Applied Biosystems 7900HT Fast Real-Time PCR System

Allelic Discrimination Getting Started Guide

Introduction

Designing an Allelic Discrimination Experiment

Preparing the Samples and Reaction Plate

Performing an Allelic Discrimination Pre-Read Run

Performing an Amplification Run

Performing and Analyzing an Allelic Discrimination Post-Read Run

 Copyright 2007, 2010 Applied Biosystems. All rights reserved. For Research Use Only. Not for use in diagnostic procedures. Notice to Purchaser The Applied Biosystems 7900HT Fast Real-Time PCR System is a real-time thermal cycler covered by US patents and corresponding claims in their non-US counterparts, owned by Applied Biosystems. No right is conveyed expressly, by implication or by estoppel under any other patent claim, such as claims to apparatus, reagents, kits, or methods such as 5 nuclease methods. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. TRADEMARKS: ABI PRISM, Applied Biosystems, MicroAmp, Primer Express, and VIC are registered trademarks and AB (Design), Applera, BloodPrep, FAM, NucPrep, ROX, and TAMRA are trademarks of Applied Biosystems or its subsidiaries in the U.S. and/or certain other countries. SYBR is a registered trademark of Molecular Probes, Inc. AmpliTaq Gold, AmpErase, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners.

Part Number 4364015 Rev. D 06/2010

Contents

Preface

How to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v How to Obtain More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii How to Obtain Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Chapter 1

Introduction

About the 7900HT Fast System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 About Allelic Discrimination Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 About Allelic Discrimination Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Before You Begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Chapter 2

Designing an Allelic Discrimination Assay Experiment

Using TaqMan Probe-Based Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Selecting the Probes and Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Chapter 3

Preparing the Samples and Reaction Plate

13

Preparing DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Preparing the Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Preparing the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Chapter 4

Performing an Allelic Discrimination Pre-Read Run

21

About Allelic Discrimination (AD) Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Creating an Allelic Discrimination (AD) Plate Document . . . . . . . . . . . . . . . . . . . . . . 23 Performing the Pre-Read Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Chapter 5

Performing an Amplification Run

37

About Standard Curve (AQ) Plate Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Creating a Standard Curve (AQ) Plate Document . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Performing the Amplification Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

iii

Chapter 6

Performing and Analyzing an Allelic Discrimination Post-Read Run

51

Performing the Allelic Discrimination Post-Read Run . . . . . . . . . . . . . . . . . . . . . . . . 52 Analyzing the Run and Evaluating the Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Assigning Calls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 Saving the Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 Post-Analysis Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Appendix A Sample Experiment

73

About the Sample Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Appendix B SDS Automation Controller Software

83

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Using the Automation Controller Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Appendix C Analyzing and Viewing Amplification Data

89

Terms Used in Quantitation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 Analyzing the Amplification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Viewing the Amplification Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Appendix D Flags and Filtering for Allelic Discrimination (AD) Plate Documents

99

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 Viewing Flags . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

References Index

103 105

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Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Preface
How to Use This Guide
Purpose of This Guide Audience Assumptions
This guide provides procedures for conducting allelic discrimination assays using the Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT Fast System). This guide is intended for principal investigators and laboratory staff who conduct allelic discrimination assays using the 7900HT 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 plan to integrate the 7900HT Fast System into your existing laboratory data flow

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.

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.

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Preface
How to Use This Guide

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.

Safety

Follow specific safety practices when using this instrument. For safety guidelines, refer to the Safety and EMC Compliance section in the Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation Guide (PN 4351923). For any chemical manufactured or distributed by Applied Biosystems, you can obtain the MSDS from Applied Biosystems. This service is available free 24 hours a day. To obtain MSDSs: The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day. To obtain MSDSs:

1. Go to www.appliedbiosystems.com, click Support, then click MSDS Search. 2. In the Keyword Search field, enter the chemical name, product name, MSDS 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 For the MSDSs of chemicals not distributed by Applied Biosystems, contact the chemical manufacturer.

4. To have a copy of the document sent by fax or e-mail:

a. Select the Fax or Email checkbox beneath the document title. b. Click RETRIEVE DOCUMENTS at the end of the document list. c. Enter the required information. d. Click View/Deliver Selected Documents Now.

For chemicals not manufactured or distributed by Applied Biosystems, contact the chemical manufacturer.

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Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Preface
How to Obtain More Information

How to Obtain More Information

Related Documentation
For more information about using the 7900HT Fast System and performing assays, refer to: Applied Biosystems 7900HT Fast Real-Time PCR System Allelic Discrimination Getting Started Guide (PN 4364015) Applied Biosystems 7900HT Fast Real-Time PCR System Absolute Quantitation Using Standard Curve Getting Started Guide (PN 4364014) Applied Biosystems 7900HT Fast Real-Time PCR System Plus-Minus Getting Started Guide (PN 4364017) Applied Biosystems 7900HT Fast Real-Time PCR System Relative Quantitation Using Comparative CT Getting Started Guide (PN 4364016) Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help) Applied Biosystems 7900HT Fast Real-Time PCR System Maintenance and Troubleshooting Guide (PN 4365542) Applied Biosystems 7900HT Fast Real-Time PCR System Site Preparation Guide (PN 4351923) ABI PRISM 6100 Nucleic Acid PrepStation Users Guide (PN 4326242) Assays-by-Design Service for SNP Assays Protocol (PN 4334431) DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and Buccal Swabs Protocol (PN 4343586) NucPrep Chemistry Protocol: Isolation of Genomic DNA from Animal and Plant Tissue (PN 4333959) Primer Express Software v3.0 Getting Started Guide (PN 4362460) Real-Time PCR Systems Chemistry Guide (PN 4348358) TaqMan Low Density Array Getting Started Guide (PN 4319399) TaqMan SNP Genotyping Assays Protocol (PN 4332856) TaqMan Universal PCR Master Mix Protocol (PN 4304449) TransPrep Chemistry Protocol: Purification of gDNA from Filtrates Obtained After the Isolation of RNA from Homogenized Animal or Plant Tissue Samples (PN 4326965)

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]

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

vii

Preface
How to Obtain Support

How to Obtain Support

To contact Applied Biosystems Technical Support from North America by telephone, call 1.800.762.4001. 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

viii

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1

Introduction

Introduction

About the 7900HT Fast System

See page 2

Designing an Allelic Discrimination Assay Experiment

Preparing the Samples and Reaction Plate

About Allelic Discrimination Assays

See page 3

Performing an Alleic Discriminiation Pre-Read Run

About Allelic Discrimination Experiments

See page 4

Performing an Amplification Run

Before You Begin

See page 7

Performing and Analyzing an Allelic Discrimination Post-Read Run

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
About the 7900HT Fast System

About the 7900HT Fast System

System Description
The Applied Biosystems 7900HT Fast Real-Time PCR System (7900HT Fast System) uses fluorescent-based PCR chemistries to provide: Quantitative detection of nucleic acid sequences using real-time analysis Qualitative detection of nucleic acid sequences using end-point and dissociation-curve analysis For more information on the 7900HT Fast System, refer to the Sequence Detection Systems Software version 2.3 Online Help (SDS Online Help) and the Applied Biosystems 7900HT Fast Real-Time PCR System Maintenance and Troubleshooting Guide (PN 4365542).
Note: To access the SDS Online Help, select Help > SDS Online Help from the

SDS software menu bar.

Supported Assays and Consumables

You can perform several assay types on the 7900HT Fast System using reactions plates in the 96-well, 384-well, or TaqMan Low Density Array format. This guide describes the allelic discrimination assay.
Reaction Plate and System Block Options Assay Type Standard curve (AQ) Comparative CT (RQ) Allelic discrimination Plus/minus Fast 96-well Yes Yes No No Standard 96-well Yes Yes Yes Yes Standard 384-well Yes Yes Yes Yes TaqMan Low Density Array (TLDA) No Yes No No

Allelic Discrimination Assay Configuration

Allelic discrimination assays can be run on the 7900HT Fast System using standard 96well and standard 384-well reaction plates with standard reagents and standard protocols.
IMPORTANT! Allelic discrimination assays are not supported using Fast reaction plates,

Fast reagents, Fast protocols, or the TaqMan Low Density Array.

IMPORTANT! Be sure you use standard reaction plates on the 7900HT Fast System with a standard block. Fast 96-well reaction plates do not fit into the standard 96-well block and standard 96-well reaction plates do not fit into the Fast 96-well block.

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
About Allelic Discrimination Assays

About Allelic Discrimination Assays

1
Definition
An allelic discrimination assay is a multiplexed (more than one primer/probe pair per reaction) end-point (data is collected at the end of the PCR process) assay that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the single-nucleic polymorphism (SNP) site in a target template sequence. The actual quantity of target sequence is not determined. For each sample in an allelic discrimination assay, a unique pair of fluorescent dye detectors is used, for example, two TaqMan MGB (minor groove binder) probes that target an SNP site (Afonina et al., 1997; Kutyavin et al., 1997). One fluorescent dye detector is a perfect match to the wild type (allele 1) and the other fluorescent dye detector is a perfect match to the mutation (allele 2). The allelic discrimination assay classifies unknown samples as: Homozygotes (samples having only allele 1 or allele 2) Heterozygotes (samples having both allele 1 and allele 2) The allelic discrimination assay measures the change in fluorescence of the dyes associated with the probes. The figure below illustrates results from matches and mismatches between target and probe sequences in TaqMan SNP Genotyping Assays (Livak et al., 1995).
Allele 1

V Q
Match

F Q

Legend

V
Mismatch

VIC dye FAMTM dye Quencher

AmpliTaq Gold DNA Polymerase
GR1556

F Q

Allele 2

F Q
Match

V Q
Mismatch

The table below shows the correlation between fluorescence signals and sequences in the sample.
A substantial increase in VIC dye fluorescence only FAM dye fluorescence only Both fluorescence signals

Indicates Homozygosity for allele 1 Homozygosity for allele 2 Heterozygosity allele 1-allele 2

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
About Allelic Discrimination Experiments

Terms Used in Allelic Discrimination Analysis

No template control (NTC)

A sample that does not contain template. Shows background signal and is used as the negative control. Provides a means of measuring contamination that might give a false positive signal (Kwok and Higuchi, 1989). Nucleotide sequence that you want to genotype. The sample for which you want to determine the genotype of a specific target A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescence fluctuations caused by changes in concentration or in volume. The dye attached to the 5 end of a TaqMan probe. Provides a fluorescence signal that indicates specific amplification. The ratio of the fluorescence emission intensity of the reporter dye to the fluorescence emission intensity of the passive reference dye.

Nucleic acid target (also called target template or target) Unknown sample (U; also called sample of interest) Passive reference

Reporter dye

Normalized reporter (Rn)

About Allelic Discrimination Experiments

Allelic Discrimination Experiment Workflow
After you design an allelic discrimination experiment and prepare the DNA samples, you need to perform: An amplification run using a Standard Curve (AQ) plate document to generate real-time PCR data. The real-time PCR data can be used to analyze and troubleshoot the PCR data for the allelic discrimination assay, if needed.
Note: The amplification run can be performed either on the 7900HT Fast System or

offline using any thermal cycler.

An allelic discrimination run using an Allelic Discrimination (AD) plate document. The SDS software analyzes the data, then you assign allele calls (automatically or manually). The following figure illustrates the complete process:

Prepare the Samples

Perform Amplification Run

Perform and Analyze an AD Run

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
About Allelic Discrimination Experiments

Sample Experiment

A sample experiment representing a typical allelic discrimination experiment is provided in Appendix A on page 73. You can use the summarized procedures of the sample experiment in Appendix A to familiarize yourself with the entire allelic discrimination assay workflow. References to the sample experiment are provided in Chapter 2 through Chapter 6, where applicable.

Required UserSupplied Materials

Chemistry/Reagents
Item Any of the following DNA isolation and purification chemistry systems: ABI PRISM 6100 Nucleic Acid PrepStation TransPrep System (purification of ABI gDNA after isolation of RNA from animal and plant tissue) BloodPrep Chemistry (genomic DNA from fresh or frozen blood) NucPrep Chemistry (DNA from animal and plant tissue) PRISM Applied Biosystems (PN 6100-01) Applied Biosystems web site Source

Applied Biosystems (PN 4346860) Applied Biosystems (PN 4340274)

TaqMan reagents appropriate for your probes and primers: For TaqMan SNP Genotyping Assays, Custom TaqMan SNP Genotyping Assays, and custom probes/primer design with Primer Express Software, use either: TaqMan Universal PCR Master Mix, No AmpErase UNG, 200 reactions TaqMan Universal PCR Master Mix For TaqMan Pre-Developed Assay Reagents for Allelic Discrimination (TaqMan PDARs for Allelic Discrimination) use: TaqMan Universal PCR Master Mix Applied Biosystems (PN 4304437) Applied Biosystems (PN 4324018) Applied Biosystems (PN 4304437)

Labeled primers and probes from one of the following sources: TaqMan SNP Genotyping Assays (predesigned primers and probes) TaqMan PDARs for Allelic Discrimination SNP Genotyping Assays Custom (predesigned primers and probes) Primer Express Software (custom-designed primers and probes) TaqMan Applied Biosystems web site Applied Biosystems web site Applied Biosystems web site Contact your Applied Biosystems sales representative.

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
About Allelic Discrimination Experiments

Reaction Plates and Covers IMPORTANT! Do not use MicroAmp caps (domed) or Optical Tubes with the 7900HT

Fast System. You can use Optical Caps (PN 4323032) only on the standard 96-well plates with the 7900HT Fast System.

Item Standard 96-well reaction plates MicroAmp 96-Well Optical Reaction Plate with Barcode (code 128), 20 plates MicroAmp 96-Well Optical Reaction Plate with Barcode (code 128), 25-Pack, 500 plates Includes 25 of PN 4306737, MicroAmp 96-Well Optical Reaction Plates with Barcode MicroAmp 96-Well Optical Reaction Plate with Barcode (code 128) and ABI PRISM Optical Adhesive Covers, 100 plates/100 covers Includes 100 ABI PRISM Optical Adhesive Covers (PN 4311971) and 5 of PN 4306737, MicroAmp 96-Well Optical Reaction Plates with Barcode MicroAmp Splash Free Support Base for 96Well Reaction Plates, 10 bases Standard 384-well reaction plates 384-Well Clear Optical Reaction Plate with Barcode (code 128), 50 plates 384-Well Clear Optical Reaction Plate with Barcode (code 128), 10-Pack, 500 plates Includes 10 of PN 4309849, 384-Well Clear Optical Reaction Plates with Barcode Optical adhesive covers ABI PRISM Optical Adhesive Cover Starter Kit, 20 covers Includes 20 ABI PRISM Optical Adhesive Covers, an Applicator, and an ABI PRISM Optical Cover Compression Pad. ABI PRISM Optical Adhesive Covers, 100 covers ABI PRISM Optical Adhesive Covers, 25 covers Optical Caps, 8 Caps/Strip, 2400 Caps/300 Strips

Source

Applied Biosystems (PN 4306737) Applied Biosystems (PN 4326659)

Applied Biosystems (PN 4314320)

Applied Biosystems (PN 4312063)

Applied Biosystems (PN 4309849) Applied Biosystems (PN 4326270)

Applied Biosystems (PN 4313663)

Applied Biosystems (PN 4311971) Applied Biosystems (PN 4360954) Applied Biosystems (PN 4323032)

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
Before You Begin

Other Consumables and Equipment

Item Centrifuge with adapter for standard 96-well or standard 384-well reaction plates Gloves Microcentrifuge Microcentrifuge tubes, sterile 1.5-mL Nuclease-free water Pipette tips, with filter plugs Pipettors, positive-displacement Tris-EDTA (TE) Buffer, pH 8.0 Vortexer Source Major laboratory supplier (MLS) MLS MLS MLS MLS MLS MLS MLS MLS

Before You Begin

Background and Pure Dye Calibrations
Check that background and pure dye calibrations have been performed regularly to ensure optimal performance of the 7900HT Fast System. For more information about background and pure dye calibrations, refer to the Sequence Detection Systems version 2.3 Software Online Help (SDS Online Help) and the Applied Biosystems 7900HT Fast Real-Time PCR System Maintenance and Troubleshooting Guide (PN 4365542). Some steps in this chapter refer you to the SDS Online Help for more information. To access the SDS Online Help, select Help > SDS Online Help from the SDS software menu bar. The 7900HT Fast System can run prepared reaction plates individually or in groups using the Automation Accessory with the Zymark Twister Microplate Handler. If you are not using the Automation Accessory, you must run reaction plates individually. For clarity, this chapter only illustrates running an individual reaction plate. For information on running multiple reaction plates, see Appendix B on page 83. For information on automated operation of the 7900HT Fast System using the Automation Accessory, see the SDS Online Help.

Accessing the SDS Online Help Automation Options

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 1 Introduction
Before You Begin

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 2

Designing an Allelic Discrimination Assay Experiment

Introduction

Designing an Allelic Discrimination Assay Experiment

Preparing the Samples and Reaction Plate

Using TaqMan Probe-Based Chemistry

See page 10

Performing an Alleic Discriminiation Pre-Read Run

Selecting the Probes and Primers

See page 11

Performing an Amplification Run

Performing and Analyzing an Allelic Discrimination Post-Read Run

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 2 Designing an Allelic Discrimination Assay Experiment

Using TaqMan Probe-Based Chemistry

Using TaqMan Probe-Based Chemistry

About the Chemistry
Allelic discrimination assays use the fluorogenic 5 nuclease chemistry (also known as TaqMan probe-based chemistry).
IMPORTANT! The SYBR Green I dye chemistry and Fast chemistry are not supported

for allelic discrimination assays.

Description TaqMan probe-based chemistry uses a fluorogenic probe to detect specific PCR product as it accumulates during PCR cycles (Lee et al., 1993).
5 3 5
REVERSE PRIMER

Process
Step 1: Polymerization A reporter (R) and a quencher (Q) are attached to the 5' and 3' ends of a TaqMan probe.
FORWARD PRIMER

Step 2: Strand Displacement When both dyes are attached to the probe, reporter dye emission is quenched.
R
Q

PROBE

Q 3

R = REPORTER Q = QUENCHER

5 3 5

5 3 5

3 5 3 5

Step 3: Cleavage During each extension cycle, the Applied Biosystems hot-start DNA polymerase system cleaves the reporter dye from the probe.
R Q 5 3 5 3 5 3 5 5 3 5

Step 4: Polymerization Completed After being separated from the quencher, the reporter dye emits its characteristic fluorescence.
Q 3 5 3 5

Two Types of TaqMan Probes

Applied Biosystems offers two types of TaqMan probes: TaqMan probes with TAMRA dye as a quencher TaqMan MGB (minor groove binder) probes with non-fluorescent quencher (NFQ) For more information about TaqMan probe-based chemistry, refer to the Real-Time PCR Systems Chemistry Guide (PN 4348358).

Chemistry Kits for Allelic Discrimination Assays

TaqMan Universal PCR Master Mix, No AmpErase UNG, 200 reactions (PN 4324018) TaqMan Universal PCR Master Mix (PN 4304437, contains AmpErase UNG)

Notes

10

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 2 Designing an Allelic Discrimination Assay Experiment

Selecting the Probes and Primers

Selecting the Probes and Primers

Each allelic discrimination primer/probe set contains two probes, one probe for allele 1 and one probe for allele 2. Applied Biosystems provides three options for selecting primers and probes:

1. TaqMan SNP Genotyping Assays Provide biologically informative, fully

validated, or predesigned TaqMan MGB-probe-based assays for genotyping single nucleotide polymorphisms (SNPs). For information on available TaqMan SNP Genotyping Assays:
a. Go to http://www.appliedbiosystems.com. b. Click the myScience tab at the top of the page to go to the myScience

Genomic Products page.

c. In the Genotyping section, click the TaqMan SNP Genotyping Assays Search

option.
d. Use specific filtering criteria to search for the assay of interest.

2. Custom TaqMan SNP Genotyping Assays Designs, synthesizes, formulates,

and delivers quality-controlled primer and probe sets. Use this service if the assay you need is not currently available. For ordering information:
a. Go to http://www.appliedbiosystems.com. b. Click the myScience tab at the top of the page to go to the myScience

Genomic Products page.

c. In the Genotyping section, click the Custom TaqMan SNP Genotyping

Assays Info option.

3. TaqMan Pre-Developed Assay Reagents for Allelic Discrimination (TaqMan

PDARs for Allelic Discrimination) Provide optimized assays for the discrimination of specific alleles. For ordering information:
a. Go to http://www.appliedbiosystems.com. b. In the Search section, select All Sections, type PDAR, then click Go. c. In the Search Results section, click TaqMan Pre-Developed Assay

Reagents for Allelic Discrimination.

d. Click the Ordering Information tab.

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

11

Chapter 2 Designing an Allelic Discrimination Assay Experiment

Selecting the Probes and Primers

4. Primer Express Software Helps you design primers and probes for your own
assays. For more information about using this software, refer to the Primer Express Software v3.0 Getting Started Guide (PN 4362460). Applied Biosystems provides Assay Design Guidelines, which have been developed specifically for quantification assays (pertinent to the amplification step in allelic discrimination assays). When used in their entirety, these steps provide a rapid and reliable system for assay design and optimization. For information about the Assay Design Guidelines, refer to the Real-Time PCR Systems Chemistry Guide (PN 4348358).

Sample Experiment
The objective of the sample experiment was to determine the genotype of 92 individuals at the SNP site of a desired target sequence. The primers and probes were ordered from TaqMan SNP Genotyping Assays (AB Assay ID C 2984390 10). The probe for allele 1 (AL-1) was labeled with VIC dye; the probe for allele 2 (AL-2) was labeled with FAM dye.

Notes

12

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 3

Preparing the Samples and Reaction Plate

Introduction

Preparing DNA

See page 14

Designing an Allelic Discrimination Assay Experiment

3
Preparing the Samples and Reaction Plate Preparing the Reaction Mix
Performing an Alleic Discriminiation Pre-Read Run

See page 15

Performing an Amplification Run

Preparing the Reaction Plate

See page 17

Performing and Analyzing an Allelic Discrimination Post-Read Run

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

13

Chapter 3 Preparing the Samples and Reaction Plate

Preparing DNA

Preparing DNA
Systems and Chemistries for DNA Isolation
Applied Biosystems supplies several instrument systems and chemistries for DNA isolation from a variety of starting materials, such as blood, tissue, cell cultures, and plant material.
System ABI PRISM 6100 Nucleic Acid PrepStation ABI PRISM TransPrep System (purification of gDNA after isolation of RNA from animal and plant tissue) BloodPrep Chemistry (genomic DNA from fresh or frozen blood) NucPrep Chemistry (DNA from animal and plant tissue)

Part Number 6100-01 Applied Biosystems web site 4346860 4340274

For more information, refer to: ABI PRISM 6100 Nucleic Acid PrepStation Users Guide (PN 4326242) TransPrep Chemistry Protocol: Purification of gDNA from Filtrates Obtained After the Isolation of RNA from Homogenized Animal or Plant Tissue Samples (PN 4326965) DNA Isolation from Fresh and Frozen Blood, Tissue Culture Cells, and Buccal Swabs Protocol (using BloodPrep Chemistry, PN 4343586) NucPrep Chemistry Protocol: Isolation of Genomic DNA from Animal and Plant Tissue (PN 4333959)

Quality of DNA

Ensure that the DNA you use for allelic discrimination assay experiments: Is extracted from the raw material you are testing with an optimized protocol Does not contain PCR inhibitors Has an A260/280 ratio greater than 1.7 Is intact as visualized by gel electrophoresis Has not been heated above 60 C, which can cause degradation

Sample Experiment
In the sample experiment, genomic DNA was isolated from blood using: A BloodPrep Chemistry Kit The recommended template for TaqMan SNP Genotyping Assays: purified genomic DNA (1 to 20 ng). The final concentration of genomic DNA for all samples in the sample experiment was 10 ng/L.

Notes

14

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 3 Preparing the Samples and Reaction Plate

Preparing the Reaction Mix

Preparing the Reaction Mix

Reagents and Protocols
Allelic discrimination assays can be run on the 7900HT Fast System using standard reagents and standard protocols.
IMPORTANT! Allelic discrimination assays are not supported using Fast reagents or Fast

protocols.

Custom-Designed Assays
If you use the Primer Express Software to design probes and primers for your SNP genotyping assay, follow instructions in the TaqMan Universal PCR Master Mix Protocol (PN 4304449) and the Real-Time PCR Systems Chemistry Guide (PN 4348358) to optimize primer and probe concentrations. If you obtain your assay from the Custom TaqMan SNP Genotyping Assays, follow instructions in the protocol for Custom TaqMan SNP Genotyping Assays: Assays-byDesign Service for SNP Assays Protocol (PN 4334431).

TaqMan PDARs for Allelic Discrimination

TaqMan PDARs for Allelic Discrimination require only three components: Genomic DNA sample Allelic Discrimination Assay Mix (10), specific for each polymorphism TaqMan Universal PCR Master Mix (2)
Note: Allele 1 and 2 control DNA is included with each assay, allowing each

homozygote signal to be generated on each run. For instructions on how to use TaqMan PDARs for Allelic Discrimination, refer to the Pre-Developed TaqMan Assay Reagents Allelic Discrimination Protocol (PN 4312214).

TaqMan SNP Genotyping Assays

The allelic discrimination reaction mix contains: SNP Genotyping Assay Mix (20) Master mix: TaqMan Universal PCR Master Mix, No AmpErase UNG, or TaqMan Universal PCR Master Mix

Notes

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15

Chapter 3 Preparing the Samples and Reaction Plate

Preparing the Reaction Mix

The reagents, volumes, and final concentrations provided below are for wet DNA samples and are excerpted from the TaqMan SNP Genotyping Assays Protocol (PN 4332856).
Note: If you are using dried-down DNA samples, refer to the TaqMan SNP Genotyping

Assays Protocol (PN 4332856) for instructions on preparing the reaction mix.

Preparing the Reaction Mix

1. Calculate the number of reactions to be performed for each assay. 2. Calculate the volume of components needed for all wells on the reaction plate:
Volume (L/reaction) Component Standard 96-Well Reaction Plate 12.50 Standard 384-Well Reaction Plate 2.50

TaqMan Universal PCR Master Mix (2), No AmpErase UNG, or TaqMan Universal PCR Master Mix (2) SNP Genotyping Assay Mix (20) Total

1.25 13.75

0.25 2.75

Note: Prepare extra volume to account for pipetting losses.

3. Swirl the bottle of master mix gently to resuspend.

CHEMICAL HAZARD. TaqMan Universal PCR Master Mix (2), No AmpErase UNG, may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

CHEMICAL HAZARD. TaqMan Universal PCR Master Mix (2) may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves

4. Vortex and centrifuge the SNP Genotyping Assay Mix (20) briefly.
CHEMICAL HAZARD. SNP Genotyping Assay Mix (20) contains formamide. Exposure causes eye, skin, and respiratory tract irritation. It is a possible developmental and birth defect hazard. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.
Notes

16

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Chapter 3 Preparing the Samples and Reaction Plate

Preparing the Reaction Plate

5. Pipette the volumes required for all wells on the reaction plate (plus extra volume to
account for pipetting losses) of master mix and SNP Genotyping Assay Mix (20) into a microcentrifuge tube. Cap the tube.
Sample Experiment
For the sample experiment: The reaction mix was prepared with TaqMan Universal PCR Master Mix (2), No AmpErase UNG. Volumes were calculated for a standard 384-well reaction plate. Enough reaction mix was prepared for 4 NTCs and 92 samples/unknowns, plus 10% extra to account for pipetting losses. Component TaqMan Universal PCR Master Mix (2), No AmpErase UNG SNP Genotyping Assay Mix (20) Total Volume (L) for One Reaction 2.5 0.25 2.75 Volume (L) for 106 Reactionsa 265.0 26.5 291.5

a. Extra volume was included to account for pipetting losses.

Preparing the Reaction Plate

Reaction Plate Formats
Allelic discrimination assays can be run on the 7900HT Fast System using standard 96well and standard 384-well reaction plates.
IMPORTANT! Allelic discrimination assays are not supported using Fast reaction plates

or the TaqMan Low Density Array.

Reaction Plate Components

A reaction plate for an allelic discrimination assay contains the following: Reaction mix No Template Controls (NTCs) Samples: Unknown genomic DNA Nuclease-free water Controls (optional): Known genomic DNA

The recommended final reaction volumes are: 25 L for a standard 96-well reaction plate 5 L for a standard 384-well reaction plate

Notes

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17

Chapter 3 Preparing the Samples and Reaction Plate

Preparing the Reaction Plate

Preparing the Reaction Plate

1. Invert the reaction mix tube prepared in the previous section to mix. 2. Centrifuge the tube briefly to spin down the contents and to eliminate air bubbles. 3. Pipette the appropriate reaction mix volume into each well of the reaction plate:
13.75 L per well for a standard 96-well reaction plate 2.75 L per well for a standard 384-well reaction plate

4. Dilute 1 to 20 ng of each sample into nuclease-free water for a total sample volume
of: 11.25 L per well for a standard 96-well reaction plate 2.25 L per well for a standard 384-well reaction plate

Sample Experiment
For the sample experiment, the recommended template for TaqMan SNP Genotyping Assays was used: purified genomic DNA (1 to 20 ng). The final concentration of genomic DNA for all samples in the sample experiment was 10 ng/L.

5. Pipette the following volumes of NTC, sample, and (if using) control into the
appropriate wells of your reaction plate:
Volume (L/reaction) Component Standard 96-Well Reaction Plate 11.25 11.25 11.25 Standard 384-Well Reaction Plate 2.25 2.25 2.25

NTC: Nuclease-free water or TE (Tris-EDTA) buffer Sample: Unknown genomic DNA Optional Control: Known genomic DNA

Note: Include NTCs on each reaction plate for both manual evaluation and optimal auto-calling of allelic discrimination assay data. If available, known genomic DNA controls may be included and used to corroborate assay performance.

IMPORTANT! Use a calibrated, positive-displacement pipettor to minimize contamination and error. Change tips between samples to prevent crosscontamination.

Notes

18

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Chapter 3 Preparing the Samples and Reaction Plate

Preparing the Reaction Plate

Sample Experiment
To set up the reaction plate for the sample experiment: 2.75 L of reaction mix were pipetted into each well of a standard 384-well reaction plate. 2.25 L of NTC or sample/unknown were added to the wells, as shown below. To Prepare... NTC Sample or Unknown We added... 2.25 L of nuclease-free water or TE buffer 2.25 L of diluted purified genomic DNA To wells... A1, B1, C1, and D1 A2 to A24 B2 to B24 C2 to C24 D2 to D24

NTC

Sample

3
GR2526 7900HT Fast 384-well plate one sample A1-D1
GR2526

6. Cover the reaction plate with an optical adhesive cover or Optical Caps.
IMPORTANT! Do not use MicroAmp caps (domed) or Optical tubes with the

7900HT Fast System. You can use Optical Caps (PN 4323032) only on the standard 96-well plates with the 7900HT Fast System.

7. Keep the reaction plate on ice until loading in the 7900HT Fast System.

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

19

Chapter 3 Preparing the Samples and Reaction Plate

Preparing the Reaction Plate

Notes

20

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 4

Performing an Allelic Discrimination Pre-Read Run

Introduction

About Allelic Discrimination (AD) Plate Documents

Designing an Allelic Discrimination Assay Experiment

See page 22

Preparing the Samples and Reaction Plate

Creating an Allelic Discrimination (AD) Plate Document Performing an Alleic Discriminiation Pre-Read Run

See page 23

Performing an Amplification Run

Performing the Pre-Read Run

See page 34

Performing and Analyzing an Allelic Discrimination Post-Read Run

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

21

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

About Allelic Discrimination (AD) Plate Documents

About Allelic Discrimination (AD) Plate Documents

An Allelic Discrimination (AD) plate document is an SDS software document that stores data collected from an allelic discrimination assay run for a single reaction plate (standard 96-well or standard 384-well reaction plate). Allelic Discrimination (AD) plate documents also store other information about the run, including sample names and detectors.

Plate Document Parameters

When you create an Allelic Discrimination (AD) plate document, you define specific parameters for each allelic discrimination assay reaction plate: Detectors A virtual representation in the SDS software of a TaqMan probe and primer set and associated fluorescent dye that detects a single target nucleic acid sequence. Markers A set of two detectors that discriminate between different alleles of a common locus. Allele 1 is detected by one detector (for example, FAM dye), and allele 2 is detected by the second detector (for example, VIC dye). Tasks A setting that you apply to the markers in a well of a plate document, which determines the way the SDS software uses the data collected from the well during analysis. Allelic Discrimination (AD) plate document markers use two types of tasks:
Task Unknown No Template Control (NTC) Symbol Apply to markers of wells that contain... PCR reagents for the amplification of target sequences no target template

Options for Creating a Plate Document Reaction Plate Options

This section describes how to create a plate document using the New Plate Document Wizard. For information about other ways to create plate documents, see the SDS Online Help. The allelic discrimination assay can be run on either a standard 96-well reaction plate or a standard 384-well reaction plate. For clarity, this chapter only illustrates running a standard 384-well reaction plate.

Notes

22

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Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

Creating an Allelic Discrimination (AD) Plate Document

1. If it is not already running, double-click
on the desktop (or select Start > All Programs > Applied Biosystems > SDS 2.3 > SDS 2.3) to start the SDS software. login option, the Login dialog box appears. Enter your User Name and Password, then click OK.
Note: If the login option is not enabled, no

2. If your System Administrator has enabled the

Login dialog box appears. Skip to step 3 below.

3. In the SDS software menu bar, click

(or select File > New Plate Wizard) to open the Create Plate Document Wizard.

4. Select the assay type:

a. Select Allelic Discrimination (AD). b. Click Next>.
4a

4
4b

5. Enter the plate information:

a. Select a Plate Type: 384 Wells Clear Plate

or 96 Wells Clear Plate.

b. Optional. In the Barcode field, scan or enter
5a

the barcode for the reaction plate.

Note: The Automation Accessory must be
5c

installed in order to use barcodes. A barcode is required if you are adding the plate document to the automation queue. See step f on page 24.
5g

Notes

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Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

c. In the Create Document From pane, select

your preferred source.

Note: You can create a plate document

from scratch (select Blank Document) or you can create a plate document from a template and/or an existing plate document (select Template, Setup File, or Template & Setup File). For more information, see the SDS Online Help.
d. Optional. Check Save Settings As My

Default. Check this option if you want the SDS software to automatically apply the current page settings every time you use the Create Plate Document Wizard.
e. Optional. Check Specify optional setup

information. Check this option if you want to enter a name, study, or comments for the plate document.
f. Optional. Check Add document to

automation queue. Check this option if you want to add the finished plate document to the Automation Controller software plate queue.
Note: This checkbox is only enabled if you are connected to an Automation Accessory. For more information, see Appendix B on page 83.

Note: This checkbox appears on

subsequent pages of the Create Plate Document Wizard. You only need to select this option on one page to enable it.
g. Click Next>.

Notes

24

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

6. Enter the samples to use in the plate:

a. Double-click in the Sample field to activate

the cursor.
b. Type a sample name (for example, NTC),

then press Enter. The sample row is numbered and another row appears.
c. Repeat steps a through b for your remaining

6a

samples.
Note: You may also copy sample names from an

existing plate document (click Existing Plate) or enter multiple sample names at once (click AutoName Selected). For more information, see the SDS Online Help.
6b and 6c

7. Enter markers to use in the plate:

a. Click Create Marker to open the Marker

Information dialog box.

Note: You can also add existing markers

from other sources (click Existing Plate, Marker Manager, or Assay Information File). For more information, see the SDS Online Help.

7a

Notes

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25

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

b. Enter a Marker name (for example,

C_2984390_10).
Note: The marker name must be unique and

it should reflect the locus or polymorphism it targets.

c. Leave the Assay ID field blank. d. Click the Color box to open the Marker

7b 7c 7d 7e

Color dialog box, select a color to represent the marker, then click OK.
e. Click the Allele X Browse button (

) to

open the Detector Manager.

8. Enter detectors to use in the plate:

a. Click Create Detector to open the Add

Detector dialog box. other sources (click Existing Plate, Detector Manager, or Assay Information File). For more information, see the SDS Online Help.
Note: You may also add detectors from

8a

b. Enter a Name for the detector (for example,

AL-1).
Note: The name of the detector must be

unique and it should reflect the target locus of the assay. Do not use the same name for multiple detectors. The SDS software does not distinguish between detectors of the same name, even if they use a different dye set.

8b 8c 8d 8e 8f 8g 8h

8i

Notes

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Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

c. Optional. In the Group field, enter or select

a group for the detector.

Note: A detector group is an optional

feature of the SDS software designed to help you organize your detectors.
d. Optional. In the Description field, enter a

brief description of the assay (up to 32 characters).

e. Leave the AIF Assay ID field blank. Note: For more information on Assay

Information Files (AIF), see the SDS Online Help.

f. From the Reporter/Quencher drop-down

lists, select the appropriate dyes for the detector.

Note: For TaqMan probes, select

TAMRA as the quencher; for TaqMan MGB probes select None as the quencher.

Note: If you are using a custom dye not

manufactured by Applied Biosystems, you must create and run a pure dye plate for the dye before applying it to a detector. For more information, see the SDS Online Help.
g. Click the Color box to open the Select

Detector Color dialog box, select a color to represent the detector, then click OK.

Notes

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27

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

h. Optional. In the Notes field, enter any

additional comments for the detector (up to 200 characters).

i. Click OK. The software saves the new

detector and displays it in the Create Plate Document Wizard.

j. Repeat steps 8a through 8e for Allele Y,

then skip to step 8k.

IMPORTANT! The detector for Allele X and

the detector for Allele Y should have different reporter dyes (for example, VIC dye for detector AL-1 and FAM dye for detector AL-2).
k. Click Next>.

8k

Sample Experiment
In the sample experiment, there were two detectors: AL-1 and AL-2. For your own experiment, enter the appropriate number of detectors. Use detector names that represent the detectors in your experiment.

Notes

28

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

l. Optional. Select a base (A, C, G, or T) from

the Allele X and Allele Y Base Name dropdown lists.

m. Click OK. The Marker Information dialog

box closes and the new marker is displayed in the Create Plate Document Wizard.

8l

9. Repeat step 7 to create markers for any

remaining assays on the plate.

10. Click Next>.

8m

10

4
Sample Experiment
In the sample experiment, only one marker was used: C_2984390_10. The detectors assigned to it were AL-1 and AL-2. For your own experiment, enter the appropriate number of markers with the appropriate detectors assigned. Use marker and detector names that represent the markers and detectors in your experiment.

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

29

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

11. Assign samples and markers to the wells:

a. From the Plate Layout drop-down list,

11b

11a

11d 11c

11f

select Individual Wells.

IMPORTANT! If you change the plate

layout after assigning samples or markers to the wells, all assignments are cleared from the Plate Grid.
b. In the Plate Grid, select the well(s)

containing the first sample.

Note: To select more than one well at a

time, hold down the ctrl key or shift key while selecting the wells.
c. Select the Add Samples and Markers tab. d. In the Markers in Selected Wells pane,

11e

check the appropriate marker for the selected well(s).

e. In the Samples in Selected Wells pane,

check the appropriate sample for the selected well(s).

f. Click in the Task field and select a task for

the marker from the drop-down list: Unknown or NTC.

g. Repeat steps 11b through 11f for the

remaining samples, then go on to step 12.

Notes

30

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Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

12. Verify the sample setup information for each

well:
a. Select the Well Inspector tab. b. Select the desired well(s) in the Plate Grid. Note: To select more than one well at a

12b

12a

time, hold down the ctrl key or shift key while selecting the wells.
c. View the sample setup information for the

selected well(s) in the Well Inspector table.

Note: If you need to correct the sample

setup information for a well, select the well in the Well Inspector table, click Clear Selected, then assign the correct setup information.

12c. The sample setup information for the selected well(s) is displayed here.

13. Click Finish. The Create Plate Document

Wizard closes and the new plate document is displayed.
Note: If your experiment does not use all the

wells in a reaction plate, do not omit the wells from use at this time. You can omit unused wells after the run. For information about omitting unused wells, see the SDS Online Help.

Note: If necessary, you can change the sample setup information (sample name, detector, task) after a run is complete.

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

31

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

Sample Experiment
For the sample experiment, the Allelic Discrimination (AD) plate document looked like this:

14. Save the new plate document:

IMPORTANT! Before the plate document can be

run, you must save it as an SDS 7900HT Document (*.sds). If you close the plate document without first saving it, your information will be lost.

a. Select File > Save to open the Save As

dialog box.

Notes

32

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Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Creating an Allelic Discrimination (AD) Plate Document

b. In the Save in field, navigate to and select a

14b

directory for the new plate document.

c. In the File name field, either:

Enter a file name for the plate document, or Enter or scan the barcode number for the reaction plate
Note: The SDS software does not require

that the plate document name match the barcode of the corresponding reaction plate.
d. From the Files of type drop-down list, select
14c 14d

14e

SDS 7900HT Document (*.sds).

e. Click Save. The software saves the plate

document to the specified directory.

Notes

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33

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Performing the Pre-Read Run

Performing the Pre-Read Run

About the Pre-Read Run
A pre-read run records the background fluorescence of each well of the allelic discrimination plate before performing PCR amplification (see Chapter 5). Then, during the post-read run (see Chapter 6), the pre-read fluorescence is subtracted from the post-read fluorescence to ensure that fluorescence due only to amplification is recorded.

1. To run an individual plate:

a. In the Allelic Discrimination (AD) plate
1a 1b

document, select the Instrument > Plate Read tabs.

b. Click Connect to connect the plate

document to the instrument.

c. Click Open/Close. The instrument tray

rotates to the OUT position.

1c

Notes

34

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Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Performing the Pre-Read Run

d. Place the prepared reaction plate into the

Standard 384-well reaction plate

instrument tray as shown.

IMPORTANT! For the standard 384-well and

standard 96-well reaction plates, the A1 position is located in the top-left side of the instrument.

Well A1

Notched corner

Barcode

e. Click Pre Read. The instrument tray rotates

1e

to the IN position. During the pre-read run, the instrument collects one fluorescence scan per well. As the instrument performs the run, it displays status information in the Plate Read tab.

2. After the pre-read run is finished:

A message indicates whether or not the run was successful. Click OK to close the dialog box. The instrument tray rotates to the OUT position. The date and time of completion is recorded in the Date Collection Stamp pane and the Pre Read button is disabled.
2

Notes

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35

Chapter 4 Performing an Allelic Discrimination Pre-Read Run

Performing the Pre-Read Run

3. Optional. Review the fluorescence data

generated during the pre-read run:
a. Click

(Hide/Show System Raw Data

Pane).
b. In the Plate Grid, select the well(s) you want

to view.
c. Select the Raw Data Plot tab, then select

Pre PCR Read from the Spectra drop-down list to view the pre-read run data.

4. Select File > Close. All raw data generated

during the pre-read run and any changes to the plate document settings are saved to the file that you specified in step 14 on page 32.

Notes

36

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

Chapter 5

Performing an Amplification Run

Introduction

About Standard Curve (AQ) Plate Documents

Designing an Allelic Discrimination Assay Experiment

See page 38

Preparing the Samples and Reaction Plate

Creating a Standard Curve (AQ) Plate Document

Performing an Alleic Discriminiation Pre-Read Run

See page 39

Performing an Amplification Run Performing the Amplification Run

See page 47

Performing and Analyzing an Allelic Discrimination Post-Read Run

Notes

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37

Chapter 5 Performing an Amplification Run

About Standard Curve (AQ) Plate Documents

About Standard Curve (AQ) Plate Documents

Using Standard Curve (AQ) Plate Documents for Amplification Benefits of Real-Time Amplification
You create and use Standard Curve (AQ) plate documents to store real-time data for allelic discrimination assays. Because the Standard Curve (AQ) plate document is used only to amplify target sequences (not to quantify the PCR data), you do not need a standard curve for the plate. Because the allelic discrimination assay is an end-point assay, you can amplify the target sequences offline using any thermal cycler. However, using the 7900HT Fast System to amplify the target sequences provides real-time PCR data. When you perform allele-calling (described in Assigning Calls on page 62), you can study the amplification plots if you observe questionable calls or do not observe data for a well. This section describes how to create a plate document using the New Plate Document Wizard. For information about other ways to create plate documents, see the SDS Online Help. The allelic discrimination assay can be run on either a standard 96-well reaction plate or a standard 384-well reaction plate. For clarity, this chapter only illustrates running a standard 384-well reaction plate.

Options for Creating a Plate Document Reaction Plate Options

Notes

38

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Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

Creating a Standard Curve (AQ) Plate Document

1. Double-click
on the desktop (or select Start > All Programs > Applied Biosystems > SDS 2.3 > SDS 2.3) to start the SDS software. login option, the Login dialog box appears. Enter your User Name and Password, then click OK.
Note: If the login option is not enabled, no

2. If your System Administrator has enabled the

Login dialog box appears. Skip to step 3 below.

3. In the SDS software menu bar, click

(or select File > New Plate Wizard) to open the Create Plate Document Wizard.

4. Select the assay type:

a. Select Standard Curve (AQ).
4a

Note: A standard curve is not necessary for

a non-quantitation amplification run.

b. Click Next>.

4b

5. Enter the plate information:

a. Select a Plate Type: 384 Wells Clear Plate

or 96 Wells Clear Plate.

b. Optional. In the Barcode field, scan or enter
5a

the barcode for the reaction plate.

Note: The Automation Accessory must be
5c

installed in order to use barcodes. A barcode is required if you are adding the plate document to the automation queue. See step f on page 40.
5g

Notes

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39

Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

c. In the Create Document From pane, select

your preferred source.

Note: You can create a plate document

from scratch (select Blank Document) or you can create a plate document from a template and/or an existing plate document (select Template, Setup File, or Template & Setup File). For more information, see the SDS Online Help.
d. Optional. Check Save Settings As My

Default. Check this option if you want the SDS software to automatically apply the current page settings every time you use the Create Plate Document Wizard.
e. Optional. Check Specify optional setup

information. Check this option if you want to enter a name, study, or comments for the plate document.
f. Optional. Check Add document to

automation queue. Check this option if you want to add the finished plate document to the Automation Controller software plate queue.
Note: This checkbox is only enabled if you are connected to an Automation Accessory. For more information, see Appendix B on page 83.

Note: This checkbox appears on

subsequent pages of the Create Plate Document Wizard. You only need to select this option on one page to enable it.
g. Click Next>.

Notes

40

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Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

6. Import the sample names from the Allelic

Discrimination (AD) plate document you created in Chapter 4 (see step 6 on page 25).
a. Click Existing Plate.

6a

b. In the Open dialog box, navigate to and

6b

select the Standard Curve (AQ) plate document.

c. Click Open. The sample names appear in

the Create Plate Document Wizard.

6c

Notes

Allelic Discrimination Assay Getting Started Guide for the 7900HT Fast System

41

Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

7. In the Detector Manager, locate the appropriate

detector from the Allelic Discrimination (AD) plate document you created in Chapter 4 (see step 8 on page 26), click once on the detector to highlight it, then click Select.

7. Click once on the

appropriate detector to highlight it, then click Select.

8. Repeat step 7 for any remaining detectors. 9. Click Next>.

Sample Experiment
In the sample experiment, there were two detectors: AL-1 and AL-2. For your own experiment, enter the appropriate number of detectors. Use detector names that represent the detectors in your experiment.

Notes

42

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Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

10. Assign samples and detectors to the wells, using

the same assignments you used for the Allelic Discrimination (AD) plate document (see step 11 on page 30):
a. From the Plate Layout drop-down list,

10b

10a

10d

10c

10f

select Individual Wells.

IMPORTANT! If you change the plate

layout after assigning samples or detectors to the wells, all assignments are cleared from the Plate Grid.
b. In the Plate Grid, select the well(s)

containing the first sample.

Note: To select more than one well at a

10e

time, hold down the ctrl key or shift key while selecting the wells.
c. Select the Add Samples and Detectors tab. d. In the Detectors in Selected Wells pane,

check the appropriate detector(s) for the selected well(s).

e. In the Samples in Selected Wells pane,

check the appropriate sample for the selected well(s).

f. Click in the Task field and select a task for

the detector from the drop-down list: Unknown or NTC. Do not select Standard.
g. Repeat steps 10b through 10d for the

remaining samples.

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Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

11. Optional. Verify the sample setup information

for each well:
a. Select the Well Inspector tab. b. Select the desired well(s) in the Plate Grid. Note: To select more than one well at a

11b

11a

time, hold down the ctrl key or shift key while selecting the wells.
c. View the sample setup information for the

selected well(s) in the Well Inspector table.

Note: If you need to correct the sample

setup information for a well, select the well in the Well Inspector table, click Clear Selected, then assign the correct setup information.

11c. The sample setup information for the selected wells is displayed here.

12. Click Finish. The Create Plate Document

Wizard closes and the new plate document is displayed.
Note: If your experiment does not use all the

wells in a reaction plate, do not omit the wells from use at this time. You can omit unused wells after the run. For information about omitting unused wells, see the SDS Online Help.

Note: If necessary, you can change the sample setup information (sample name, detector, task) after a run is complete.

Notes

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Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

Sample Experiment
For the sample experiment, the Standard Curve (AQ) plate document looked like this:

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Chapter 5 Performing an Amplification Run

Creating a Standard Curve (AQ) Plate Document

13. Save the new plate document:

IMPORTANT! Before the plate document can be

run, you must save it as an SDS 7900HT Document (*.sds). If you close the plate document without first saving it, your information will be lost.

a. Select File > Save to open the Save As

13b

dialog box.
b. In the Save in field, navigate to and select a

directory for the new plate document.

c. In the File name field, either:

Enter a file name for the plate document, or Enter or scan the barcode number for the reaction plate
Note: The SDS software does not require

13e

that the plate document name match the barcode of the corresponding reaction plate.
d. From the Files of type drop-down list, select

13c

13d

SDS 7900HT Document (*.sds).

e. Click Save. The software saves the plate

document to the specified directory.

Notes

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Chapter 5 Performing an Amplification Run

Performing the Amplification Run

Performing the Amplification Run

1. In the Standard Curve (AQ) plate document you
just created, select the Setup tab.
1

2. Verify that the Passive Reference setting is ROX

dye. (ROX dye is the default.)
Note: If your experiment does not use all the

wells in a reaction plate, do not omit the wells from use at this time. You can omit unused wells after the run. For information about omitting unused wells, see the SDS Online Help.

Note: If necessary, you can change the sample setup information (sample name, detector, task) after a run is complete.

3. Select the Instrument > Thermal Cycler tabs. 4. Select a Mode: Standard or 9600 Emulation.
Note: When Standard mode is selected, the SDS

software uses the 9700 thermal cycler ramp rate in the 7900HT Fast System for standard PCR reactions. When the 9600 Emulation mode is selected, the SDS software reduces the 7900HT Fast System ramp rate to match that of the 9600 thermal cycler in the ABI PRISM 7700 Sequence Detection System.

5. Enter a Sample Volume.

Note: The default sample volumes are 50 L for

a standard 96-well reaction plate and 20 L for a standard 384-well reaction plate.

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Chapter 5 Performing an Amplification Run

Performing the Amplification Run

6. Select the Thermal Profile tab.

If you are using TaqMan Universal PCR Master Mix (PN 4304437, which contains AmpErase UNG), accept the default times and temperatures (shown at right).

Times and Temperatures Initial Steps AmpErase UNG Activation HOLD 2 min @ 50 C AmpliTaq Gold DNA Polymerase Activation HOLD 10 min @ 95 C PCR (Each of 40 cycles) Melt Anneal/ Extend

CYCLE 15 sec @ 95 C 1 min @ 60 C

If you are using TaqMan Universal PCR Master Mix, No AmpErase UNG (PN 4324018):
a. Delete the default Stage 1 by clicking

inside the stage to select it (the ramp line turns red), then clicking Delete Step.

6a. Click inside Stage 1 to select it, then click Delete Step.

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Chapter 5 Performing an Amplification Run

Performing the Amplification Run

b. Change the temperature for the new Stage 2 from 95.0 to 92.0 by doubleclicking inside the top box, then typing 92.0.

6b

c. Accept the remaining default times and

temperatures.

Times and Temperatures Initial Steps AmpliTaq Gold DNA Polymerase Activation HOLD 10 min @ 95 C

PCR (Each of 40 cycles) Melt Anneal/Extend

CYCLE 15 sec @ 92 C 1 min @ 60 C

Sample Experiment
For the sample experiment, the following parameters were selected for the amplification run: Mode: Standard. Sample Volume: 5 L (This is the recommended volume for TaqMan SNP Genotyping Assays run on a standard 384well reaction plate.) Thermal Profile: The thermal profile was changed per steps 6a through 6c above for TaqMan Universal PCR Master Mix, No AmpErase UNG.

7. Select the Real-Time tab. 8. Click Open/Close. The instrument tray rotates to
the OUT position.

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Chapter 5 Performing an Amplification Run

Performing the Amplification Run

9. Place the prepared reaction plate into the

instrument tray as shown.
IMPORTANT! For standard 384-well and

Standard 384-well reaction plate

standard 96-well reaction plates, the A1 position is located in the top-left side of the instrument.

Well A1

Notched corner

Barcode

10. Click Start Run. The instrument tray rotates to

the IN position and the instrument performs the amplification run. As the instrument performs the amplification run, it displays real-time status information in the Real-Time tab and records the fluorescence resulting from cleavage of TaqMan probes in the presence of the target sequences. After the run, the status values and buttons are grayed-out, the Analysis button is enabled ( ), and a message indicates whether or not the run is successful.
10

11. Select File > Save. All data generated during the
run are saved to the plate document.
Note: If necessary, the amplification run data

from the Standard Curve (AQ) plate document can be analyzed later to help troubleshoot the allelic discrimination assay results. For more information, see Appendix C on page 89.

12. Close the plate document, then proceed to

Chapter 6 on page 51 to complete the allelic discrimination assay workflow.

Notes

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Chapter 6

Performing and Analyzing an Allelic Discrimination Post-Read Run

Introduction

Performing the Allelic Discrimination Post-Read Run

See page 52

Designing an Allelic Discrimination Assay Experiment

Analyzing the Run and Evaluating the Results

See page 55

Preparing the Samples and Reaction Plate

Assigning Calls
Performing an Alleic Discriminiation Pre-Read Run

See page 62

Performing an Amplification Run

Saving the Analysis Results

See page 68

Performing and Analyzing an Allelic Discrimination Post-Read Run

Post-Analysis Options

See page 69

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Performing the Allelic Discrimination Post-Read Run

Performing the Allelic Discrimination Post-Read Run

About the Post-Read Run
A post-read run records the fluorescence of each well of the allelic discrimination plate after performing PCR amplification (see Chapter 5). During the postread run, the pre-read background fluorescence (see Chapter 4) is subtracted from the post-read fluorescence to ensure that fluorescence due only to amplification is recorded.

Reaction Plate Options

The plus/minus assay can be run on both standard 96well and standard 384-well reaction plates. For clarity, this chapter only illustrates the running of a standard 96-well reaction plate.
To perform the post-read run:

1. Select File > Open, browse to open the

previously saved pre-read plate document, then click Open. The allelic discrimination plate document opens in the main SDS software window.

2. In the Allelic Discrimination (AD) plate

document, select the Instrument > Plate Read tabs.

3. Click Connect to connect the plate document to

the instrument.

Notes

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Performing the Allelic Discrimination Post-Read Run

4. Click Open/Close. The instrument tray rotates to

the OUT position.
4

5. Place the prepared reaction plate into the

instrument tray as shown.
IMPORTANT! For the standard 384-well and

Standard 384-well reaction plate

standard 96-well reaction plates, the A1 position is located in the top-left side of the instrument.

Well A1

Notched corner

Barcode

6. Click Post Read. The instrument tray rotates to

the IN position and the instrument performs the run. As the instrument performs the run, it displays status information in the Plate Read tab. After the run, the status values and buttons are grayed-out, the Analysis button is enabled ( ), and a message indicates whether or not the run is successful.

7. When the run is complete, click Open/Close to

eject the reaction plate.

6
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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Performing the Allelic Discrimination Post-Read Run

8. Optional. Review the fluorescence data

generated during the post-read run:
a. Click

(Hide/Show System Raw Data

Pane).
b. In the Plate Grid, select the well(s) you want

to view.
c. Select the Raw Data Plot tab, then select

Post PCR Read from the Spectra dropdown list to view the post-read run data.

9. Select File > Close. All raw data generated

during the post-read run and any changes to the plate document settings are saved to the file that you specified in step 1 on page 52.

Notes

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Analyzing the Run and Evaluating the Results

Analyzing the Run and Evaluating the Results

Analyzing the Run
1. In the Allelic Discrimination (AD) plate
document, click (or select Analysis > Analyze). The SDS software analyzes the run data.
Tip: Reanalyzing Data Once you click the Analyze button, it becomes disabled. To reanalyze the data, you can: Reenable the Analyze button by changing the setup information in the plate document (for example, removing a well or omitting a marker assignment), then click (or select Analysis > Analyze) again. Change analysis settings in the Analysis Settings dialog box, then click OK. The SDS software automatically reanalyzes the data when you click OK. (For information on using the Analysis Settings dialog box, see Assigning Calls on page 62.)

Note: In order for the SDS software to automatically reanalyze the data via the Analysis
Settings dialog box, you must have already analyzed the data once by clicking the Analyze button.

2. Select the Results tab, then proceed to

Evaluating the Results below.

Evaluating the Results

To evaluate the analysis results, you can use the: Allelic Discrimination Plot (page 57) Results Grid (page 60) Results Table (page 61)
Note: The results displays are actively synchronized.

For example, selecting a well in the Results Grid also selects the corresponding well in the Results Table and Allelic Discrimination Plot. After evaluating the initial analysis results:

If you are satisfied with the initial analysis results, proceed to Saving the Analysis Results on page 68.

6
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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Analyzing the Run and Evaluating the Results

If you are not satisfied with the initial analysis results, you can assign calls (see page 62) or scrutinize the allele calls.
Note: For information on scrutinizing the allele calls, see the SDS Online Help.

Notes

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Analyzing the Run and Evaluating the Results

Using the Allelic Discrimination Plot

The SDS software graphs the results of allelic discrimination runs on a scatterplot (the Allelic Discrimination Plot) that contrasts reporter dye fluorescence. After signal normalization and multicomponent analysis, the SDS software graphs the normalized data from each well as a single datapoint on the scatterplot. The figure below illustrates the components of the Allelic Discrimination Plot.
Marker drop-down list Call drop-down list Toolbar

Legend

Detector name

Scatterplot

Detector name

Marker drop-down list Determines the marker data that the SDS software displays in the scatterplot. Call drop-down list When a datapoint is selected, this menu allows you to assign an allele call to the datapoint within the scatterplot. Toolbar Contains the following tools for manipulating the scatterplot:
Icon Description
Selects: Individual datapoints by clicking, or Groups of datapoints by clicking and dragging a box across a group of datapoints.

Selects groups of datapoints by encircling them with the tool.

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Analyzing the Run and Evaluating the Results

Icon

Description
Repositions the view within the scatterplot by clicking and dragging the screen. Zooms in on the scatterplot by: Clicking the mouse button within the scatterplot, or Clicking and dragging a section of the scatterplot to view.

Zooms out on the scatterplot by clicking the mouse button within the scatterplot.

Scatterplot A scatterplot of datapoints from the run (also called the Allelic Discrimination Plot). Legend An explanation of the symbols in the scatterplot. Detector names The names you assigned to the detectors are displayed on the axes of the scatterplot.
Note: You can adjust the appearance of the Allelic Discrimination Plot or the datapoints

it contains using the Display Settings dialog box (View > Display Setting).

Note: For more information on the Allelic Discrimination Plot tools or the Display

Settings dialog box, see the SDS Online Help.

To evaluate results using the Allelic Discrimination Plot:

In the Results tab, select a marker from the Marker drop-down list. If the auto caller is enabled: Alleles are identified for the selected marker and displayed in the Allelic Discrimination Plot:

Notes

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Analyzing the Run and Evaluating the Results

The samples are grouped as follows:

Samples Containing... Are Grouped In... Symbol

Allele X Allele Y Both (Allele X and Allele Y heterozygote) No Template Control (NTC) Undetermined

Lower right corner of the plot Upper left corner of the plot Approximately midway between the Allele X and Allele Y groups Bottom left corner of the plot (Not shown in the example.) Anywhere on plot

If the auto caller is not enabled, crossmarks ( Undetermined) representing the selected marker are displayed in the Allelic Discrimination Plot.

6
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Analyzing the Run and Evaluating the Results

Using the Results Grid

The Results Grid displays the assay-specific setup and analysis properties for the plate document in a well format corresponding to the type of reaction plate used for the run. The following parameters can be displayed for each well in the plate document: Well color Flag Sample Marker Color Marker Number of markers Call: Allele Y, Allele X, Both, Undetermined. The call symbols are as follows:
Wells Containing... Symbol

Allele X Allele Y Both (Allele X and Allele Y heterozygote) NTC or Undetermined

Note: You can configure which parameters are displayed in the Results Grid by selecting View > Display Setting, then selecting Results Grid in the Display Settings dialog box. For more information, see the SDS Online Help. To evaluate results using the Results Grid:

1. Right-click inside the Plate Grid, then select Result View.

Note: The default view for the Plate Grid is the Setup View.

2. If desired, select View > Grid Zoom to zoom in on individual wells. 3. Pass the cursor over a well to see the information for that well.

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Analyzing the Run and Evaluating the Results

The Results table displays the assay-specific setup and analysis properties for the plate document in a table format. The following parameters can be displayed for each well in the plate document: Well position Flag Sample Marker Task: Unknown, NTC Call: Allele Y, Allele X, Both, NTC, Undetermined Quality Value Call Type Allele X Rn Allele Y Rn Passive Reference Rn
Note: See Appendix C for an explanation of

Using the Results Table

terms used in quantitation analysis.

Flags: HMD (the well has missing data), FOS (fluorescence is off-scale), LME (large mean squared error), EW (the well is empty), BPR (bad passive reference)
Note: You can configure which parameters are

displayed in the Results Table by double-clicking on a column header, by selecting a user-defined Table Settings profile from the drop-down list, or by clicking (Table Settings) to open the Table Settings dialog box. For more information, see the SDS Online Help.

6
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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Assigning Calls

Assigning Calls
Note: Assigning calls is optional. If you are satisfied

with the initial analysis results (page 55), you do not need to assign calls.

The analysis of SNP (or genotyping) data involves the automatic or manual calling of sample data for each marker. The calls are data labels assigned to individual samples to reflect their genomic content. You can call sample data: Automatically, using the auto caller (see page 62) Manually, using the toolbar and scatterplot (see page 65)

Assigning Calls Automatically 1. In the Allelic Discrimination (AD) plate

document, click (or select Analysis > Analysis Settings) to open the Analysis Settings dialog box.

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Assigning Calls

2. Set the marker analysis settings:

a. Select the Marker tab. b. Select All Markers from the Marker drop-

2a

down list.
Note: You can also select individual

markers from the Marker drop-down list.

c. Select the Auto caller enabled check box to

2b 2c 2d 2e

activate automatic analysis.

d. If you expect the analyzed data to consist of

only two clusters, select the 2-cluster calling enabled check box.
e. In the Quality Value field, enter a

2f

percentage value to apply as the quality interval for auto-calling samples. (The greater the value, the more stringent the allele calling.)
f. Click Apply to save your settings in the

Marker tab.

3. Optional. Assign flags:

a. Select the Plate tab. b. Assign flags as desired. Note: When you assign flags, you can flag

3a

conditions and omit wells when certain criteria are met (for example, when a well has missing data). For more information, see Appendix D on page 99.
c. Click Apply to save your settings in the

Plate tab.

4. Click OK to close the Analysis Settings dialog

box. The SDS software automatically reanalyzes the data using your new analysis settings.
Note: In order for the SDS software to
3c

automatically reanalyze the data via the Analysis Settings dialog box, you must have already analyzed the data once by clicking the Analyze button (see step 1 on page 55).

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Assigning Calls

5. Select the Results tab to display the Allelic

Discrimination Plot.
Homozygous Allele Y

6. Select a marker from the Marker drop-down list.

For the selected marker: Alleles are identified in the Allelic Discrimination Plot Samples are grouped as follows:
Samples Containing... Are Grouped In... Symbol
Undetermined Heterozygous Allele XY

Homozygous Allele X

Allele X Allele Y Both (Allele X and Allele Y heterozygote) No Template Control (NTC) Undetermined

Lower right corner of the plot Upper left corner of the plot Approximately midway between the Allele X and Allele Y groups Bottom left corner of the plot (Not shown in the example.) Anywhere on plot

Notes

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Assigning Calls

Assigning Calls Manually 1. In the Allelic Discrimination (AD) plate

document, click (or select Analysis > Analysis Settings) to open the Analysis Settings dialog box.

2. Set the marker analysis settings:

a. Select the Marker tab. b. Select All Markers from the Marker drop-

2a

down list.
c. Deselect the Auto caller enabled check

2b 2c

box.
d. Click Apply to save your settings in the

Marker tab.

2d

3. Optional. Assign flags:

a. Select the Plate tab. b. Assign flags as desired. Note: When you assign flags, you can flag

3a

conditions and omit wells when certain criteria are met (for example, when a well has missing data). For more information, see Appendix D on page 99.
c. Click Apply to save your settings in the

Plate tab.

3c

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Assigning Calls

4. Click OK to close the Analysis Settings dialog

box. The SDS software automatically reanalyzes the data using your new analysis settings.
Note: In order for the SDS software to

automatically reanalyze the data via the Analysis Settings dialog box, you must have already analyzed the data once by clicking the Analyze button (see step 1 on page 55).

5. Select the Results tab to display the Allelic

Discrimination Plot.

5 6

6. Select a marker from the Marker drop-down list.

Crossmarks ( Undetermined) representing the selected marker are displayed in the Allelic Discrimination Plot.

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Assigning Calls

7. To assign calls:
a. Click the

7c

7a

selection tool.

b. Click-drag a box around the allele data

points in the lower-right of the plot.

c. In the Call drop-down list, select Allele X. d. Click-drag a box around the allele data

points in the upper-left of the plot.

e. In the Call drop-down list, select Allele Y. f. Click-drag a box around the allele data

points in the center of the plot.

g. In the Call drop-down list, select Both. h. Click-drag a box around any allele data
7b

points that are not included in any of the grouped data points.
i. In the Call drop-down list, select

Undetermined.

8. If you are assigning calls for multiple markers,

select a different marker from the Marker dropdown list and repeat step 7.

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Saving the Analysis Results

Saving the Analysis Results

IMPORTANT! Although the SDS software saves any

changes made to the appearance of a plate document and to the analysis settings, it does not save the calls made during the analysis.

1. Select File > Save As to open the Save As dialog

box.

2. In the Save in field, navigate to and select a

directory for the analyzed plate document.

3. In the File name field, either:

Enter a file name for the plate document, or Enter or scan the barcode number for the reaction plate
Note: The SDS software does not require that

the plate document name match the barcode of the corresponding reaction plate.

4. From the Files of type drop-down list, select

SDS 7900HT Document (*.sds).

5. Click Save. The software saves the plate

document to the specified directory.

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Chapter 6 Performing and Analyzing an Allelic Discrimination Post-Read Run

Post-Analysis Options

Post-Analysis Options
The following options are available after the analysis: Exporting data as text files (below) Exporting data as graphic files (page 70) Printing a data report (page 72)

Exporting Data as Text Files

You can export raw or analyzed data from plate documents into tab-delimited text files (*.txt). The text files can then be imported into spreadsheet software, such as Microsoft Excel software.

1. Open the plate document from which you wish to

export data.

2. Select File > Export to open the Export dialog

box.

3. In the Look in field, navigate to and select a

directory for the new file.
4

4. From the Export drop-down list, select the data

type to export.
Note: For more information about the data type

options, see the SDS Online Help.

5. Select to export data from All Wells or a select

group of wells (Selected Wells).
Note: To use the Selected Wells option, you
5 6

must select a subset of wells in the plate document before opening the Export dialog box.

6. From the Format options, select the appropriate

software version.
10

7. Optional. Check Save Settings As My Default.

Check this option if you want the SDS software to automatically apply the current settings every time you export data as a text file.
8 9

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Post-Analysis Options

8. In the File name field, type a name for the new

file.

9. From the Files of type drop-down list, select

Tab-delimited Text (*.txt).

10. Click Export. The software exports the new file

to the specified directory.

Exporting Data as Graphic Files

You can export the Plate Grid and plot images from plate documents as Joint Photographic Experts Group (JPEG) graphic files (*.jpeg). The JPEG files can then be viewed in most common word processing, spreadsheet, and HTML-based software.

1. Open the plate document from which you wish to

export data.

2. To export the:
Plate Grid, right-click in the Plate Grid, then select Save Grid to Image File. The Save dialog box appears.

Plot, right-click in the plot, then select Save Plot to Image File. The Save dialog box appears.
Note: If desired, adjust the plot dimensions

(length and width) as you want them to appear in the exported file. The exported file retains the dimensions of the original screen element.

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Post-Analysis Options

3. Complete the Save dialog box:

a. In the Save in field, navigate to and select a

3a

directory for the new file.

b. In the File name field, type a name for the

new file.
c. From the Files of type drop-down list, select

JPEG File.
d. Click Save. The software saves the new file

to the specified directory.

3d

3b

3c

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Post-Analysis Options

Printing a Data Report

You can use the SDS software to print a report of the analyzed data containing individual or multiple elements of the plate document.
Note: For more information on printing reports, see

the SDS Online Help.

1. Open the plate document from which you wish to

print a report.

2. Click

(or select File > Print Report) to open the Print Report dialog box. document element(s) to print.

3. In the Include Data pane, select the plate 4. Optional. To format the display of the report and
how the report is printed, click Page Setup.
3

5. Optional. Check Save Settings As My Default.

Check this option if you want the SDS software to automatically apply the current settings every time you print a report.
7 8

6. Optional. Click Preview to view the report

before printing it.

7. Click Print to open the Print dialog box and print

the report.

8. When the report has finished printing, click

Done to close the Print Report dialog box.

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Appendix A

Sample Experiment
About the Sample Experiment
Overview
To better illustrate how to design, perform, and analyze allelic discrimination experiments, this appendix guides you through a sample experiment. The sample experiment represents a typical allelic discrimination experiment setup that you can use as a quick-start procedure to familiarize yourself with the allelic discrimination workflow. Detailed steps in the allelic discrimination workflow are described in the preceding chapters of this guide. Also in the preceding chapters are Sample Experiment boxes, which provide details for some of the related steps in the sample experiment. The objective of the sample experiment was to determine the genotype of 92 individuals at the SNP site of a desired target sequence. The experiment used multiplex PCR. The primers and probes were ordered from TaqMan SNP Genotyping Assays (AB Assay ID C 2984390 10). Reactions were set up for PCR using the TaqMan Universal PCR Master Mix (2), No AmpErase UNG, and appropriate primers and probes. The sample experiment data and results were generated using a 7900HT Fast System by performing: An amplification run using a Standard Curve (AQ) plate document to generate real-time PCR data. The real-time PCR data could then be used to analyze and troubleshoot the PCR data for the allelic discrimination assay, if needed. An allelic discrimination run using an Allelic Discrimination (AD) plate document. The SDS software analyzed the data, then we assigned allele calls (automatically or manually).

Description

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Appendix A Sample Experiment

About the Sample Experiment

Sample Allelic Discrimination Experiment Procedure 1. Design the experiment (for more detail, see
Chapter 2 on page 9):
a. Order the appropriate master mix.

The sample experiment used TaqMan Universal PCR Master Mix, No AmpErase UNG.
b. Select and order the probes and primers.

2. Extract the DNA from samples (for more detail,

see Preparing DNA on page 14). The sample DNA for the sample experiment was extracted using the BloodPrep Chemistry Kit (PN 4346860) to obtain a final concentration of 10 ng/L of DNA for each sample.

3. Prepare the reaction mix (for more detail, see

Preparing the Reaction Mix on page 15). The sample experiment was run on a standard 384-well reaction plate. As shown below, enough reaction mix was prepared for 4 NTCs and 92 samples/unknowns, plus 10% extra to account for pipetting losses.
Volume (L) Component One Reaction 2.50 106 Reactions 265

CHEMICAL HAZARD. TaqMan Universal PCR Master Mix (2), No AmpErase UNG, may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

TaqMan Universal PCR Master Mix (2), No AmpErase UNG SNP Genotyping Assay Mix (20) Total

0.25 2.75

26.5 291.5

CHEMICAL HAZARD. TaqMan Universal PCR Master Mix (2) may cause eye and skin irritation. Exposure may cause discomfort if swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves.

Extra volume was included to account for pipetting losses.

CHEMICAL HAZARD. SNP Genotyping Assay Mix (20) contains formamide. Exposure causes eye, skin, and respiratory tract irritation. It is a possible developmental and birth defect hazard. Read the MSDS, and follow the handling instructions. Wear appropriate protective eyewear, clothing, and

Notes

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Appendix A Sample Experiment

About the Sample Experiment

4. Prepare the reaction plate (for more detail, see

Preparing the Reaction Plate on page 17). A reaction plate for an allelic discrimination assay contains the following: Reaction mix No Template Controls (NTCs) Unknown genomic DNA samples Known genomic DNA controls (optional, not included in the sample experiment)

For the sample experiment, a standard 384-well reaction plate was used. 2.75 L of reaction mix were pipetted into each well. 2.25 L of the following solutions were pipetted into the indicated wells:
To wells... A1, B1, C1, and D1 (NTC, No Template Control) A2 through A24 B2 through B24 C2 through C24 D2 through D24 (Sample or Unknown) We added... Nuclease-free water or TE (Tris-EDTA) buffer

NTC

Sample

GR2526 7900HT Fast 384-well plate one sample A1-D1

GR2526

Diluted purified genomic DNA

5. Create a Standard Curve (AQ) plate document

for amplifying samples (for more detail, see Creating a Standard Curve (AQ) Plate Document on page 39):

Notes

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Appendix A Sample Experiment

About the Sample Experiment

a. Select File > New Plate Wizard. b. For the assay type, select Standard Curve

(AQ), then click Next.

Note: A standard curve is not necessary for

a non-quantitation amplification run.

c. Select 96 Wells Clear Plate or 384 Wells

Clear Plate, select Blank Document, then click Next.

d. Enter the samples and detectors to use in the

plate, then click Next.

Notes

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About the Sample Experiment

e. Assign detectors, samples, and tasks to the

wells.

f. Select the Well Inspector tab to verify the

sample setup information for each well, then click Finish.

g. Save the new plate document.

6. Perform the amplification run (for more detail,

see Performing the Amplification Run on page 47):
a. In the Standard Curve (AQ) plate document,

select the Instrument > Thermal Cycler tabs.

b. For the Mode, select Standard or 9600

Emulation.
c. Enter a Sample Volume.

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Appendix A Sample Experiment

About the Sample Experiment

d. Select the Thermal Profile tab; accept the

default stages (shown at right) or modify the thermal profile as needed.

Times and Temperatures Initial Steps AmpErase UNG Activation HOLD 2 min @ 50 C AmpliTaq Gold DNA Polymerase Activation HOLD 10 min @ 95 C PCR (Each of 40 cycles) Melt Anneal/ Extend

CYCLE 15 sec @ 95 C 1 min @ 60 C

For the sample experiment (using TaqMan Universal PCR Master Mix, No AmpErase UNG), the thermal profile was modified as shown at right.

Times and Temperatures Initial Steps AmpliTaq Gold DNA Polymerase Activation HOLD 10 min @ 95 C

PCR (Each of 40 cycles) Melt Anneal/Extend

CYCLE 15 sec @ 92 C 1 min @ 60 C

e. Select the Real-Time tab, click

Open/Close, then load the reaction plate into the instrument

f. Click Start Run. As the instrument

performs the amplification run, it displays real-time status information in the RealTime tab.
g. When the amplification run is complete,

select File > Save, then close the plate document.

7. Create an Allelic Discrimination (AD) plate

document (for more detail, see Creating an Allelic Discrimination (AD) Plate Document on page 23):

Notes

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Appendix A Sample Experiment

About the Sample Experiment

a. Select File > New Plate Wizard. b. For the assay type, select Allelic

Discrimination (AD), then click Next.

c. Select 96 Wells Clear Plate or 384 Wells

Clear Plate, select Blank Document, then click Next.

d. Import the sample names from the Standard

Curve (AQ) plate document you just created.

Notes

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Appendix A Sample Experiment

About the Sample Experiment

e. Enter the markers to use in the plate, then

click Next.

f. Assign markers, samples, and tasks to the

wells.

g. Select the Well Inspector tab to verify the

sample setup information for each well, then click Finish.

h. Save the new plate document.

Notes

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Appendix A Sample Experiment

About the Sample Experiment

8. Perform the allelic discrimination run (for more

detail, see Performing the Allelic Discrimination Post-Read Run on page 52):
a. In the Allelic Discrimination (AD) plate

document, select the Instrument > Plate Read tabs.

b. Click Open/Close, then load the reaction

plate into the instrument.

c. Click Post Read. As the instrument

performs the run, it displays status information in the Plate Read tab.

9. Analyze and evaluate the allelic discrimination

run (for more detail, see Analyzing the Run and Evaluating the Results on page 55):
a. Click

(or select Analysis > Analyze). The SDS software analyzes the run data.

b. Select the Results tab. c. View the analyzed run data in the Plate

Grid, Results Table, and Allelic Discrimination Plot.

10. If desired, assign calls automatically or manually

(for more detail, see Assigning Calls on page 62).

11. Save the plate document (for more detail, see

Saving the Analysis Results on page 68).

12. If desired, export or print the results data (for

more detail, see Post-Analysis Options on page 69).

Notes

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Appendix A Sample Experiment

About the Sample Experiment

Notes

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Appendix B

SDS Automation Controller Software

Overview
The Sequence Detection Systems (SDS) Automation Controller Software v2.3 provides an interface between the Zymark Twister Microplate Handler, the 7900HT Fast System, the fixed-position bar code reader, and the plate documents created in main Sequence Detection Systems Software v2.3. This Automation Controller software controls and coordinates the action of the 7900HT instrument and the automation module. It initiates and controls the sequence detection run and acquires data during the run. This option allows for plates to be run as part of a group or batch allowing for highthroughput unattended operation. For more information about the Automation Controller, refer to the SDS Online Help.

Using the Automation Controller Software

Note: Before launching the Automation Controller software, you must close the main

Sequence Detection Systems Software v2.3 application. Failure to do so will result in an instrument connection failure.

1. Double click the Automation Controller shortcut icon. An initialization pop-up will
appear displaying the status of instrument connection and other verifications. If you encounter an error, refer to the SDS Online Help.

Notes

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Appendix B SDS Automation Controller Software

Using the Automation Controller Software

2. If you have already created and saved the desired plates (sent them to the queue) in
the main SDS application, then the plates will appear in the Plate Queue tab. For more information about creating plate documents and sending them to the queue, refer to Creating a Standard Curve (AQ) Plate Document on page 39 or Creating an Allelic Discrimination (AD) Plate Document on page 23.

3. You can add plates by clicking Add Plates or (or click

located.

).

a. At the Open dialog box, navigate to the location where the plate files are

b. Use the barcode reader to scan the Plate IDs. Note that you can automatically

scan the next plate without having to select Add Plates again. You can also save the list of queued plates, by clicking Save List.
Note: To remove a plate, click on the plate name then click Remove. To

remove all plates, click Remove All.

Notes

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Appendix B SDS Automation Controller Software

Using the Automation Controller Software

4. Place the plates in the input stack of the Zymark Twister Microplate Handler. The
order of the plates may vary from the order they were scanned in (the Handlers fixed barcode reader will verify the location of each plate). The software matches the plate document and the method before it starts the run. Orient the plates inside the stacks so that the well A1 of each plate corresponds to the locations shown in the figure below.

5. Click into the boxes next to each stack location where the plates are located.

6. Select Tools > Batch Settings to specify the desired output settings.
a. In the Batch Settings dialog box, at the Auto-Analysis Workflow tab, click into

a box to select that export option. You can also change the export directory by clicking the Browse button (located in the Export Destination box) and specifying a new location for the file data to be exported to. Note that after analysis exported data will be saved as *.txt files.
6a

GR2104

6b

b. Click on Change Format to assign attributes to be included in the export file

name.
Notes

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Appendix B SDS Automation Controller Software

Using the Automation Controller Software

c. At the Filename dialog box, click into a field box (under the Include column)

to deselect an attribute.
6c

6d

d. Click OK. e. Back at the Batch Settings dialog box, click on the Email Notifications tab to

specify desired email notification options. For more information on email settings, see your network administrator.
6e

6f

f. Click OK.

7. Verify that the plate adapter and the output stack of the Handler are empty.

Notes

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Appendix B SDS Automation Controller Software

Using the Automation Controller Software

8. Click Start Batch.

Note: Note that if the plate type does not match the plate block installed on the instrument, the Automation Controller will not start. You can assign plate types by selecting Tools > Options and selecting the desired plate format. The handler loads the plates and the instrument starts the run.

Note: To stop the run, click Stop Batch. You will see a warning message asking

you to verify stopping the run.

Notes

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Appendix B SDS Automation Controller Software

Using the Automation Controller Software

9. Click on the Run Status tab to view the plate run status. Note that once a plate is
being processed, it will not appear in the Processed Plates tab or be listed in the Queued Plates section of the Batch Status box. The plate information will be available under the Currently Running Plate section.
9 10

Note: For standard curve (AQ) and ddCt (RQ) plates, a view of the amplification and

temperature plot will be available and can be selected by clicking on the radial button.

10. Click on Processed Plates to view information about the processed plates.

Notes

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Appendix C

Analyzing and Viewing Amplification Data

Terms Used in Quantitation Analysis
The following are terms commonly used in quantitation analysis.
Term Baseline Threshold cycle (CT) Passive reference Definition A line fit to fluorescence intensity values during the initial cycles of PCR, in which there is little change in fluorescence signal. The fractional cycle number at which the fluorescence intensity exceeds the threshold intensity. A dye that provides an internal fluorescence reference to which the reporter dye signal can be normalized during data analysis. Normalization is necessary to correct for fluorescence fluctuations caused by changes in concentration or volume. The dye attached to the 5 end of a TaqMan probe. The dye provides a signal that indicates specific amplification. The ratio of the fluorescence intensity of the reporter dye signal to the fluorescence intensity of the passive reference dye signal. The magnitude of the signal generated by a set of PCR conditions. (Rn= Rn baseline)

Reporter dye Normalized reporter (Rn) Delta Rn (Rn)

The figure below is a representative DNA amplification plot and includes some of the terms defined above.

Sample

Rn Rn
Threshold

Baseline

No Template Control

CT

10

15

20

25

30

35

40

Cycle Number

Notes

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Appendix C Analyzing and Viewing Amplification Data

Analyzing the Amplification Data

Analyzing the Amplification Data

Before you can analyze the amplification data from the standard curve (AQ) plate document you generated in Chapter 5, you must specify parameter values (baseline and threshold settings or CT) for the analysis. Unless you have already determined the optimal baseline and threshold settings (CT) for your experiment, use the automatic baseline and threshold feature of the SDS software (Automatic CT) to analyze the amplification run. If the baseline and threshold are called correctly for each well, you can proceed to view the results (see Reviewing the Automatic CT Results on page 91). Otherwise, you must manually set the baseline and threshold (see Reviewing the Automatic CT Results on page 91). For more information about determining CT, refer to the Real-Time PCR Systems Chemistry Guide (PN 4348358). For more information about manually adjusting CT, refer to the SDS Online Help.

Starting the Analysis

1. Open the AQ plate document you want to analyze. 2. Click

(or select Analysis > Analysis Settings). The Analysis Settings dialog box opens.

3 4

3. In the Detector drop-down list of the Detector tab, select All Detectors. 4. In the Ct Analysis window, select Automatic Ct. The SDS software will
automatically generate baseline values for each well and threshold values for each detector.

5. Click OK to apply any changes and close the Analysis Settings dialog box. 6. Click
(or select Analysis > Analyze). The Results and QC Summary tabs are available after analysis completes.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Analyzing the Amplification Data

Reviewing the Automatic CT Results

Click the Results tab to view results of the amplification run for each well. The SDS software Automatic CT analysis process calculates baseline and threshold values for a detector based on the assumption that the data exhibits a typical amplification curve. A typical amplification curve has a: Plateau phase (a) Linear phase (b) Exponential (geometric) phase (c) Background (d) Baseline (e)
a b c

Threshold Rn

e Cycle

IMPORTANT! Experimental error (such as contamination, pipetting errors, etc.) can produce atypical amplification curves that can result in incorrect baseline and threshold value calculations by the SDS software. Therefore, Applied Biosystems recommends you examine the amplification plot and review the assigned baseline and threshold parameter values for each well after analysis completes, and if necessary manually adjust the baseline and threshold. For more information, see the SDS Online Help.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Viewing the Amplification Data

The following options are available for further determination of the analysis results: Using the Plate Grid Results View (below) Using the Raw Data Plot (page 93) Using the Multicomponent Data Plot (page 93) Using the Amplification Plot Views (page 94)

Using the Plate Grid Results View

Before analysis, the plate grid displays the setup information for each well, including: detector and sample information. After analysis, the plate grid displays the category and number of data flags generated for each well of the plate, as illustrated below.

Two data flags are assigned to well C12 (flagged well) Three data flags are assigned to well F12 (omitted well)

Note: A list of the data flags generated for the plate document is also displayed in both

the QC Summary tab and Results Table. See the SDS Online Help for more information about viewing and configuring the plate data flags.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Using the Raw Data Plot

The Raw Data Plot (click ) displays the fluorescence spectra and temperature profile of selected wells. The vertical slider in the Temperature plot allows you to see the spectra for each run cycle by dragging it with the pointer.

C
Click and drag the slider to view temporal changes in the raw data profile

Using the Multicomponent Data Plot

The Multicomponent Data Plot (click ) displays the complete spectral contribution of each dye in a selected well over the duration of the PCR run.

Note: Only one well can be displayed at a time.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Using the Amplification Plot Views

The Amplification Plot views allow you to review the post-run amplification results for selected wells from the plate grid. Several different views are available from the Plot drop-down list: Rn vs. Cycle (below) Rn vs. Cycle (page 95) CT vs. Well Position (page 96)
Note: For more information on using the Amplification Plot views to evaluate

amplification results, see the SDS Online Help.

Rn vs. Cycle (linear scale)

This plot displays normalized reporter dye fluorescence signal (as Rn) in linear scale as a function of run cycle. You can use this plot to identify and examine irregular amplification.

Note: For more information about Rn, refer to the Real-Time PCR Systems Chemistry

Guide (PN 4348358).

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Rn vs.Cycle (log scale)

This plot displays the reporter dye fluorescence signal (as Rn) in log scale as a function of run cycle, and is the default view after analysis completes. You can use this plot to identify and examine irregular amplification and to manually set the threshold and baseline parameters for the run.

C
Threshold

IMPORTANT! Applied Biosystems recommends verifying that the baseline and threshold were called correctly for each well. If necessary, adjust the values manually as described in the SDS Online Help.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Ct vs. Well Position Plot

This plot displays threshold run cycle (CT) as a function of well position. You can use this plot to locate outliers from detector data sets. See Omitting Samples below.

Omitting Samples

Experimental error (such as contamination, pipetting errors, etc.) can produce atypical amplification data for some wells. These wells typically produce CT values that differ significantly from the average. If included in the baseline and threshold value calculations, these outlying data (outliers) can result in erroneous threshold run cycle (CT) measurements. To ensure precision, carefully review your data set for outliers. You can remove outliers manually using the Ct vs. Well Position plot and the plate grid. For more information about omitting wells, refer to the SDS Online Help.
Note: You will need to re-analyze the data each time you remove samples from the data

set.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Adjusting Plot Settings

Click or select View > Display Setting to open the Display Settings dialog box and adjust the selected plot settings.

Note: The adjustable settings depend on which plot you are viewing. Refer to the SDS

Online Help for more information.

Notes

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Appendix C Analyzing and Viewing Amplification Data

Viewing the Amplification Data

Notes

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Appendix D

Flags and Filtering for Allelic Discrimination (AD) Plate Documents

Overview
In Sequence Detection Systems (SDS) Software v2.3, you can automatically flag and filter results data to meet specified criteria. Flags and filters are assay-specific and are assigned at either the system level or are user-configured. System level flags are not user-configurable. These flags are displayed in the Results Table and the QC Summary tab. User-configured flags are set/defined by the user. These flags are also displayed in the Results Table and QC Summary tab. For more information about flags and filters, see the SDS Online Help.

Using the Analysis Settings Dialog Box to Assign Flags

You assign flags when you configure the analysis settings for an individual plate document or study. When you open the Analysis Settings dialog box for your current plate document, a list of the flags applicable to that plate document appears in the Plate tab.

1. Click

(or select Analysis > Analysis Settings) to open the Analysis Settings dialog box.

2. Select the Plate tab. A list of all applicable flags for the open plate document is
displayed. For more information about assigning flags, see the SDS Online Help.

Plate Tab

The contents of the Plate tab are described on page 100.

Notes

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Appendix D Flags and Filtering for Allelic Discrimination (AD) Plate Documents
Overview

No. 1

Column

Description

Flag Condition and Omit Plates When... Flag Condition Condition

Name of the flag setting. Indicates whether or not to flag a condition. If the checkbox is checked, the selected condition will be flagged. Allows you to select conditions for the flag from a drop-down list. The drop-down list contains conditions such as <, >, =, etc.

Note: The Condition drop-down lists vary per flag. If conditions are not applicable
to a flag, no drop-down list is available.
4

Flag Condition Value

Allows you to enter a numeric range or threshold for a flag. If you specify a value outside the allowed range/threshold, an error message is displayed.

Note: If ranges/thresholds are not applicable to a flag, you will not be able to enter a value.
5

Omit

Determines whether or not to omit a well if the flag condition is met. If the checkbox is checked, the well will be omitted if the condition is met.

Note: Well omission behavior is different depending on whether they are omitted
manually or by the flag and filter settings. Wells omitted here will contain result values that were generated until the wells were omitted. You will be able to view the flag data/condition for omitted wells. However, when wells are omitted manually, results data for these wells are lost.

Notes

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Appendix D Flags and Filtering for Allelic Discrimination (AD) Plate Documents
Viewing Flags

Viewing Flags
In the plate document, you can view flags in the: QC View of the Plate Grid For Allelic Discrimination (AD) plate documents, the Plate Grid automatically switches to QC View after analysis. (To zoom in on the flags, click View > Grid Zoom.) Only flags are displayed in the QC view; all other display items (sample name, detector color, etc.) are turned off. QC Summary Tab The QC Summary tab provides an overview of the plate documents quality. Results Table View flags, filter the results based on flags, and sort the table by flags.

Flag Icons

The following flag icons are used in the Results Table and QC view of the Plate Grid:
Icon Passing well (no flags are assigned) Flagged well Well omitted by the algorithm Well omitted manually Description

Note: In the Results Table, the Flag column is blank for manually omitted wells.

Notes

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Appendix D Flags and Filtering for Allelic Discrimination (AD) Plate Documents
Viewing Flags

Notes

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References
Afonina, I., Zivarts, M., Kutyavin, I., et al., 1997. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25:26572660. Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B. 1997. Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization. Nucleic Acids Res. 25:37183723. Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR. Nature 339:237238. Lee, L. G., Connell, C. R., and Block, W. 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21:37613766. Livak, K.J., Marmaro, J., and Todd, J.A. 1995. Towards fully automated genome-wide polymorphism screening [letter]. Nat. Genet. 9:341342.

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References

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Index

Numerics
7900HT Fast Real-Time PCR System 2

A
AD assay See allelic discrimination assay adding markers to a plate document 25, 41 samples to a plate document 25, 41 allele calling automatic 62 manual 65 allelic discrimination assay description 3 allelic discrimination experiment materials required 5 overview 4, 5 TaqMan chemistry 10 Allelic Discrimination Plot 57 allelic discrimination run Allelic Discrimination Plot 57 analyzing 55 evaluating the results 55 performing 52 purpose 4, 73 Results Grid 60 Results Table 61 saving analysis results 68 Amplification Plot tab 94 amplification run conditions 49 performing 47 purpose 4, 73 amplification, real-time, benefit in allelic discrimination assay 38 analysis settings 90 Analysis Settings dialog box 99 Applied Biosystems contacting viii customer feedback on documentation vii Information Development department vii Technical Support viii Applied Biosystems 7900HT Fast Real-Time PCR System 2 AQ run. See amplification run

Assay Design Guidelines 12 assay types, overview 2 assumptions for using this guide automatic allele calling 62

B
baseline 89, 90 bold text, when to use v

C
CAUTION, description vi Component tab 93 concentration of DNA 14 Connect button 52 conventions bold text v for describing menu commands v IMPORTANTS! vi in this guide v italic text v Notes v user attention words v Ct vs. Well Position view 96 Ct. See threshold cycle Custom TaqMan SNP Genotyping Assays customer feedback, on Applied Biosystems documents vii

11

D
delta Rn 89 Delta Rn vs.Cycle view 95 detectors definition 22 DNA amplifying 89 concentration 14 preparing 14 quality 14 documentation, related vii dyes 89

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Index

E
entering comments 28 markers into a plate document 25, 41 samples into a plate document 25, 41 exporting plate data graphic files 70 text files 69

P
passive reference 89 plate documents about Allelic Discrimination (AD) plate documents 22 about Standard Curve (AQ) plate documents 38 adding markers 25, 41 adding samples 25, 41 creating a Standard Curve (AQ) plate document 39 creating Allelic Discrimination (AD) plate documents 23 importing samples 25, 41 options for creating 22, 38 Plate tab 92, 99 plus/minus assay pre-read run, performing 34 result calls 91 post-analysis options 69 post-read run analysis 90 description 52 Pre Read button 35 pre-read run description 34 performing 34 Primer Express Software 12 printing a data report 72 probes, designed for specific alleles 3 vii

F
filtering 99 flags 99 viewing 101 fluorescence spectra 93

G
Graph Settings dialog box 97 guidelines assay development 12 DNA preparation 14

H
heterozygote definition 3 homozygote definition 3

I
Information Development department, contacting italic text, when to use v

R
Raw Data Plot 36, 54 reaction mix Custom TaqMan SNP Genotyping Assays 15 custom-designed assays 15 Primer Express assays 15 TaqMan SNP Genotyping Assays 15 reaction plate volume per reaction 17 real-time amplification, benefit in allelic discrimination assay 38 reference, passive 89 Report tab 72 reporter dye 89 results assigning calls automatically 62 assigning calls manually 65 Results Grid 60 Results tab 91 Results Table 61 Rn vs. Cycle view 94 Rn. See normalized reporter

M
manual allele calling 65 markers adding to a plate document 25, 41 definition 22 materials required for allelic discrimination experiment 5 menu commands, conventions for describing MSDSs, obtaining vi, viii

N
No AmpErase UNG 15 no template control 22 normalized reporter 89 NTC definition 4 task 22

106

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Index

S
sample experiment overview 5 samples adding to a plate document 25, 41 single-nucleotide polymorphism 3 SNP assay 3 software, starting 23, 39

T
TaqMan probe-based chemistry 10 TaqMan SNP Genotyping Assays 11 TaqMan Universal PCR Master Mix 15 target and probe matches and mismatches 3 target, definition 4 tasks, description 22 Technical Support, contacting viii text conventions v threshold 90 threshold cycle auto Ct 90 definition 89 training, information on viii

U
unknown definition 4 task 22 user attention words, described

W
WARNING, description vi

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Index

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Worldwide Sales and Support Applied Biosystems vast distribution and service network, composed of highly trained support and applications personnel, reaches 150 countries on six continents. For sales office locations and technical support, please call our local office or refer to our Web site at www.appliedbiosystems.com. Applied Biosystems is committed to providing the worlds leading technology and information for life scientists. Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 USA Phone: +1 650.638.5800 Toll Free (In North America): +1 800.345.5224 Fax: +1 650.638.5884

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