Bosch 480 Series Operator`s manual

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LightCycler ® 480
Gene Scanning Software
Version 1.5
www.roche-applied-science.com
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Table of Contents
Prologue
5
I
Revision History.........................................................................................................................5
II
Contact Addresses....................................................................................................................5
III
Trademarks..................................................................................................................................6
IV
Intended Use...............................................................................................................................6
V
License Statements for the LightCycler
VI
Software License Agreement..................................................................................................7
®
480 Gene Scanning Software.....................6
1
2
3
4
5
6
7
8
9
10
11
Program License Agreement.............................................................................................................................7
Grant of Software License..................................................................................................................................7
Limited Warranty....................................................................................................................................................8
Disclaimer of Warranties ...................................................................................................................................9
Limitations of Remedies......................................................................................................................................9
General Information........................................................................................................................................... 10
Intellectual Property Rights............................................................................................................................. 10
Duration and Termination................................................................................................................................ 11
Import, Export and Use of the Software..................................................................................................... 11
Miscellaneous...................................................................................................................................................... 11
Governing Law and Place of Jurisdiction.................................................................................................. 11
VII
Conventions Used in this Manual....................................................................................... 12
VIII
Warnings and Precautions.................................................................................................... 13
A
Gene Scanning with the LightCycler® 480 System
1
High Resolution Melting............................................................................................................................. 15
2
Components of the LightCycler® 480 System Gene Scanning Assay............................... 17
2.1
2.2
2.3
14
LightCycler® 480 High Resolution Melting Dye...................................................................................... 17
Data Analysis using the LightCycler® 480 Gene Scanning Software............................................. 18
LightCycler® 480 Instrument.......................................................................................................................... 20
3
Advantages of the High Resolution Melting Technique............................................................ 21
B
Preparing a LightCycler® 480 System Gene Scanning Experiment
1
Designing the Gene Scanning Assay................................................................................................... 22
2
Sample Material.............................................................................................................................................. 23
3
PCR Primers....................................................................................................................................................... 24
4
PCR Reagents................................................................................................................................................... 25
5
PCR Parameters............................................................................................................................................... 26
6
Rapid Detection of Amplification Artifacts....................................................................................... 28
C
Installing the LightCycler® 480 Gene Scanning Software
29
D
Performing Gene Scanning Analysis
31
22
3
Table of Contents
4
E
Supplementary Functions
43
1
Gene Scanning Template............................................................................................................................ 43
2
Result Control ..................................................................................................................................................44
3
Unlicensed Features...................................................................................................................................... 45
4
Ordering Information.....................................................................................................................................46
LightCycler 480® Gene Scanning Software
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.
Roche Diagnostics GmbH
Roche Applied Science
Global Customer Support
Nonnenwald 2
82372 Penzberg, Germany
Every effort has been made to ensure that all the information contained in the
LightCycler® 480 Gene Scanning Software manual is correct at the time of printing. However, Roche Diagnostics GmbH reserves the right to make any changes necessary without
notice as part of ongoing product development.
II
Contact Addresses
Manufacturer
Roche Diagnostics Ltd.
Forrenstrasse
CH-6343 Rotkreuz
Switzerland
Distribution
Roche Diagnostics GmbH
Sandhofer Straße 116
D-68305 Mannheim
Germany
Distribution in the US
Roche Diagnostics
9115 Hague Road
PO Box 50457
Indianapolis, IN 46250
USA
5
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. This assay requires only
routine PCR amplification of the sample followed by a simple, rapid High Resolution
Melting experiment in the presence of LightCycler® 480 High Resolution Melting Dye.
After the experiment, the software analyzes the shape of the melting curves and groups
those that are similar. Samples with known genotype can be included as melting standards
in the experiment, allowing the user to determine genotypes of unknown samples in the
analysis.
The LightCycler® 480 Gene Scanning Software is intended for general laboratory use in
combination with the LightCycler® 480 Instrument and LightCycler® 480 Software.
V
License Statements for the
LightCycler® 480 Gene Scanning Software
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 product is covered in-part by US 5,871,908 or any foreign equivalents, co-exclusively
licensed from Evotec OAI AG. The purchase price includes a license to practice the methods covered by US 5,871, 908 by using the product. Purchase of this product, however,
does not convey to the purchaser a license or right to
(i) commercially make, have made or sell reagents and/or kits, or
(ii) buy or use reagents and/or kits provided by a third party
used in conjunction with the product or any other thermocycler to practice the methods
covered by US 5,871,908 or any foreign equivalents.
6
LightCycler® 480 Gene Scanning Software
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. By accepting the terms and conditions
of this Agreement, the end-user (“Licensee”) assumes all responsibility and liability for
the selection of this Software to achieve the intended results, and for its installation and
subsequent use. If Licensee is not willing to be bound by the terms and conditions of this
Agreement, the Software package must be promptly returned to Roche (“Supplier”) with
a copy of the receipt against refunding of the purchase price for this Software.
1
Program License Agreement
Licensee assumes all responsibility and liability for the selection of this Software to achieve
the intended results, and for its installation and subsequent use. The Software is protected
by copyright.
2
Grant of Software License
Supplier grants to Licensee subject to continuous compliance with all the provisions hereinafter, a non-exclusive, single-use license to use the Software upon the terms and conditions contained in this Agreement.
Licensee may:
a. Use the Software on up to five workstations at a time and such workstations have to
be owned, leased or otherwise controlled by Licensee, whether in a network or other
configuration.
b. Transfer the Software by assigning the rights under this Agreement to another party,
provided that the other party agrees in writing to accept the terms and conditions of
this Agreement. In addition, Licensee must ensure that the copyright notice is maintained on the Software transferred.
7
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. Modify, adapt, translate, reverse engineer, decompile or disassemble the Software.
Supplier reserves all rights not expressly granted herein, including, but not limited to, the
rights to market the Software either directly or through affiliates, distributors and/or third
parties.
For further information, please contact your local Roche Applied Science support organization. You will find the contact information on the following webpage:
www.roche-applied-science.com.
3
Limited Warranty
The Software is provided “as is” without warranty of any kind, either expressed or implied,
including, but not limited to the implied warranties of merchantability and fitness for
a particular purpose. The entire risk as to the quality and performance of the Software
is with Licensee, should the Software prove to be defective. Licensee assumes the entire
costs of all necessary servicing, repair, or correction. However, Supplier warrants that the
program media on which the Software is furnished is free from defects in materials and
workmanship under normal use for a period of ninety (90) days from the date of delivery
as evidenced by a copy of your receipt. SUPPLIER MAKES NO FURTHER WARRANTIES
OR GUARANTEES NOR EXPLICIT NOR IMPLIED
8
LightCycler® 480 Gene Scanning Software
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.
SUPPLIER AND ANY ENTITY CONTROLLING, CONTROLLED BY OR UNDER
COMMON CONTROL WITH SUPPLIER (“SUPPLIER’S AFFILIATE”) SPECIFICALLY
DISCLAIM, WITHOUT LIMITATION, ALL WARRANTIES OF ANY KIND, WHETHER
EXPRESS OR IMPLIED, INCLUDING, WITHOUT LIMITATION, THE IMPLIED WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, AND
NON-INFRINGEMENT. SUPPLIER AND SUPPLIER’S AFFILIATES MAKE NO REPRESENTATION OR WARRANTY AS TO THE SOFTWARE OR AS TO THE RESULTS
TO BE ATTAINED BY LICENSEE OR ANY THIRD PARTY FROM THE SOFTWARE.
LICENSEE ACKNOWLEDGES THAT IT HAS NOT RELIED UPON ANY REPRESENTATIONS OR WARRANTIES MADE BY SUPPLIER OR A SUPPLIER’S AFFILIATE EXCEPT FOR THOSE EXPRESSLY AND SPECIFICALLY SET FORTH IN THIS AGREEMENT.
5
Limitations of Remedies
Supplier’s sole liability and Licensee’s sole remedy shall be:
a. The replacement of the program media not meeting Supplier’s limited warranty and
which is returned to Supplier with a copy of Licensee’s receipt;
b. If Supplier is unable to deliver replacement of program media which is free of defects
in material and workmanship, Licensee may terminate this Agreement by returning
the Software and a copy of Licensee’s receipt to Supplier, and Licensee’s money will be
refunded.
IN NO EVENT WILL SUPPLIER OR ANY OF SUPPLIER’S AFFILIATES (OR THEIR RESPECTIVE OFFICERS, EMPLOYEES, CONSULTANTS, ATTORNEYS OR AGENTS), BE
LIABLE FOR ANY SPECIAL, INDIRECT, INCIDENTAL, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, LOST PROFITS, LOST DATA OR INFORMATION, LOSS OF USE OF THE SOFTWARE, BUSINESS INTERRUPTION, LOSS
OF BUSINESS REPUTATION OR GOODWILL, OR DOWNTIME COSTS) WHICH
THE LICENSEE OR THIRD PARTIES MAY INCUR OR EXPERIENCE, DIRECTLY OR
INDIRECTLY ARISING OUT OF OR RELATING TO THE SOFTWARE, THIS AGREEMENT, OR THE TERMINATION OF THIS AGREEMENT, EVEN IF SUPPLIER OR A
SUPPLIER’S AFFILIATE HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES AND NOTWITHSTANDING ANY FAILURE OF ESSENTIAL PURPOSE. THE
AGGREGATE LIABILITY, ON A COMBINED BASIS, OF SUPPLIER AND SUPPLIER’S
AFFILIATES (AND THEIR RESPECTIVE OFFICERS, EMPLOYEES CONSULTANTS,
ATTORNEYS, AND AGENTS) FOR DAMAGES FOR ANY CAUSE WHATSOEVER DIRECTLY OR INDIRECTLY RELATING TO OR ARISING OUT OF THIS AGREEMENT
OR THE SOFTWARE, AND REGARDLESS OF THE FORM OF ACTION, SHALL BE
LIMITED TO, AT SUPPLIER’S OPTION, REPLACEMENT OF THE SOFTWARE OR REFUND OF THE FEES RECEIVED BY SUPPLIER OR A SUPPLIER’S AFFILIATE FROM
LICENSEE WITH RESPECT TO THE SOFTWARE.
9
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. Any other rights with regard to the Software, including without limitation, ownership rights and patent, copyright, trademark, trade secret and other
intellectual property rights, shall remain the sole property of Supplier. Licensee will not
remove from the Software any references to copyrights, trademarks or other ownership
rights, or cover up or alter any such references. Licensee will take all reasonable steps
to prevent any unauthorized use, reproduction, sale, or publication of the Software or
the unauthorized provision of access thereto. Licensee will indemnify and hold harmless
Supplier from any losses, damages, claims and expenses (including, without limitation,
reasonable legal expenses) relating to any infringement of the rights of Supplier caused by
Licensee, Licensee’s breach of this Agreement or Licensee’s use of the Software in a manner
not authorized under this Agreement.
10
LightCycler® 480 Gene Scanning Software
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. On any termination of this Agreement, all rights of use of the Software held by Licensee shall expire.
9
Import, Export and Use of the Software
Licensee shall be exclusively responsible for ensuring compliance with the relevant legislation relating to its rights to import, export or use the Software.
10
Miscellaneous
Should any part of this Agreement be declared void or unenforceable by a court of competent jurisdiction, the remaining terms shall remain in full force and effect.
Failure of Supplier to enforce any of its rights in this Agreement shall not be considered
a waiver of its rights, including but not limited to its rights to respond to subsequent
breaches.
By opening and using this Software Licensee acknowledges that he has read this Agreement, understands it, and agrees to be bound by its terms and conditions. Licensee further agrees that this Agreement is the complete and exclusive statement of the Agreement
between Licensee and Supplier and supersedes any proposal or prior agreement, oral or
written, any other communications between Licensee and Supplier relating to the subject
matter of this Agreement.
The headings of the several Sections of this Agreement are intended for convenience of
reference only and are not intended to be a part of or to affect the meaning or interpretation of this Agreement.
11
Governing Law and Place of Jurisdiction
This Agreement shall be governed by and construed in accordance with the laws of the
State of Indiana, without giving effect to any choice of law principles thereof. The parties agree that the United Nations Convention on Contracts for the International Sale of
Goods (1980) is specifically excluded from application to this Agreement.
11
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.
Symbols
In this Manual, these symbols are used to indicate information that deserves your special
attention.
Symbol
12
Heading
Description
IMPORTANT NOTE
Information critical to the success of the procedure or use of the product.
INFORMATION NOTE
Additional information about the current
topic or procedure.
►►►
Procedure continued on next page.
■
End of procedure.
LightCycler® 480 Gene Scanning Software
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). Roche therefore highly recommends to protect
the Product against such risks by taking appropriate and state-of-the-art
action. As the Product is not intended to be used within networks without
an appropriate firewall and has not been designed for such use, Roche assumes no liability in that regard.
Roche offers the user the cobas IT firewall to be installed prior to the first
connection of the Product to any network. For further information on this
cobas IT firewall and/or the Roche network security concept please contact
your local Roche representative.
In the event the user connects the Product with any network without using
the cobas IT Firewall, Roche cannot offer any Product support regarding
any problem resulting from such network connection. In case of a standalone use of the software of the Product on or in connection with other IT
components (e.g., installation on other PCs) Roche assumes no liability with
respect to any interference of the user’s networks and/other IT components
such use might have. Roche’s liability for the proper functioning of the
software under the respective license and/or purchase agreements with the
user shall remain unaffected.
Contact your local Roche representative for detailed information on
the cobas IT firewall.
Tests indicate that Microsoft Office and Norton Antivirus software do not
interfere with LightCycler® 480 Software and LightCycler® 480 software
modules; these programs may be installed on the LightCycler® 480
Instrument PC. Do not install any other software on the LightCycler® 480
Instrument PC. Installation of any additional software on the
LightCycler® 480 Instrument PC may interfere with the operation of the
LightCycler® 480 Software and LightCycler® 480 software modules, and
could affect data security.
Anti-virus software is not provided. Therefore, it is essential to take precautions to ensure that any software loaded onto the system is virus free.
13
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. High Resolution Melting provides high specificity,
sensi­tivity and convenience at significantly higher speed and much lower cost than other
established (e.g., gel-based) methods. For example, in a diploid genome, equivalent regions from maternal and paternal chromosomes are both amplified by the Polymerase
Chain Reaction. The PCR products can then be analyzed for completely matched hybrids
(called homoduplexes) and mismatched hybrids (heteroduplexes).
In a LightCycler® 480 System Gene Scanning experiment, sample DNA is first amplified via real-time PCR in the presence of LightCycler® 480 High Resolution Melting
Dye. Immediately after DNA amplification, a High Resolution Melting experiment can
be per­formed on the same LightCycler® 480 Instrument and analyzed with LightCycler®
480 Gene Scanning Software to identify sequence variants. Thus, the entire mutation
screening process is homogeneous. That is, the entire experiment can be done on the
LightCycler® 480 Instrument; post-PCR analysis does not require a separate device.
14
LightCycler® 480 Gene Scanning Software
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).
Before a High Resolution Melting analysis can be performed, the target sequence must be
available in high copy number. The easiest way to accomplish this is to perform a DNA
amplification reaction (PCR) before the High Resolution Melt.
Both procedures are performed in the presence of a fluorescent dye that binds only dsDNA.
The dye does not interact with ssDNA, but fluoresces strongly in the presence of dsDNA.
This change in fluorescence can be used both to measure the increase in DNA concentration during PCR and then to directly measure thermally-induced DNA dissociation during High Resolution Melting.
For detection of sequence variations, differences in the melting curves of the amplicons
are analyzed. Heterozygote DNA forms heteroduplices that begin to separate into single
strands at a lower temperature and with a different curve shape than homozygote DNA.
Depending on the individual sequence, most of the different homozygotes give distin­
guishable melting curves, too.
In a melting experiment, fluorescence is initially high because the sample starts as dsDNA,
but fluorescence diminishes as the temperature is raised and DNA dissociates into single
strands. The observed “melting” behavior is characteristic of a particular DNA sample.
Mutations in PCR products are detectable because they change the shape of the melting curve. When the mutant sample is compared to a reference “wild type” sample, these
changes are visible. Below is an example of an experiment with the LightCycler® 480
Instrument and the LightCycler® 480 High Resolution Melting Dye that identifies a single
nucleotide polymorphism (SNP).
15
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.
In heterozygous samples, melting curve shape changes because the observed melting
curve is actually a composite of both heteroduplex and homoduplex components.
­Heteroduplexes formed in the sample (i.e., between the “wild type” and variant
strands) are less stable than the homoduplexes formed, and thus, dissociate more
readily.
16
LightCycler® 480 Gene Scanning Software
Gene Scanning with the LightCycler® 480 System
Components of the LightCycler® 480 System Gene Scanning Assay
Components of the LightCycler® 480
System Gene Scanning Assay
2
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. Here is a brief
look at how these three components have been engineered to work together well in a
LightCycler® 480 System Gene Scanning experiment.
2.1
LightCycler® 480 High Resolution Melting Dye
High Resolution Melting analysis relies on a new generation of dsDNA binding dyes.
LightCycler® 480 High Resolution Melting Dye is a member of this new dye family. This
unique dye can detect the presence of heteroduplexes formed during PCR (e.g., if the
sample is heterozygous for a particular mutation). This feature is not shared with other
dyes traditionally used in real-time PCR (e.g., SYBR Green I or ethidium bromide).
LightCycler® 480 High Resolution Melting Dye is not toxic to amplification enzymes.
Thus, high concentrations of the dye do not affect the PCR. These high concentrations
completely saturate the dsDNA in the sample.
dsDNA remains dye-saturated during the subsequent melting experiment. Under these
conditions, even small changes in the melting behavior result in subtle, but reproducible
changes in High Resolution Melting Dye fluorescence.
According to Wittwer et al (2003)1), this occurs because the dye cannot redistribute
itself from denatured to non-denatured regions of the DNA during melting. Further,
the dyes no longer show a preference for products that melt at higher temperatures.
High fidelity correlation between fluorescence changes and DNA melting increases the
resolution of the recorded melt profiles.
1)
Carl T. Wittwer, Gudrun H. Reed, Cameron N. Gundry, Joshua G. Vandersteen, and Robert J. Pryor (2003).
High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen. Clinical Chemistry 49, 853–860
17
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. LightCycler® 480 Gene Scanning Software has been developed specifically to provide the most accurate analysis of High Resolution Melting curves.
The standard workflow followed by the LightCycler® 480 Gene Scanning Software has
four basic steps:
1. Detecting negatives: LightCycler® 480 Gene Scanning Software automatically uses a
negative filter to detect negative samples, i.e., samples with low fluorescence signals
that lack a prominent melting curve. The software also allows you, if you wish, to
identify the negative samples in the run manually.
2. Normalizing: The second step in the analysis is to normalize the raw melting curve
data by setting the pre-melt (initial fluorescence) and post-melt (final fluorescence)
signals of all samples to uniform values. Pre-melt signals are uniformly set to a relative
value of 100%, while post-melt signals are set to a relative value of 0%. Normalizing
the initial and final fluorescence in all samples aids interpretation and analysis of the
data. In some cases, samples with homozygous SNPs may be distinguished from the
wild type by the displacement of their melting curves, which is easier to see in the
normalized data.
18
unnormalized melting curves
normalized melting curves
LightCycler® 480 Gene Scanning Software
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.)
Now, samples with heterozygous SNPs can easily be distinguished from the wild type
by the different shapes of their melting curves.
normalized melting curves
normalized, temp-shifted melting curves
4. Difference Plot: The final step is to further analyze the differences in melting curve
shape by subtracting the curves from a reference curve (also called “base curve”), thus
generating a Difference Plot, which helps cluster samples automatically into groups
that have similar melting curves (e.g., those with the same genotype).
The way melting curves for homozygotes and heterozygotes are plotted depends on
the base curve you selected for the Difference Plot. In the example above, a homozygote sample was selected as base curve, resulting in negative melting curves for the
heterozygotes. (This is because heterozygotes melt at a lower temperature than homozygotes.) If you selected a heterozygote sample as base curve, homozygotes would
appear as positive melting curves in the example shown.
19
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.
2.3
LightCycler® 480 Instrument
High Resolution Melting analysis of nucleic acid requires that the analyzing instrument
is able to detect even small changes in fluorescence as a function of temperature-induced
DNA melting. In the LightCycler® 480 Instrument, images of DNA melting (i.e., fluorescence) are captured by a sensitive CCD camera and magnified to reveal subtle details in
DNA melting profiles. These profiles are then compared from sample to sample to determine relationships between the samples.
The optical components of the LightCycler® 480 System work together with its accurate
thermal control system to generate highly reproducible results from sample to sample.
Since the samples are held in a multiwell plate, the LightCycler® 480 System can analyze
many samples in a single run.
20
LightCycler® 480 Gene Scanning Software
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 with allele-specific probes.
►► You can design a genotyping assay on the basis of less sequence data.
►► Each reaction generally reveals more information.
In addition, the homogeneous High Resolution Melting technique can process more
samples more conveniently than traditional, non-homogeneous methods (e.g., dHPLC or
SSCP) which require amplicons to be screened for sequence variants on a separate instrument after PCR.
High Resolution Melting analysis identifies heterozygous single-base changes in PCR
products with a sensitivity and specificity that is comparable or superior to non-homoge­
neous techniques. Variants can be detected regardless of their position within the fragment.
Identification of homozygous sequence alterations is more difficult; most other mutation
scanning methods are unable to distinguish these alterations from wild type sequences.
By contrast, High Resolution Melting has identified homozygous sequence alterations in
several different types of amplicons.
21
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. Therefore, it is recommended to select PCR primers that amplify a
relatively short sequence (100 - 250 bp).
Primers should anneal at temperatures around 60°C.
Nevertheless, it is possible to target longer sequences (up to 500 bp), but keep in mind
that analysis of these products will usually have lower resolution. In addition, such
products (>250 bp) are more likely to contain multiple melting domains and generate
complicated melting curves.
►► Secondary structures can affect the efficiency of the amplification reaction. Therefore,
it might be of advantage to determine the folding characteristics of both primers and
amplicon with software that can profile secondary structures.
Make sure to set the folding temperature equal to the annealing temperature that will
be used for the reaction (e.g., 60°C).
The DINAMelt Server from Rensselaer Polytechnic Institute provides appropriate software for such secondary structure analyses; this software can make corrections for
both salt and magnesium concentration (see http://www.bioinfo.rpi.edu/applications/
hybrid/twostate.php).
Low delta-G values indicate a high level of secondary structure. Strands with high
delta-G values produce less secondary structures and so are favored in the amplifica­
tion reaction. For best results, the delta-G values should be above -1.
22
LightCycler® 480 Gene Scanning Software
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. Avoid procedures that can introduce excessive amounts of inhibitors (e.g., due to
ethanol carry-over).
►► If extraction is required, use the same extraction procedure to prepare all samples to
be analyzed via High Resolution Melting. This eliminates any subtle differences that
might be introduced by the reagent components in the final elution buffers of different
extraction procedures.
For reproducible isolation of nucleic acids use:
►► Either the MagNA Pure LC Instrument or the MagNA Pure Compact Instrument
together with a dedicated nucleic acid isolation kit (for automated isolation) or
►► A high pure nucleic acid isolation kit (for manual isolation), e.g., the High Pure
PCR Template Preparation Kit.
For details see the Roche Applied Science Biochemicals catalog or home page,
http://www.roche-applied-science.com.
►► Resuspend all DNA samples in the same buffer, quantify them using spectrophotome­
try, and adjust them to the same concentration with the resuspension buffer. Salts
affect DNA melting behavior, so it is important that the concentrations of buffer, Mg2+
and other salts in the reaction mix are as uniform as possible for all samples.
►► Use the same amount of template in each reaction. The recommended amount is
5 to 30 nanograms of template DNA in a 20 µl reaction volume, which should produce
amplification plots with a Cp value of no more than 30 cycles. Products that reach this
threshold at higher Cps (due to insufficient amounts of starting template or template
degradation) typically produce variable High Resolution Melting results due to amplification artifacts.
►► If you are using archival genomic DNA, repurify the DNA by binding it to silica (e.g.,
with the High Pure PCR Template Preparation Kit from Roche Applied Science) and
eluting it into a fresh buffer before using it in a Gene Scanning experiment. This will
eliminate artifacts caused by sublimation (the direct transition of frozen material to
gas), which frequently occurs in such samples and concentrates salts and other material that affect both amplification and High Resolution Melting.
23
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.
►► To avoid primer-dimer formation, use relatively low primer concentrations (less
than 300 nM) in the experimental reactions.
►► Depending on the amount of specific product observed in the initial experiment,
try repeating the experiment with a series of primer dilutions, increasing or decreasing the concentration in 0.1 μM steps. If initial production of the specific
product is robust, you might try lower concentrations of the primers. If initial
production of specific product is weak, try higher concentrations of the primers.
►► You do not need to use primers with GC clamps; they will not improve
High Resolution Melting.
►► BLAST the primer sequences to ensure they are specific for the target species and gene
(see http://www.ncbi.nlm.nih.gov/BLAST/).
24
LightCycler® 480 Gene Scanning Software
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. This LightCycler® 480 High Resolution Master contains FastStart Taq
DNA Polymerase and the High Resolution Melting Dye in a reaction buffer that contains
no MgCl2. The Master is compatible with additives (e.g., DMSO) that enhance amplification of GC-rich sequences.
FastStart Taq DNA Polymerase is a chemically modified form of thermostable recombinant Taq DNA polymerase that shows no activity at temperatures up to 75°C. The enzyme
is active only at high temperatures, where primers do not bind non-specifically. The enzyme is completely activated (by removal of blocking groups) during a single pre-incubation step (95°C, 5 minutes) before cycling begins.
A separate 25 mM MgCl2 stock solution, supplied with the Master, allows you to easily
optimize the Mg2+ concentration. Determining the optimal MgCl2 concentration is
essential to ensure both the specificity and robustness of the PCR. The optimum MgCl2
concentration for a LightCycler® 480 High Resolution Melting assay may vary from
1.5 to 3.5 mM. Therefore, we strongly recommend that you titrate the MgCl2 concentration in the reaction between 1.5 and 3.5 mM (in 0.5 mM steps) when establishing a new
assay. Determine the specificity of each PCR by agarose gel electrophoresis.
The amount of MgCl2 in a reaction will affect the shape and Tm of the melting profile.
In some cases, fragments that melt with a single inflection at low MgCl2 concentrations
may show multiple inflections at high MgCl2. While we observed this infrequently, you
should be aware of this possibility. As a general rule, at higher MgCl2 concentrations,
a fragment will melt with a slightly higher overall Tm.
25
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. Specific amplification product, undesired amplification product(s), and primer-dimer products all bind the dye with equal affinity; thus all
contribute to the overall fluorescence and melting profile.
To optimize the amplification, determine the best thermal cycling parameters. If you use
the LightCycler® 480 High Resolution Master to establish your LightCycler® 480 Gene
Scanning assay, you can use the following parameters in your initial experiments:
Setup
Detection Format
High Resolution Melting Dye
Programs
Program Name
Cycles
Analysis Mode
Pre-Denaturation
1
None
45 1)
Quantification
High Resolution Melting
1
Melting Curves 6)
Cooling
1
None
Amplification
Temperature Targets
Target [°C]
Acquisition Mode
Hold (hh:mm:ss)
Ramp Rate (°C/s)
(96-well / 384-well)
Acquisitions
(per °C)
None
00:10:00
4.4 / 4.8
-
95
None
00:00:10
4.4 / 4.8
-
primer dependent 2)
None
00:00:10
2.2 / 2.5
-
72
Single
00:00:10 -
Pre-Incubation
95
Amplification
00:00:20
4.4 / 4.8
3)
High Resolution Melting
95
None
00:01:00
4.4 / 4.8
40 4)
None
00:01:00
2.2 / 2.5
60 5)
None
00:00:01
1
-
95 5)
Continuous
-
-
25
00:00:10
4.4 / 4.8
-
Cooling
40
26
None
LightCycler® 480 Gene Scanning Software
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. Modify the Temperature Targets of the Amplification program
as shown in the table below:
Target
(°C)
Acquisition
Mode
95
None
65
3)
72
Ramp Rate
(°C/s)
(96-well /
384-well)
Acquisitions
(per °C)
Sec Target
(°C)
Step
Size (°C)
Step
Delay
(cycles)
00:00:10
4.4 / 4.8
-
0
0
0
None
00:00:10
2.2 / 2.5
-
53
0.5
1
Single
00:00:10 00:00:20
4.4 / 4.8
-
0
0
0
1)
Hold
(hh:mm:ss)
Number of cycles
45 cycles are suitable for most assays. If the assay is optimized and has steep amplification curves and early crossing points (even when target concentrations are low), 40 cycles
should be sufficient. Reducing the number of cycles will reduce the time required for the
assay.
2)
Annealing temperature
Annealing temperature is the parameter that most influences specificity and robustness of
amplification. For initial experiments set the target temperature (i.e., the primer annealing temperature) 2°C below the calculated primer Tm. The amount of specific product, the
presence/absence of undesirable side product, and the presence/absence of dimer product
in these experiments will dictate the best way to optimize this parameter. If the reaction
produces undesirable product, increase the annealing temperature. If amplification is not
robust, decrease the annealing temperature and/or increase the duration of the annealing
step.
3)
Elongation time
Calculate the exact elongation time required for your specific target by dividing the amplicon length by 25 (e.g., a 500 bp amplicon requires 20 s elongation time).
4)
Melting pre-hold step
This pre-hold temperature ensures that all PCR products have re-associated and encourages heteroduplex formation.
5)
Melting interval
Actual melting conditions depend upon the amplicon. For initial experiments set a wide
melting interval, e.g., from 60 to 95°C. Once you have determined where the product will
melt, reduce the melting interval to approximately 25°C. Ensure that the melt program
starts at least 10°C before and ends at least 10°C after the expected Tm value.
6)
Analysis mode
No special analysis mode for Gene Scanning assays is available. Gene Scanning experiments are performed in standard Melting Curves analysis mode.
27
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. After the
initial melting curve run, simply view the Melting Peaks plot (negative first derivative of
the sample fluorescence plotted against temperature; generated by the Tm Calling Analysis
Module of the LightCycler® 480 Software). That plot should show no peaks for the notemplate control. If initial experiments show reaction product in your no-template control, reoptimize the reaction (e.g., by designing new primers) and repeat the experiment.
For further verification, you may also want to examine the product on an electrophoretic
gel.
You can access the Melting Peaks plot directly from the LightCycler® 480 Gene ­Scanning
Software module. There is no need to run a separate Tm Calling Analysis.
28
LightCycler® 480 Gene Scanning Software
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.
To enable installation and running of the LightCycler® 480 Gene Scanning Software,
the LightCycler® 480 Software must be installed on the PC.
To install the LightCycler® 480 Gene Scanning Software:
�
Make sure that you have the administration rights to install the software.
Insert the LightCycler® 480 Gene Scanning Software CD.
If installation does not start automatically, double-click ScanningModuleInstall.exe.
The installation process transfers files, extracts the files, and prepares the installation
wizard. The InstallShield Wizard window opens.
Click Next.
�
You are prompted to agree to the license conditions. Click Yes.
►►►
29
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.
■
30
LightCycler® 480 Gene Scanning Software
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. After samples are amplified by PCR
and subjected to a melting curve experiment, the software analyzes the shapes of the individual curves and groups samples that have similar melting curves.
You can also include melting standards of known sequence variants or genotypes in your
experiment. In this case, the software compares the melting curves of the individual samples to the designated in-run melting standards.
Without the LightCycler® 480 Gene Scanning Software you can still view any gene
scanning experiment in the LightCycler® 480 Software but you cannot perform a Gene
Scanning analysis.
To perform a gene scanning experiment:
�
Set up an experiment containing an amplification program (to amplify the target
samples) and a melting curve program (to melt the samples). Whether you need to
include an internal standard in your experiment depends on the analysis mode you
want to use:
►► Auto Grouping or Common/Variants analysis: no melting standards are required
►► In-run melting standards: include melting standard samples
If you wish, you may also include control samples (e.g., negative controls or no-template controls) in your experiment.
Internal standards act as positive controls.
►►►
31
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”.
The software uses the following parameters for calculation:
Column Name
Description
Valid Values
Target Name
Name of the (PCR) target
(e.g., the name of the gene
amplified and detected)
Alphanumeric value
(≤ 25 characters);
default value is
“Target xxx-yyy”,
where xxx and yyy
are the excitation and
emission wavelengths
Sample Type
Type of sample
Choose from dropdown list:
If you want to use the
“In-run Standards”
­grouping option, you need
to include a Melting Standard for any genotype you
want to detect.
Genotype
Genotype of melting standard
or positive control samples
►► Unknown
►► Negative Control
►► Melting Standard
Alphanumeric value
(≤ 25 characters)
This field is active only
when the Sample Type is
“Melting Standard”.
�
Click Analysis in the Module bar. In the Create New Analysis list, select Gene Scanning.
►►►
32
LightCycler® 480 Gene Scanning Software
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.
■
33
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.
If the Auto Group analysis mode is selected, the software will assign all samples to a well-defined group (up to 6 variant groups
can be assigned on the basis of melting curve shape); samples that
cannot be assigned to such a group will be placed in either the
Unknown or the Negative group.
Comn/Vars
Groups automatically assigned by the software are named
with sequential numbers (1, 2, etc.). Some groups may contain no samples.
Applies automated grouping in the absence of in-run melting
standard samples.
If the Common/Variants analysis mode is selected, the software will
place all samples into either group 1 (the largest group of samples
with similar melting curve shapes, the so-called Common group),
the Variants group, the Unknown group or the Negative group.
No further grouping will be done.
Standards
(In Run)
Applies grouping based on melting standard samples included in
the run.
�
If, in the Sample Editor, you define samples as melting standards, the program will select this option by default after it
creates the analysis.
Groups based on internal standards are given names that
correspond to the appropriate standards (assigned in the
Sample Editor).
►► Deselecting Negatives
If you wish, click the Negatives tab to designate samples as known negatives and
remove them from the analysis (i.e., deselect them).
Although LightCycler® 480 Gene Scanning Software uses a negative filter to
automatically deselect negative samples, it is still possible that all negative
samples will not be detected. You will have another opportunity to correct this at
the end of the run. You can do this by using the New Call option to assign known
negative samples to the Negatives group (see below).
►►►
34
LightCycler® 480 Gene Scanning Software
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. In normalization, two regions of
each curve are selected (one before and one after the major transition); these
are arbitrarily defined, respectively, as 100% fluorescence and 0% (baseline)
fluorescence.
The upper graph, the Melting Curves graph, contains two pairs of movable vertical
sliders that correspond to Pre-melt Low and High temperatures (colored green) and
to Post-melt Low and High temperatures (colored blue). The grey area between the
sliders indicates the area used for normalization.
Below the Melting Curves chart you will see two groups of values that you can set:
The left group is used to define the Pre-melt Temperature Range, while the right group
is used to set the Post-melt Temperature Range.
►►►
35
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). Examine the upper graph to make sure that the
Pre-melt Temperature Range lies in an area where the background fluorescence of all
the samples is dropping consistently but no temperature transitions have occurred,
and that the Post-melt Temperature Range is placed in an area where melting is complete for all samples.
If necessary, adjust the temperature settings by either dragging the vertical sliders
on the upper graph or by changing the values in the Slider Settings fields. For most
purposes, the recommended temperature interval between each set of vertical sliders
is about one degree.
If the Pre-melt and Post-melt Temperature Ranges are set correctly, you should then
obtain a set of normalized melting curves that have a flat section before and after
the major transition; no curve should rise very far above the upper line or fall very far
below the lower line.
►►►
36
LightCycler® 480 Gene Scanning Software
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.
The display on the Temperature Shift tab contains two graphs: the upper graph is
identical to the Normalized Melting Curves graph displayed on the Normalization tab,
the lower graph shows melting curves that are both normalized and temperature
shifted.
The upper graph has a movable horizontal slider that corresponds to the amount
of the Temperature Shift. Left-hand of the graph you will see a control field, labeled
Threshold, which can also be used to set the Temperature Shift level.
The default value of the Temperature Shift level is 5. You can change the value of the
Temperature Shift either by entering a value in the Threshold control field or by dragging the horizontal slider on the upper graph.
Select a shift level that makes the curves form tight groups with the maximum amount
of distance between groups (as shown in the image below). For most applications, the
default level of 5 produces acceptable results.
►►►
37
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.
�
Results of a Gene Scanning analysis include the Sample Selector with Legend ­Property
Selector and Legend Property buttons and the Results table. Use the Legend Property
Selector to display colors by result, by sample types, by sample preferences or by
replicate groups. Use the colored Legend Property buttons to select the display of
samples with certain properties in the MWP image, the Results table and in the
charts.
If you choose Scanning results in the Legend Property Selector, the Results table of a
Gene Scanning analysis displays the following results. The Legend Property buttons
enable you to select samples depending on the variant group:
►► Either 6 genotype groups (if you selected the Auto Group analysis mode) or 2
genotype groups (if you selected the Comn/Vars analysis mode).
►► Negative and Unknown groups.
Checkboxes will be shown even if the corresponding groups are not found in the
data.
►► Group: Name of the genotype group to which this sample is assigned.
Variant/genotype groups that are automatically assigned by the software are
given sequential numbers (1, 2, etc.), while groups based on in-run melting standards are given names that correspond to the appropriate standards (assigned in
the Sample Editor).
►►►
38
LightCycler® 480 Gene Scanning Software
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. The difference between the reference and each remaining curve is plotted against Temperature.
By checking the Show Standards box, you can also display the curves of the melting
standards in the chart. These appear as black lines.
In Auto Group analysis mode, the median curve of each variant/genotype group
is defined as the standard for that group.
Click Select base curve to manually define the reference curve for the Difference Plot.
By default, the standard of the largest variant/genotype is used as base curve.
■
39
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.,
it shows the unnormalized and non-temperature shifted curves
►► Normalized Curves: displays the normalized melting curves
►► Normalized and Temp-Shifted Curves: corresponds to the Normalized and Shifted
Melting Curves chart in the Temperature Shift tab.
The normalized and shifted melting curves are basis for calculating the Difference Plot.
For the lower chart you have the following choices:
►► Melting Peaks: displays the Melting Peaks plot (negative first derivative of the
sample fluorescence plotted against temperature, corresponds to the result of the
Tm Calling Analysis Module)
►► Normalized and Temp-Shifted Difference Plot: displays the normalized and
temperature-shifted Difference Plot (see Step 8 above)
■
To change the sensitivity setting:
You can adjust the Sensitivity value to refine the Gene Scanning result. The Sensitivity
function lets you influence the stringency with which melting curves are classified into different groups. A high Sensitivity value generally produces more groups than a low value.
Click the Sensitivity tab in the Results table to modify the analysis setting:
Slide the Sensitivity slider bar to the left to reduce stringency, or to the right to increase
stringency.
The default value is 0.3 (recommended for initial analysis). This value provides a reasonable balance between producing too many unknowns and generating potentially wrong
calls.
Please note, that in LightCycler® 480 Gene Scanning Software version 1.5 the sensitivity
calculation has changed. This means that an analysis defined in an earlier version with a
specific sensitivity value (e.g., “0,95”) will not have the same results as an analysis defined
in ­version 1.5 with the same sensitivity value.
40
LightCycler® 480 Gene Scanning Software
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.
�
On the Groups tab, double-click the field for the group name you want to
change.
�
Type a new name in the field. The new name is immediately shown on both the
Sample Selector and the Results table.
■
To change variant/genotype calls:
If you believe the software has assigned a sample to the wrong group, you can manually
change the assigned group by using the New Call option.
�
In the Results table, select the samples to be changed. For details see the
LightCycler® 480 Instrument Operator’s Manual, section “Working with Samples in the
Analysis”.
�
From the New Call drop-down list below the chart area, select a new group name
(genotype, Unknown, or Negative).
�
Click the Apply button.
►►►
41
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.
You cannot assign a new call to negative controls. When in-run melting standards are used, you cannot assign a new call to them. If you attempt to apply a
new call to a selection that includes an in-run melting standard or control, the
software will warn you that the selection includes in-run standards or controls
that cannot be changed, and asks you if you want to continue. If you choose to
continue, the software will apply the changes to all samples except the in-run
standards and controls. If you choose not to continue, the operation will be cancelled.
■
42
LightCycler® 480 Gene Scanning Software
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.
►► If the current experiment does contain a subset with the same name, but the subset
does not contain the same well positions as the subset in the template, the template
cannot be used. You can delete the respective subset in the experiment, and then
you are able to apply the template.
►► Filter combination
►► Analysis name
►► Analysis mode setting (Auto Group, In-run Standards, or Common/Variants)
►► Analysis notes
►► Pre-melt and post-melt temperature settings
►► Temperature shift setting
►► Sensitivity setting
43
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.
►► If any Negative Control is not designated negative, the run fails this control check.
►► If any Negative Controls fail, the software will report no results and will inform the
user that the Negative Control check has failed. The error dialogue also indicates
which controls have failed this check.
►► Melting Standards
►► If every internal standard is designated positive, the run passes this control check.
►► If all internal standards with the same group name are put into the same group,
and standards with distinct group names are placed into distinct groups, the run
passes this control check.
►► If any two internal standards with the same group name are not grouped together,
the run fails this control check.
►► If any two internal standards with distinct group names are grouped together, the
run fails this control check.
►► If any internal standard is designated negative the run fails this control check.
44
LightCycler® 480 Gene Scanning Software
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. However, in this case,
the Gene Scanning analysis and the analysis-specific Sample Editor will be set to readonly.
►► When a macro that contains a Gene Scanning analysis is run on a system where the
LightCycler® 480 Gene Scanning Software is not installed, the Gene Scanning analysis
will not be performed.
►► A template for a Gene Scanning analysis cannot be used on a system where the
LightCycler® 480 Gene Scanning Software is not installed.
►► When a report is generated from an experiment that contains a Gene Scanning analysis on a system where the LightCycler® 480 Gene Scanning Software is not installed,
the report will contain the read-only information from the Gene Scanning analysis.
45
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.com
►► DNA & RNA preparation – Versatile Tools for Nucleic Acid Purification:
http://www.roche-applied-science.com/napure
►► Redefining Real-Time qPCR Assays with prevalidated UPL-probes:
http://www.universalprobelibrary.com
Instruments
LightCycler® 480 Instrument II, 96-well
1 instrument with control unit and
accessories
05 015 278 001
LightCycler® 480 Instrument II, 384-well
1 instrument with control unit and
accessories
05 015 243 001
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
LightCycler® 480 Thermal Block Cycler Unit
(96-well) Silver
96-well thermal block cycler unit,
including block cycler cover, storage
box and loading device
05 015 219 001
LightCycler® 480 Thermal Block Cycler Unit
(384-well) Silver
384-well thermal block cycler unit,
including block cycler cover, storage
box and loading device
05 015 197 001
4 filters
04 686 128 001
1 lamp
04 686 136 001
50 plates with 50 sealing foils
04 729 692 001
LightCycler 480 Multiwell Plate 384
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
LightCycler® 480 Sealing Foil
1 x 50 foils
04 729 757 001
Software
®
Accessories
Spare Parts
LightCycler® 480 Dust Filters
®
LightCycler 480 Xenon Lamp
Disposables
LightCycler® 480 Multiwell Plate 96
®
®
®
LightCycler 480 Sealing Foil Applicator
46
04 706 170 001
LightCycler® 480 Gene Scanning Software
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 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
LightCycler® 480 High Resolution Melting Dye
1 ml
04 909 640 001
SimpleProbe 519 Labeling Reagent
100 µmol
04 687 132 001
LightCycler® 480 CYAN 500 Labeling Reagent
1 vial (100 µmol)
04 764 153 001
1g
03 138 178 001
5 columns
03 113 906 001
LightCycler Red 640-N-hydroxysuccinimide ester
1 vial
12 015 161 001
LightCycler® Red 610-N-hydroxysuccinimide ester
1 vial
03 561 488 001
1 kit (3 instrument runs)
04 710 924 001
100 purifications
11 796 828 001
LightCycler® 480 Probes Master
Labeling Reagents
®
LightCycler Fluorescein CPG
®
®
LightCycler 480 Demo Kit
Isolation of Nucleic Acids
High Pure PCR Template Preparation Kit
47
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www.roche-applied-science.com
Published by
Roche Diagnostics GmbH
Roche Applied Science
68298 Mannheim
Germany
2008 Roche Diagnostics GmbH
All rights reserved.
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