Applied Biosystems
7300, 7500 and 7500 Plus Minus Assay Getting Started Guide Rev B June 2006
Getting Started Guide
78 Pages

Preview
Page 1
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System
Plus/Minus Assay Getting Started Guide
Introduction
Designing a Plus/Minus Experiment
Setting Up the Reaction Plate
Performing the Plus/Minus Pre-Read Run
STANDARD
Generating Amplification Data
Performing the Plus/Minus Post-Read Run
© Copyright 2006, Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. NOTICE TO PURCHASER: Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that may appear in this document. In no event shall Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use of this document. TRADEMARKS: Applera, Applied Biosystems, AB (Design), ABI PRISM, BloodPrep, MicroAmp, NucPrep, Primer Express, and VIC are registered trademarks and FAM, PrepMan, ROX, and TAMRA are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries. AmpErase, AmpliTaq Gold, TaqMan are registered trademarks of Roche Molecular Systems, Inc. SYBR is a registered trademark of Molecular Probes, Inc. All other trademarks are the sole property of their respective owners.
Part Number 4378652 Rev. A 09/2006
Plus/Minus Experiment Workflow
Chapter 1
Introduction
About the 7300/7500/7500 Fast System
About Plus/Minus Assays Using an IPC
Chapter 2
Designing a Plus/Minus Experiment
Using TaqMan ® Probe-Based Reagent Configuration
Designing the Probe and Primers
Chapter 3
Setting Up the Reaction Plate
Preparing DNA
Setting Up the Reaction Plate
Chapter 4
Performing the Plus/Minus Pre-ReadRun
The Pre-Read Run
Before You Begin
Creating a Plate Document for Sample Amplification
Performing the Amplification Run
Performing the Post-Read Run
Viewing Plus/Minus Results
Chapter 5
Chapter 6
Generating STANDARD Amplification Data
Performing the Plus/Minus Post-Read Run
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
About Plus/Minus Assays
Creating a Plus/Minus Plate Document
Performing the Pre-Read Run
STANDARD
Exporting Plus/Minus Plate Data
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Contents
Plus/Minus Experiment Workflow
iii
Preface
vii
How to Use This Guide... vii How to Obtain More Information... ix How to Obtain Support... x
Chapter 1
Introduction
1
About the 7300/7500/7500 Fast System... 2 About Plus/Minus Assays Using an IPC... 2 About Plus/Minus Assays... 4
Chapter 2
Designing a Plus/Minus Experiment
7
Using TaqMan® Probe-Based Reagent Configuration... 8 Designing the Probe and Primers... 9
Chapter 3
Setting Up the Reaction Plate
11
Preparing DNA... 12 Setting Up the Reaction Plate... 13
Chapter 4
Performing the Plus/Minus Pre-Read Run
17
The Pre-Read Run... 18 Before You Begin... 18 Creating a Plus/Minus Plate Document... 18 Performing the Pre-Read Run... 24
Chapter 5
Generating Amplification Data
27
Creating a Plate Document for Sample Amplification... 28 Performing the Amplification Run... 32
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
v
Chapter 6
Performing the Plus/Minus Post-Read Run
35
Performing the Post-Read Run... 36 Viewing Plus/Minus Results... 39 Exporting Plus/Minus Plate Data... 43
Appendix A Creating Detectors
45
Appendix B Viewing Amplification Data
47
Specifying Analysis Settings... 47 Analyzing the Plus/Minus Amplification Data (AQ Plate)... 48 Viewing the Amplification Data... 49
Appendix C Example Plus/Minus Experiment
vi
55
References
61
Index
63
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Preface How to Use This Guide Purpose of This Guide Assumptions
This manual is written for principal investigators and laboratory staff who run plus/minus assays using the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System (7300/7500/7500 Fast system). This guide assumes that you have: • Familiarity with Microsoft® Windows® XP operating system. • Knowledge of general techniques for handling DNA samples and preparing them for PCR. • A general understanding of hard drives and data storage, file transfers, and copying and pasting. • Networking experience if you want to integrate the 7300/7500/7500 Fast system into your existing laboratory data flow system.
Text Conventions
This guide uses the following conventions: • Bold indicates user action. For example: Type 0, then press Enter for each of the remaining fields. • Italic text indicates new or important words and is also used for emphasis. For example: Before analyzing, always prepare fresh matrix. • A right arrow bracket (>) separates successive commands you select from a dropdown or shortcut menu. For example: Select File > Open > Spot Set.
User Attention Words
The following user attention words appear in Applied Biosystems user documentation. Each word implies a particular level of observation or action as described below: Note – Provides information that may be of interest or help but is not critical to the use of the product. IMPORTANT! – Provides information that is necessary for proper instrument operation,
accurate chemistry kit use, or safe use of a chemical. Indicates a potentially hazardous situation that, if not avoided, may result in minor or moderate injury. It may also be used to alert against unsafe practices. Indicates a potentially hazardous situation that, if not avoided, could result in death or serious injury.
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
vii
Preface How to Use This Guide
Safety
Chemical manufacturers supply current Material Safety Data Sheets (MSDSs) with shipments of hazardous chemicals to new customers. They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated. MSDSs provide the safety information you need to store, handle, transport, and dispose of the chemicals safely. Each time you receive a new MSDS packaged with a hazardous chemical, be sure to replace the appropriate MSDS in your files. You can obtain from Applied Biosystems the MSDS for any chemical supplied by Applied Biosystems. This service is free and available 24 hours a day. To obtain MSDSs:
1. Go to https://docs.appliedbiosystems.com/msdssearch.html 2. In the Search field, type in the chemical name, part number, or other information that appears in the MSDS of interest. Select the language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any of the following: • Open – To view the document • Print Target – To print the document • Save Target As – To download a PDF version of the document to a destination that you choose
4. To have a copy of a document sent by fax or e-mail, select Fax or Email to the left of the document title in the Search Results page, then click RETRIEVE DOCUMENTS at the end of the document list.
5. After you enter the required information, click View/Deliver Selected Documents Now. Refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Getting Started Guide (PN 4378657) and the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Site Preparation Guide (PN 4378654) for important safety information.
viii
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Preface How to Obtain More Information
How to Obtain More Information Related Documentation
For more information about using the 7300/7500/7500 Fast system, refer to the Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Online Help or the documents shown below. Online Help P/N
P/N
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Allelic Discrimination Getting Started Guide
4347822
4378653
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Relative Quantification Getting Started Guide
4347824
4378655
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Absolute Quantification Getting Started Guide
4347825
4378656
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Site Preparation Guide
4347823
4378654
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Installation and Maintenance Guide
4347828
4378657
Real-Time PCR Systems Chemistry Guide
4348358
4378658
Applied Biosystems 7500 FAST Real-Time PCR System, QRC
4362285
4378659
Applied Biosystems Real-Time System Computer Set Up Guide, QRC
4365367
4378660
Document Title
Send Us Your Comments
Applied Biosystems welcomes your comments and suggestions for improving its user documents. You can e-mail your comments to: [email protected]
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
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Preface How to Obtain Support
How to Obtain Support For the latest services and support information for all locations, go to http://www.appliedbiosystems.com, then click the link for Support. At the Support page, you can: • Obtain worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities • Search through frequently asked questions (FAQs) • Submit a question directly to Technical Support • Order Applied Biosystems user documents, MSDSs, certificates of analysis, and other related documents • Download PDF documents • Obtain information about customer training • Download software updates and patches
x
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Chapter 1
Introduction
Introduction About the 7300/7500/7500 Fast System
See page 2
Designing a Plus/Minus Experiment
Setting Up the Reaction Plate
About Plus/Minus Assays Using an IPC
See page 2
About Plus/Minus Assays
See page 4
Performing the Plus/Minus Pre-ReadRun
STANDARD D
Generating Amplification Data
Performing the Plus/Minus Post-Read Run
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
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Chapter 1 Introduction About the 7300/7500/7500 Fast System
About the 7300/7500/7500 Fast System Description
The Applied Biosystems Real Time PCR System (7300/7500/7500 Fast system) uses fluorescent-based PCR chemistries to provide: • Quantitative detection of nucleic acid sequence using real-time analysis. • Qualitative detection of nucleic acid sequence using end-point and dissociationcurve analysis.
Plus/Minus Assay
The 7300/7500/7500 Fast system allows you to perform several assay types with plates in the 96-well format. This guide describes the plus/minus assay, which determines whether or not a specific target sequence is present in a sample. Note: For information about the other assay types, refer to the Real-Time PCR Systems
Chemistry Guide (PN 4378658) and the Online Help for the 7300/7500/7500 Fast system.
Note: Plus/Minus Assays may be run on a 7500 Fast system using standard reagents;
Plus/Minus Assays are not supported using Fast reagents and protocols.
About Plus/Minus Assays Using an IPC Definition
A plus/minus assay is an end point assay that determines if a specific target sequence is present (plus) or not present (minus) in a sample. In an end-point assay, data are collected at the end of the PCR process.
What Is An IPC?
An IPC is an internal positive control (see TaqMan® Exogenous Internal Positive Control Reagents kit, PN 4308323) that is used in plus/minus assays to monitor the PCR process and to ensure that a negative result is not due to failed PCR. The IPC consists of a template, a primer set, and a dye-labeled (VIC®) probe added to each well of a reaction plate (the IPC is part of the reaction mix, see “Preparing the PCR Reaction Mix” on page 14). Plus/Minus assays with an IPC use fluorogenic 5′ nuclease chemistry (also known as “TaqMan probe-based chemistry”). During amplification, the sample target and the IPC target generate reporter fluorescence signals, so that positive or negative calls may be made on unknown samples. Note: The SYBR® Green I dye chemistry is not supported for plus/minus assays using
an IPC.
Notes
2
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Chapter 1 Introduction About Plus/Minus Assays Using an IPC
Terms Used in Plus/Minus Analysis
Term
Definition
Internal positive control (IPC)
A second TaqMan® probe and primer set added to the reaction plate to monitor the PCR process and to ensure that a negative result is not due to failed PCR in the sample
No amplification control (NAC)
Wells that contain no target template and blocked IPC (the IPC target template has been blocked by a blocking agent)
No template control (NTC)
A sample that contains no target template
Nucleic acid target
Nucleotide sequence that you want to identify as present or absent
Unknown sample (U)
The sample for which you want to determine the presence or absence of a specific target
1
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
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Chapter 1 Introduction About Plus/Minus Assays
About Plus/Minus Assays Plus/Minus Experiment Workflow
This document uses the term “plus/minus assay” to refer to the entire process of analyzing samples of extracted DNA from data collected at the end of the PCR process. Design the experiment and isolate the DNA, then conduct a plus/minus assay by performing: • A pre-read run on a plus/minus plate document to determine the baseline fluorescence associated with primers and probes before amplification. • An amplification run using an AQ plate document to generate real-time PCR data, which can be used to analyze and troubleshoot the PCR data for the plus/minus assay, if needed. • A post-read run using the original plus/minus plate document, which automatically subtracts the baseline fluorescence determined during the pre-read run to calculate the result. The following figure illustrates the complete process.
Reaction Plate
Required User-Supplied Materials
7300/7500/7500 Fast System
Pre-Read Run
Item
Amplification Run
Post-Read Run
Source
DNA isolation and purification chemistry systems: • ABI PRISM™ 6100 Nucleic Acid PrepStation
• Applied Biosystems (PN 6100-01)
• BloodPrep™ Chemistry (genomic DNA from fresh or frozen blood or cells)
• Applied Biosystems (PN 4346860)
• NucPrep™ Chemistry (DNA from animal and plant tissue)
• Applied Biosystems (PN 4340274)
• PrepMan™ Ultra Sample Preparation Reagent Kit
• Applied Biosystems (PN 4322547)
Labeled primers and probes source: • Primer Express® Software (customdesigned primers and probes)
• PN 4330710 (1-user license) PN 4330709 (10-user license) PN 4330708 (50-user license)
MicroAmp® Optical 96-Well Reaction Plates
Applied Biosystems (PN 4306757)
Optical Adhesive Covers
Applied Biosystems (PN 4311971)
Reagent tubes with caps, 10-mL
Applied Biosystems (PN 4305932)
Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Chapter 1 Introduction About Plus/Minus Assays
Item
Source
TaqMan® Exogenous Internal Positive Control Reagents (VIC® Probe)
Applied Biosystems (PN 4308323)
TaqMan® Universal PCR Master Mix
Applied Biosystems (PN 4304437)
Centrifuge with adapter for 96-well plates
Major Laboratory Supplier (MLS)
Gloves
MLS
Microcentrifuge
MLS
Microcentrifuge tubes, sterile 1.5-mL
MLS
Nuclease-free water
MLS
Pipette tips, with filter plugs
MLS
Pipettors, positive-displacement
MLS
Tris-EDTA (TE) Buffer, pH 8.0
MLS
Vortexer
MLS
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Example Plus/Minus Experiment To better illustrate how to design, perform, and analyze plus/minus experiments, this document provides an example experiment. The example experiment represents a typical plus/minus experiment that you can use as a quick-start procedure to familiarize yourself with the plus/minus workflow. Details about the plus/minus workflow are described in the subsequent chapters of this guide. Example Experiment boxes appear in subsequent chapters to illustrate workflow details. Refer to Appendix C, “Example Plus/Minus Experiment,” for more information.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
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Chapter 1 Introduction About Plus/Minus Assays
Notes
6
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Chapter 2
Designing a Plus/Minus Experiment
Introduction
2
Designing a Plus/Minus Experiment Using TaqMan ® Probe-Based Reagent Configuration
See page 8
Designing the Probe and Primers
See page 9
Setting Up the Reaction Plate
Performing the Plus/Minus Pre-Read Run
STANDARD D
Generating Amplification Data
Performing the Plus/Minus Post-Read Run
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
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Chapter 2 Designing a Plus/Minus Experiment Using TaqMan® Probe-Based Reagent Configuration
Using TaqMan® Probe-Based Reagent Configuration About the Chemistry
Plus/minus assays with an IPC use the fluorogenic 5′ nuclease chemistry (also known as TaqMan® probe-based chemistry).
Chemistry
Process
TaqMan® reagents or kits
Polymerization
Description
FORWARD PRIMER
TaqMan probe-based chemistry uses a fluorogenic probe to detect a specific PCR product as it accumulates during PCR cycles.
Strand Displacement R
5′ 3′
PROBE
R = REPORTER
Q 3′
R
Q = QUENCHER
5′
5′
3′ 5′ REVERSE PRIMER
Q 5′ 3′
3′ 5′
5′
3′ 5′
Step 1: A reporter (R) and a quencher (Q) are attached to the 5' and 3' ends of a TaqMan probe.
Step 1 (continued): When both dyes are attached to the probe, reporter dye emission is quenched.
Cleavage
Polymerization Completed R
R Q 5′ 3′
3′ 5′
5′
3′ 5′
Step 2: During each extension cycle, the AmpliTaq Gold® DNA polymerase cleaves the reporter dye from the probe.
5′ 3′
Q 3′ 5′
5′
3′ 5′
Step 3: After being separated from the quencher, the reporter dye emits its characteristic fluorescence.
For more information about the TaqMan probe-based chemistry, refer to the Real-Time PCR Systems Chemistry Guide (PN 4378658).
Chemistry Kits for Plus/Minus Assay
The following reagents are available from Applied Biosystems for designing and running plus/minus assays. Kit
Part Number
TaqMan® Exogenous Internal Positive Control Reagents with TaqMan Universal PCR Master Mix
4308323
TaqMan® Universal PCR Master Mix
4304437
Note: The IPC DNA, primers, and probe supplied in these reagents can be used with all
sample target systems. Refer to the TaqMan® Universal PCR Master Mix Protocol (PN 4304449) for instructions on optimizing amplification of your target. Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
Chapter 2 Designing a Plus/Minus Experiment Designing the Probe and Primers
Designing the Probe and Primers Design a probe and primer set for your target sequence. Applied Biosystems provides the Primer Express® software for this purpose. For more information about using this software, refer to the Primer Express Software v3.0 Getting Started Guide (PN 4362460).
Sample Experiment In the example experiment, we extracted DNA from 84 batches of hamburger meat and tested them for the presence of E. coli using the plus/minus assay on the 7300/7500 Real Time PCR System. Six no IPC/no target template controls, six IPC/no target template controls, and 84 unknown samples were run.
2
For the example experiment, the TaqMan® Exogenous Internal Positive Control Reagents Kit supplies one 1-mL tube of 10✕ Exo IPC Mix. This mix contains the IPC primers and VIC®-labeled probe. The primers/probe set for E. coli was designed by Applied Biosystems Primer Express software, and contained a FAM™-labeled probe with TAMRA™ as the quencher.
Notes
Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide
9
Chapter 2 Designing a Plus/Minus Experiment Designing the Probe and Primers
Notes
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Applied Biosystems 7300/7500/7500 Fast Real-Time PCR System Plus/Minus Assay Getting Started Guide