System A Gene Scanning with the LightCycler® 480 System “Gene scanning” or “mutation scanning” techniques detect the presence of sequence variation in target-gene derived PCR amplicons. “Gene Scanning” is based on “High Resolution Melting”, a novel, closed-tube post-PCR (Polymerase Chain Reaction) method enabling genomic researchers to analyze genetic variations in PCR amplicons prior to or as an alternative to sequencing.
Gene Scanning with the LightCycler® 480 System High Resolution Melting 1 High Resolution Melting The key technique in gene scanning, High Resolution Melting, is a refinement of earlier, well-established DNA dissociation (or “melting”) techniques (e.g., to determine the Tm of a DNA hybrid). Like all melting analyses, the technique subjects DNA samples to increasing temperatures and records the details of their dissociation from double-stranded (dsDNA) to single-stranded form (ssDNA).
Gene Scanning with the LightCycler® 480 System High Resolution Melting This figure shows how a High Resolution Melting experiment can detect both homozygous and heterozygous allelic variants in a sample. Homozygous variants are detectable because their melting curves are displaced along the temperature axis (x-axis) relative to homozygous “wild type” samples. Heterozygous variants have melting curves that differ even more dramatically in shape from “wild type” curves.
Gene Scanning with the LightCycler® 480 System Components of the LightCycler® 480 System Gene Scanning Assay 2 Components of the LightCycler® 480 System Gene Scanning Assay To obtain meaningful gene scanning results from High Resolution Melting analysis, three components (the DNA-binding dye, the analytical software, and the real-time PCR instrument itself) of the LightCycler® 480 System must work optimally.
Gene Scanning with the LightCycler® 480 System Components of the LightCycler® 480 System Gene Scanning Assay 2.2 Data Analysis using the LightCycler® 480 Gene Scanning Software LightCycler® 480 Gene Scanning Software analyzes the High Resolution Melting curve data to identify changes in the shape of the curve, which indicate the presence of sequence variations in the PCR product. Correct interpretation of the data depends to a large extent on the software algorithms used.
Gene Scanning with the LightCycler® 480 System Components of the LightCycler® 480 System Gene Scanning Assay 3. Temperature shifting: The next step is to shift the temperature axis of the normalized melting curves at the point where the entire double-stranded DNA is completely denatured. For this, the software automatically applies a default Temp Shift Threshold of 5% to all data. (If you wish, you can set this threshold manually to a different value.
Gene Scanning with the LightCycler® 480 System Components of the LightCycler® 480 System Gene Scanning Assay The following examples demonstrate gene scanning: ►► For the single-nucleotide polymorphism (SNP) G→T in the LPLH3 gene (163 bp amplicon), resulting in 3 main variant groups. ►► For sequence variations in the MBL2 gene (219 bp amplicon), resulting in 4 main variant groups corresponding to the 4 most frequent haplotypes described for this gene in literature and 3 samples of a further genetic variant.
Gene Scanning with the LightCycler® 480 System Advantages of the High Resolution Melting Technique 3 Advantages of the High Resolution Melting Technique Melting curve analysis is based on a robust, post-PCR physical measurement and, therefore, offers several advantages over mutation detection methods that derive information from the amplification process itself: ►► Any amplicon can be screened for unknown sequence variants with a single highresolution dye; you do not need to target a specific variant wit
Preparing a LightCycler® 480 System Gene Scanning Experiment Designing the Gene Scanning Assay B Preparing a LightCycler® 480 System Gene Scanning Experiment 1 Designing the Gene Scanning Assay These guidelines will help you design an effective Gene Scanning assay: ►► A single base variation affects the melting behavior of a 100 bp amplicon more than a 500 bp amplicon; thus, short amplicons are more likely to show the effects of small sequence changes.
Preparing a LightCycler® 480 System Gene Scanning Experiment Sample Material 2 Sample Material Because a LightCycler® 480 System Gene Scanning experiment involves comparing melting profiles from independent PCR reactions, it is crucial to minimize reaction-to-reaction variability. Standardizing the template DNA is one means of minimizing variability. Follow these guidelines when preparing or handling template DNA: ►► Use isolation and storage procedures that minimize the potential for sample degradation.
Preparing a LightCycler® 480 System Gene Scanning Experiment PCR Primers 3 PCR Primers ►► Design PCR primers that have annealing temperatures around 60°C and produce short amplicons (100–250 bp). Use a software package like Primer3 (see http://frodo. wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) or LightCycler® Probe Design Software 2.0 for designing the primers. Use primers that have been purified by HPLC.
Preparing a LightCycler® 480 System Gene Scanning Experiment PCR Reagents 4 PCR Reagents Hot-start PCR techniques are strongly recommended for High Resolution Melting applications, since they avoid the formation of non-specific amplification products at the beginning of the reaction. Roche Applied Science provides a convenient, 2× concentrated master mix for such hot start procedures.
Preparing a LightCycler® 480 System Gene Scanning Experiment PCR Parameters 5 PCR Parameters Data obtained from High Resolution Melting is only as good as the amplification product being analyzed. Nothing is more critical to High Resolution Melting data than having robust amplification of a single product. The High Resolution Melting Dye binds all doublestranded DNA present in a reaction.
Preparing a LightCycler® 480 System Gene Scanning Experiment PCR Parameters In case you do not know the melting temperatures of your PCR primers exactly, it is recommended to apply a touchdown PCR protocol covering a range of annealing temperature from 65 to 53°C.
Preparing a LightCycler® 480 System Gene Scanning Experiment Rapid Detection of Amplification Artifacts 6 Rapid Detection of Amplification Artifacts The goal of a LightCycler® 480 System Gene Scanning assay is to generate a single pure PCR product. Any amplification artifacts (e.g., primer-dimers) may lead to misleading results. On the LightCycler® 480 System, you can easily determine whether the PCR products include primer-dimers by comparing your samples to a non-template control.
Installing the LightCycler® 480 Gene Scanning Software C Installing the LightCycler® 480 Gene Scanning Software The LightCycler® 480 System Gene Scanning Software is provided as an additional module on a separate software CD. Software installation is performed using a self-extracting installation program. To install the software on a LightCycler® 480 control unit or on a non-Roche PC follow the steps below.
Installing the LightCycler® 480 Gene Scanning Software � The LightCycler® 480 Gene Scanning Software is installed. When the installation process has finished, the InstallShield Wizard Complete window is displayed. Click Finish.
Performing Gene Scanning Analysis D Performing Gene Scanning Analysis You can perform a Gene Scanning analysis on any experiment that contains a melting curve program. The LightCycler® 480 Gene Scanning Software determines the heterodu plex structures in samples by analyzing experimental data generated in the presence of the LightCycler® 480 High Resolution Melting Dye.
Performing Gene Scanning Analysis � Click Sample Editor in the Module bar, then select the workflow Scanning. After the workflow is selected, the LightCycler 480 Software automatically reconfigures the Sample Editor. � Define the properties of the samples. For detailed information on the Sample Editor see the LightCycler® 480 Instrument Operator’s Manual, section “Entering Sample Information”.
Performing Gene Scanning Analysis � The Create new analysis dialog opens. Select an analysis subset and an experimental program from the Program list (usually, you will select the melting curve program). If you wish, you can change the analysis name (default name = “analysis type for subset name”). Click . � The Gene Scanning Analysis screen opens.
Performing Gene Scanning Analysis To perform a Gene Scanning analysis: � Using the Standards multi-select button, select the analysis mode (grouping method) you want to apply: Auto Group Applies automated grouping in the absence of in-run melting standard samples. If, in the Sample Editor, you have not defined any sample as a melting standard, the program will select this option by default after it creates the analysis.
Performing Gene Scanning Analysis � ►► Normalization Click the Normalization tab to normalize the melting curves. Fluorescence values acquired during High Resolution Melting will vary in magnitude, e.g., due to differences in the starting amount of template in each sample. This variability can mask differences between genotypes. Therefore the first step in the analysis process is to normalize the data.
Performing Gene Scanning Analysis � LightCycler® 480 Gene Scanning Software automatically places the temperature sliders in a suitable region for normalization and displays the normalized data in the lower graph (Normalized Melting Curves).
Performing Gene Scanning Analysis � ►► Temperature Shifting Click the Temperature Shift tab to reset the temperature axis (x-axis) of the melting curves. Eliminating the temperature offsets between samples can provide clearer separation of samples that have subtle changes in their melting profiles. The temperature axis of each curve is shifted in the region of low fluorescence (at the end of homoduplex melting) allowing heteroduplexes to be identified by their early drop in fluorescence.
Performing Gene Scanning Analysis � In the Results table, deselect any samples you do not want included in analysis of the results. � Now click the Calculate button to analyze the results and determine the grouping. The software determines variant (or genotype) groups and assigns a color and name to each group. Simultaneously, the software calculates the Normalized and Temperature Shifted Difference Plot.
Performing Gene Scanning Analysis � ►► Difference Plot Click the Difference Plot tab in the charts area to view ►► the Normalized and Shifted Melting Curves and ►► the Normalized and Temperature Shifted Difference Plot: The Difference Plot is determined as follows: ►► First the software selects the group that contains the most samples. ►► Then the standard of that group (the Base Curve) is designated the reference genotype.
Performing Gene Scanning Analysis To customize the Difference Plot display: � In the Difference Plot tab, click either above the upper or lower chart. The chart’s options toolbar is displayed, containing a chart menu. � To change the chart type, select the new chart type from the respective Chart menu. For the upper chart you have the following choices: ►► Melting Curves: corresponds to the Melting Curve display in the Negatives tab, i.e.
Performing Gene Scanning Analysis To rename groups: On the Groups tab in the Results table, you can change the name associated with each variant/genotype group. You can edit group names for groups generated by Auto Group analysis or generated by Comn/Vars analysis, not for groups based on in-run melting standards. Further, you cannot change the names of the Negative and Unknown groups or the number of group boxes that are displayed.
Performing Gene Scanning Analysis � Selecting a new genotype group from the New Call list and clicking Apply changes all affected samples currently displayed in the graphs to the new name. Selecting Auto Call from the New Call list (and clicking Apply) causes all affected samples currently displayed in the charts to revert to their previous (automatically determined) name. All manually modified calls are marked with an asterisk in the Results table and in reports.
Supplementary Functions Gene Scanning Template E Supplementary Functions 1 Gene Scanning Template A Gene Scanning analysis template contains the following settings: ►► Subset and program When you select a template, the software determines whether the current experiment contains a subset with the same name and the same well positions as the subset in the template. ►► If the current experiment does not contain a subset with the same name, the software creates the subset.
Supplementary Functions Result Control 2 Result Control The LightCycler® 480 Gene Scanning Software uses a control routine to check whether the analysis has passed or failed. The result control check is only applied to a run that contains internal melting standards; it is not applied to runs in Auto Group or Comn/Vars analysis mode. ►► Negative Controls ►► If every Negative Control is designated negative, the run passes this control check.
Supplementary Functions Unlicensed Features 3 Unlicensed Features The following statements define the behavior of the LightCycler® 480 Software when a gene scanning feature is used in the application, but a license for the L ightCycler® 480 Gene Scanning Software is not available: ►► An experiment that contains a Gene Scanning analysis can be imported into a system where the additional software module has not been installed.
Supplementary Functions Ordering Information 4 Ordering Information Roche Applied Science offers a large selection of reagents and systems for life science research. For a complete overview of related products and manuals, please visit and bookmark our home page, http://www.roche-applied-science.com, and visit our special interest site for ►► the LightCycler® 480 System: http://www.lightcycler480.com ►► The MagNA Pure System family for automated nucleic acid isolation: http://www.magnapure.
Supplementary Functions Ordering Information PCR Reagents LightCycler® 480 High Resolution Melting Master 5 × 100 µl (500 reactions, 20 µl each) 04 909 631 001 LightCycler® 480 PCR Master SYBR Green 1 kit (5 × 100 reactions, 20 µl each) 04 707 516 001 1 kit (10 × 500 reactions, 20 µl each) 04 887 352 001 1 kit (5 × 100 reactions, 20 µl each) 04 707 494 001 1 kit (10 × 500 reactions, 20 µl each) 04 887 301 001 1 kit (1 × 5000 reactions, 20 µl each) 04 902 343 001 LightCycler® 480 Genotyping Ma
C M Y CM MY CY CMY K www.roche-applied-science.com Published by Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany 2008 Roche Diagnostics GmbH All rights reserved.
