MAN0009593 TaqManSNP UG

TaqMan® SNP Genotyping Assays
USER GUIDE
Single-tube assays
for use with:

TaqMan® Predesigned SNP Genotyping Assays
TaqMan® Drug Metabolism Enzyme Genotyping Assays
TaqMan® Custom SNP Genotyping Assays
Publication Number MAN0009593
Revision B.0
For Research Use Only. Not for use in diagnostic procedures.

Manufacturer: Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated with
the workflow covered in this guide.
Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288
The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,
INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR
USE OF IT.
Revision history: Revision history of Pub. No. MAN0009593

Revision Date Description
B.0 29 September 2017 • Updates to supported instruments, required materials, and document delivery information
® ™
• Added information about TaqMan QSY probes
• Updates to analysis options
• Corrections to instrument accessories
• Updates to Troubleshooting
• Updated to the current template, with associated updates to warranty, trademarks, and
logos
A.0 30 January 2014 New document
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan and AmpliTaq
Gold are trademarks of Roche Molecular Systems, Inc., TaqMan is used under permission and license. DNAzol is a trademark of Molecular Research
Center, Inc. Oragene is a trademark of DNA Genotek, Inc. NanoDrop is a trademark of NanoDrop Technologies, LLC. Microsoft Excel is a trademark of
Microsoft Corporation.
©2017 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

®
TaqMan SNP Genotyping Assay overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Context sequence representation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Compatible master mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
®
Predesigned TaqMan SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
®
TaqMan Drug Metabolism Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
®
Custom TaqMan SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Assay format, size, and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Predesigned assays: single‑tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
DME assays: single‑tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Custom assays: single‑tube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Plate products and formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Shipment contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Storage and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■ CHAPTER 2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Extract and purify genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Quantify the sample gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Prepare assays, DNA samples, and master mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Set up the PCR reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Select the DNA delivery method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Set up the PCR reactions: wet DNA method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Set up the PCR reactions: dried‑down DNA method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
(Recommended) Perform a pre‑PCR plate read . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Perform PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Perform the post-PCR plate read and analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
®
Data analysis with TaqMan Genotyper Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Data analysis with the Thermo Fisher Cloud Genotyping Application . . . . . . . . . . . . . . 25
Allelic discrimination plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
TaqMan® SNP Genotyping Assays User Guide 3

Contents
■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Troubleshooting the AD plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Troubleshooting the AD plot: genetic characteristics of the assay and/or the samples . . 35
Low allele frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Deletion alleles in an individual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Additional SNP present under a probe or primer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Gene has a copy number polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
SNP is triallelic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Gene on the X chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Gene on the Y chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Troubleshooting the AD plot: sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Degraded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
PCR inhibitors in sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Troubleshooting the AD plot: assay preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Reagents mishandled or expired . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
™
Using a master mix without ROX dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
DNA or assay reagent not added to the reaction well . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Insufficient DNA added to the reaction well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Evaporation from the reaction well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Pipetting errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Inefficient mixing or centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Assay has high background fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
More than one sample in the well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
More than one assay in the well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Troubleshooting the AD plot: instrument set-up and maintenance . . . . . . . . . . . . . . . . . . . . 51
Routine instrument maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Instrument calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Types of calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Troubleshooting the AD plot: data analysis (software) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Empty allelic discrimination plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
No alleles called in the AD plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Homozygous allele frequencies reversed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Too many alleles called in AD plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

®
TaqMan SNP Genotyping Assays chemistry overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
® ™
TaqMan MGB and QSY probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
About the 5' nuclease assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
UV spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Absolute quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Fluorometric analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Good laboratory practices for PCR and RT-PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
4 TaqMan® SNP Genotyping Assays User Guide

Contents
■ APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
■ APPENDIX D Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
®
TaqMan Assays qPCR Guarantee . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

1 Product information
IMPORTANT! Before using this product, read and understand the information in the
“Safety” appendix in this document.
TaqMan® SNP Genotyping Assay overview

Applied Biosystems™ TaqMan® SNP Genotyping Assays use TaqMan® 5´‑nuclease
chemistry for amplifying and detecting specific polymorphisms in purified genomic
DNA samples. Each assay allows genotyping of individuals for a single nucleotide
polymorphism (SNP). All assays are developed using our bioinformatics assay design
processes that apply heuristic rules that are deduced from both manufacturing and
assay performance data.
Each TaqMan® SNP Genotyping Assay contains:
• Sequence-specific forward and reverse primers to amplify the polymorphic
sequence of interest
• Two TaqMan® minor groove binder (MGB) probes with nonfluorescent quenchers
(NFQ):
– One VIC™‑labeled probe to detect Allele 1 sequence
– One FAM™‑labeled probe to detect Allele 2 sequence
MGB probes at the 3' end bind to the DNA helix minor groove, improving
hybridization‑based assays by stabilizing the MGB probe‑template complex. This
increased binding stability allows the use of probes as short as 13 bases for superior
allelic discrimination and assay design flexibility. All MGB probes also include a
nonfluorescent quencher (NFQ) that virtually eliminates background fluorescence
and provides excellent signal‑to‑noise ratio for superior assay sensitivity.
TaqMan® Assays QPCR Guarantee (see “TaqMan® Assays qPCR Guarantee“ on
page 68) ensures performance and satisfaction with results that are obtained with all
Predesigned TaqMan® SNP Genotyping Assays and TaqMan® Drug Metabolism
Genotyping Assays (DME).

Chapter 1 Product information
TaqMan® SNP Genotyping Assay overview 1
Context sequence Reporter dye information for the TaqMan® SNP and DME Genotyping Assays are
representation represented in the assay context sequence. The context sequence is the nucleotide
sequence surrounding the SNP site and is provided in the (+) genome strand
orientation relative to the NCBI reference genome. The SNP alleles are included in
brackets, where the order of the alleles corresponds to the association with probe
reporter dyes, where [Allele 1 = VIC™ dye / Allele 2 = FAM™ dye].
If the context sequence is ...XXXXX[A/B]XXXXX...:
• A allele always represents the VIC™ dye
• B allele always represents the FAM™ dye
Table 2 Example allele-to-dye associations
VIC™‑associated FAM™‑associated
Context sequence clip SNP alleles
allele allele
...CCCACCCTTC[G/A]ACACTATTAC... [G/A] G A
...ATTAACCCAT[T/C]AGTGATGGGG... [T/C] T C
...AAGCAACTAT[G/A]TTCATAACTT... [G/A] G A
...AACCGTGTGA[T/C]GGCAGTGATT... [T/C] T C
The context sequence for your assay is provided in the Assay Information File,
available at thermofisher.com/taqmanfiles.

1 Chapter 1 Product information
Predesigned TaqMan® SNP Genotyping Assays
Compatible All TaqMan® SNP Genotyping Assays are optimized for use with TaqMan®
master mixes Genotyping Master Mix and require only three components for PCR.
• 1–20 ng purified genomic DNA per well (final concentration: ³0.2 ng/µL)
• 20X, 40X, or 80X TaqMan® SNP Genotyping Assay (depending on product and
assay scale)
• TaqMan® Genotyping Master Mix, or another compatible master mix
Table 3 Compatible master mixes for use with TaqMan® SNP Genotyping Assays
Master mix Guidelines for use

®
TaqMan Genotyping Recommended master mix for use with DNA samples
Master Mix not prepared with TaqMan® Sample-to-SNP™.
Use standard thermal cycling conditions only.
TaqMan® GTXpress™ Recommended master mix for use with DNA samples
Master Mix[1] prepared with TaqMan® Sample-to-SNP™.
Fast thermal cycling conditions are not recommended
for TaqMan® DME SNP Genotyping Assays due to the
overall longer length of many DME amplicons.
TaqMan® Universal Master TaqMan® Genotyping Master Mix is recommended over
Mix II TaqMan® Universal Master Mix II due to overall better
performance and stronger signal.
TaqPath™ ProAmp™ Master Recommended for samples with PCR inhibitors such as
Mix heparin and hematin.
Not recommended for use with DME and CFTR
TaqMan® SNP Genotyping Assays due to high
background signal associated with some sensitive
assays in these collections.
[1] Provided with the TaqMan® Sample-to-SNP™ Kit
Predesigned TaqMan® SNP Genotyping Assays

The Predesigned TaqMan® SNP Genotyping Assays collection includes millions of
genome‑wide human assays:
• Common 1,000 Genome SNPs—The 1,000 Genomes Project created the largest
and most detailed catalog of human variation and genotypes.
• HapMap SNPs—SNPs genotyped by the HapMap Project across several
populations to develop a haplotype map of the human genome.
• Coding SNP assays—Assays for the detection of informative and putative
functional, nonsynonymous cSNPs in gene‑coding regions.
The library also includes over 10,000 predesigned mouse assays. For targets of interest
that are not covered by the predesigned assays collections, Custom TaqMan® SNP
Genotyping Assays are available (see “CustomTaqMan® SNP Genotyping Assays“ on
page 9).

TaqMan® Drug Metabolism Genotyping Assays 1
Predesigned TaqMan® SNP Genotyping Assays are made‑to‑order, available in

multiple scales, and available in the following types:
• Validated—Validated assays are tested using genomic DNA from 45 individuals
from each of four ethnic populations (African American, Caucasian, Chinese, and
Japanese). The minor allele frequency (MAF) for validated assays is >5% in at
least one test population. MAF data is available on the product web page and in
the order Assay Information Files (AIF).
• Functionally Tested—Functionally Tested assays meet minimum functionality
criteria in testing of 20 genomic DNA samples from three ethnic populations
(African American, Caucasian, and Asian) and both genders. Minor allele
frequency is not calculated. To ensure assay amplification and sample clustering,
all human SNP genotyping assays undergo functional testing before shipment of
the first order. Quality of repeat orders is verified by mass spectrometry.
• Qualified—Qualified Assays are pre‑tested using cell line and tissue genomic
DNA and/or synthetic templates to meet specific functionality requirements.
• DME—See “TaqMan® Drug Metabolism Genotyping Assays“ on page 9.
See thermofisher.com/taqmansnp for more information.
®
TaqMan Drug Metabolism Genotyping Assays
TaqMan® Drug Metabolism Genotyping Assays (DME Genotyping Assays) offer a
comprehensive collection of inventoried assays that are optimized for genotyping
SNPs, insertions and deletions (indels), and multi‑nucleotide polymorphisms (MNPs)
in drug metabolism‑related genes. TaqMan® DME Genotyping Assays are available in
small‑scale and are inventoried for fast availability.
TaqMan® DME Genotyping Assays detect potentially causative polymorphisms in 221
drug metabolism enzyme and associated transporter genes. Assays are tested using
genomic DNA from 45 individuals from each of four ethnicities. The minor allele
frequency for each population is available, but rare and mutant alleles may not be
present in the test DNA.
See thermofisher.com/taqmandme for more information.
Custom TaqMan® SNP Genotyping Assays

Create Custom TaqMan® SNP Genotyping Assays by submitting confidential target
sequences to our secure assay design pipeline using the Custom TaqMan® Assay
Design Tool (thermofisher.com/taqmansnpdesign). All human SNP genotyping
assays are functionally tested using 20 unique genomic DNA samples to ensure assay
amplification and sample clustering. Custom assays are designed, synthesized,
formulated, optimized, and quality control tested. See thermofisher.com/
taqmancustomsnp for more information.

Assay format, size, and storage
Custom TaqMan® SNP Genotyping Assays allow you to:

• Perform genotyping studies with any possible SNP in any organism.
For example, AGTTCATCCATGGTCA u AGTTCATACATGGTCA, annotated as
AGTTCAT[C/A]CATGGTCA.
• Detect indels of up to six bases for genotyping studies.
For example, AGTTCATCCATGGTCA u AGTTCATGGTCA, annotated as
AGTTCAT[CCAT/*]GGTCA.
• Detect MNPs of up to six bases for genotyping studies.
For example, AGTTCATCCATGGTCA u AGTTCATATATGGTCA, annotated as
AGTTCAT[CC/AT]ATGGTCA.
For maximal efficiency in a multiplex format, Custom TaqMan® SNP Genotyping

Assays are available with TaqMan® probes using a QSY™ quencher. QSY™ probes can
be ordered with FAM™ and VIC™ dyes and with ABY™ and JUN™ dyes, allowing
amplification of up to four targets in a single reaction. These reporter dyes are
optimized to work together with minimal spectral overlap for optimal performance.
The QSY™ quencher is fully compatible with probes that have MGB quenchers. See
thermofisher.com/multiplexqpcr for compatible master mixes and ordering
information.
SNP primer and probe sequences can also be submitted directly to the Custom
TaqMan® Assay Design Tool to manufacture a known assay sequence.

Predesigned For the list of available Predesigned TaqMan® SNP Genotyping Assays, visit
assays: thermofisher.com/taqmansnp.
single‑tube Search the assay database by:
• Gene name
• Gene symbol
• SNP ID
• Assay ID
• Genomic location
Table 4 Predesigned TaqMan® SNP Genotyping Assays: single‑tube
Number of reactions Cat. No.

Assay mix
Scale 96‑well Non-
384‑well[1] 96‑well[3] formulation Human
Fast[2] human[4]
Small 1,500 750 300 40X 4351379 4351384
Medium 5,000 2,500 1,000 40X 4351376 4351382
Large 12,000 6,000 2,400 80X 4351374 4351380

[1] 5‑µL reaction volume
[4] Non-human TaqMan® Predesigned SNP Genotyping Assays are designed to amplify and detect specific
polymorphisms in specified non-human genomic DNA samples.

Assay format, size, and storage 1
DME assays: For the list of inventoried TaqMan® DME Genotyping Assays, visit thermofisher.com/
single‑tube taqmandme.
Table 5 TaqMan® DME Genotyping Assays: single‑tube

Assay mix
Scale Non-
human[3]
Small 750 150 20X 4362691 —

TaqMan® DME Genotyping Assays can also be ordered through the DME Index
(thermofisher.com/taqmandme). The DME Index contains a comprehensive list of the
DME assays with annotations that include:
• The gene to which the polymorphism location is mapped
• Allele nomenclature (from public allele nomenclature sites, when available)
• Polymorphism (for example, A/G)
• Context sequence
• Amino acid change (if applicable)
• Thermo Fisher Scientific MAF data
Custom assays: Custom TaqMan® SNP Genotyping Assays are designed, synthesized, and receive
single‑tube analytical quality control based on sequence information supplied. All information
supplied is secure and confidential. For more information, visit thermofisher.com/
taqmancustomsnp.
Note: For detailed instructions on how to design and order Custom TaqMan® SNP
Genotyping Assays, consult the Custom TaqMan® Assays Design and Ordering Guide
(Pub. No. 4367671). Custom SNP Assays are designed using the Custom Assay Design
Tool (CADT)(thermofisher.com/taqmansnpdesign). This tool may also be used to
upload assays that have already been designed outside the system.
Table 6 Custom TaqMan® SNP Genotyping Assays: single‑tube

Assay mix
Scale 96‑well Non-
Fast[2] human[4]
Small 1,500 750 300 40X 4331349 4332077
Medium 5,000 2,500 1,000 40X 4332072 4332075
Large 12,000 6,000 2,400 80X 4332073 4332076


Plate products For projects that require analyzing many samples or for evaluating samples for many
and formats SNP assays can be ordered in plated formats.
Table 7 TaqMan® SNP Genotyping Assays plate products and formats
Format Source
OpenArray™ plates[1] thermofisher.com/openarray
96- or 384-well plates (fast or standard) thermofisher.com/customplating

[1] For use with the QuantStudio™ 12K Flex Real-Time PCR System.
Shipment contents Single‑tube shipments include:

• Dispatch Note (Packing List)—Contains your order information, a list of the
assays that were ordered, and the quantity of the assays shipped.
• Assay tube(s)—Each TaqMan® SNP Genotyping Assay tube is identified with a
label and a 2‑D barcode.
• Data Sheet—Provides a summary of the assays in your shipment, the assay
details, and the storage conditions.
Plate shipment contents depend on the assay format. Regardless of format, each
shipment includes:
• Assay plate(s) identified with a label, including 2‑D barcodes, on the bottom.
• Data Sheet—Provides a summary of the assays in your shipment, the assay
details, and the storage conditions.
Go to thermofisher.com/taqmanfiles to download the following supporting
documents.
• Assay information files (AIFs)
• User Instruction Documents (Protocols, User Guides, and Quick Reference Cards)
• Certificates of Analysis
• Safety Data Sheets
For detailed information about the shipment and assay information files (AIFs), see
Understanding Your Shipment (Pub. No. MAN0017153).

Assay format, size, and storage 1
Storage and Note: For plate formats, see the documentation included with your shipment for
stability storage and stability information.
• Store single‑tubeTaqMan® SNP Genotyping Assays at –25 to –15°C in the dark.
IMPORTANT! Protect TaqMan® Assays from direct exposure to light. Excessive
exposure to light may affect the fluorescent probes.
• Do not perform more than 10 freeze‑thaw cycles. If you expect to freeze‑thaw

TaqMan® Assays more than three times, consider aliquoting to minimize the
number of freeze‑thaw cycles.
• We recommend diluting the 40X and 80X predesigned and CustomTaqMan® SNP
Genotyping Assays to a 20X working stock (see “Dilute, then dispense aliquots of
TaqMan® SNP Genotyping Assays“ on page 13).
• TaqMan® SNP Genotyping Assays are stable for up to 5 years past their
manufacturing date. The manufacturing date is printed on each assay tube or
plate.
Dilute, then dispense aliquots of TaqMan® SNP Genotyping Assays

We recommend diluting TaqMan® SNP Genotyping Assays to a 20X working stock,
then dispensing aliquots into tubes for routine use, to minimize freeze‑thaw cycles
and exposure to light.
1. Dilute 40X or 80X TaqMan® SNP Genotyping Assays to a 20X working stock with
1X TE buffer.
Note: 1X TE buffer composition: 10‑mM Tris‑HCl, 1 mM EDTA, pH 8.0, in
DNase‑free, sterile-filtered water.
2. Vortex, then centrifuge the mixture.
3. Store multiple aliquots of the TaqMan® SNP Genotyping Assays at –25 to –15°C
in the dark.

Required materials not supplied
Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.
Table 8 Kits for genomic DNA extraction
Item Sample type Source
Select the appropriate kit for your

Kits for genomic DNA extraction thermofisher.com/gdnaprep
sample type.
• Buccal samples
• Blood
TaqMan Sample-to-SNP Kit[1]
® ™ • Cells cultures 4403081
• Plant samples
• Tissue
[1] We recommend pre-amplifying all samples prepared with the TaqMan® Sample-to-SNP™ Kit when using OpenArray™ for subsequent
genotyping.
Table 9 Supported instruments
Item Block modules Source
96-well, 96-well Fast, 384-well, and 8-strip

Veriti™ Thermal Cycler[1]
tubes
96-well, 2 × 96-well, 2 × 384-well, and 8-strip

GeneAmp™ PCR System 9700[1]
tubes
3 × 32-well, 96-well, 2 × 96-well, 2 × 384-well,

ProFlex™ PCR System[1]
2 × Flat, and 8-strip tubes
SimpliAmp™ Thermal Cycler[1] 96-well and 8-strip tubes
7500 Real-Time PCR System 96-well and 8-strip tubes
7500 Fast Real-Time PCR System 96-well and 8-strip tubes
7900HT Fast Real-Time PCR Instrument 96-well, 96-well Fast, and 384-well Contact your local
sales office.
StepOne™ Real-Time PCR
System/StepOnePlus™ Real-Time PCR StepOne™ 48-well and 8-strip tubes
System

ViiA™ 7 Real-Time PCR System
tubes
QuantStudio™ 3 Real-Time PCR System 96-well, 96-well Fast, and 8-strip tubes

QuantStudio™ 5 Real-Time PCR System
tubes

QuantStudio™ 6 Flex Real-Time PCR System
tubes

Required materials not supplied 1
Item Block modules Source

QuantStudio™ 7 Flex Real-Time PCR System
tubes Contact your local
™
QuantStudio 12K Flex Real-Time PCR 96-well, 96-well Fast, 384-well, OpenArray , ™ sales office.
System and 8-strip tubes
[1] If a thermal cycler is used for PCR amplification, the optional pre-read and the post-read must be performed separately on a real-time PCR
system in order to detect and record fluorescent signals.
Table 10 Equipment, tubes, plates and consumables
Item Source
Equipment
Centrifuge with plate adapter MLS
Microcentrifuge MLS
Adjustable pipettors MLS
Laboratory mixer (vortex or equivalent) MLS
Tubes, plates, and other consumables
RNase-Free Microfuge Tubes (1.5 mL) AM12400
Plastics consumables[1] thermofisher.com/plastics
Pipette tips thermofisher.com/pipettetips
Disposable gloves MLS

[1] Click Plastics selection guide to select the appropriate plates and seals for use with your instrument.
Table 11 Reagents for PCR
Item Source
Master mix, one of the following[1]:
TaqMan® Genotyping Master Mix (2X) 4371355
TaqMan® Sample-to-SNP™ Kit[2] 4403081
TaqMan® Universal Master Mix II 4440038
TaqMan® GTXpress™ Master Mix 4401892
TaqPath™ ProAmp™ Master Mix A30866
Additional reagents
Nuclease-Free Water (not DEPC-Treated) AM9938

[1] See “Compatible master mixes“ on page 8
[2] TaqMan® Sample-to-SNP™ Kit contains all necessary reagents for sample preparation and amplification, including TaqMan® GTXpress™ Master
Mix.

Workflow
Workflow
Start with high-quality gDNA, quantified 18
Set up the PCR reactions: wet DNA OR Set up the PCR reactions:
method (page 19) dried‑down DNA method (page 21)
(Recommended) Perform a pre‑PCR plate read (page 22)
Perform PCR (page 22)
Perform the post-PCR plate read and analysis (page 23)

2 Methods
Procedural guidelines
• Store TaqMan® SNP Genotyping Assays frozen and away from light until use.
Excessive exposure to light may affect the fluorescent probes.
• Do not perform more than 10 freeze‑thaw cycles.
• To ensure optimal analysis and troubleshooting of the TaqMan® SNP Genotyping
Assays, prepare an optical reaction plate containing the following for each assay.
Table 12 Recommended reaction types for each assay
Reaction type Component

Test sample DNA samples with unknown genotype at the
polymorphism of interest
No‑template control[1] DNase‑free water
(Optional) Positive control DNA sample with known genotype at the polymorphism
of interest
[1] Use two no‑template controls per assay to allow the genotyping algorithm to correct for background
signal from fluorescent probes and to enable the detection of DNA contamination.
• Mix thoroughly after adding reagents to DNA samples to avoid stratification of
the reagents and/or air bubbles in the well, which can lead to "stringy" clusters
(see Appendix A, “Troubleshooting“).
• This procedure is optimized for TaqMan® Genotyping Master Mix. Use the
appropriate thermal cycling conditions for your assay type and the master mix
used.
Table 13 Alternative thermal cycling conditions
Assay or master mix Thermal cycling conditions

®
TaqMan DME Genotyping (Recommended) Use standard mode thermal cycling
Assays conditions and a longer extension time with additional
cycles due to the longer average amplicon lengths.
TaqMan® Genotyping Use standard mode thermal cycling conditions only.
Master Mix
Other compatible master Use the protocol that is provided with the master mix.
mix[1] For TaqMan® DME Genotyping Assays, use standard
mode thermal cycling conditions only.
[1] See “Compatible master mixes“ on page 8.
• See “Good laboratory practices for PCR and RT‑PCR“ on page 63 for additional
guidelines.

2 Chapter 2 Methods
Before you begin
Before you begin

Extract and purify Extract and purify genomic DNA (gDNA) according to standard practices. For
genomic DNA recommended kits and ordering information, see Table 8 on page 14.
Quantify the Use one of the following methods to quantify sample gDNA before using TaqMan®
sample gDNA SNP Genotyping Assays:
• UV/Vis spectrophotometry [A260 /A280 measurement (see “UV spectroscopy“ on
page 62)
• NanoDrop™ spectrophotometry
• RNase P method (see “Absolute quantification“ on page 63)
• Qubit™ 3.0 Fluorometer (Cat. No. Q33216) with Qubit™ Assay Tubes (Cat.
No. Q32856) and Qubit™ dsDNA HS Assay Kit (Cat. Nos. Q32851 and Q32854)
See “DNA quantification“ on page 61 for more information.
Prepare assays, • Dilute the 40X or 80X Predesigned and Custom TaqMan® SNP Genotyping
DNA samples, and Assays to a 20X working stock solution (see “Dilute, then dispense aliquots of
TaqMan® SNP Genotyping Assays“ on page 13).
master mix
Note: TaqMan® DME Genotyping Assays are supplied at a 20X concentration.
• Before each PCR run:

– Resuspend the assay by vortexing, then centrifuge the tube briefly.
– (For wet DNA only) Resuspend thawed frozen DNA samples by vortexing,
then centrifuge the tubes briefly.
– Thoroughly mix the TaqMan® Genotyping Master Mix by swirling the bottle.

Chapter 2 Methods
Set up the PCR reactions 2
Set up the PCR reactions
Select the DNA TaqMan® SNP Genotyping Assays can be used with either wet or dried‑down DNA.
delivery method Drying down the gDNA in plates is the most convenient method for an experiment
that requires multiple plates using the same gDNA or for multiple experiments that
use the same gDNA.
Note: For both methods, it is recommended to use a calibrated, positive‑displacement
pipette to minimize contamination and error.
Table 14 DNA delivery methods
Method Description Experimental uses
1. SNP reaction mix is Large number of DNA templates

dispensed to an optical tested on few SNP targets.
reaction plate.
2. gDNA is delivered to the
Wet DNA delivery final reaction mix.
Note: In this method the DNA is
already dissolved in buffer or
water and is used as a
component in the final reaction.
1. gDNA is applied to the • Low DNA concentration

bottom surface of an results in large sample
optical reaction plate. volumes (2–5 μL) to run the
assay.
2. DNA sample is dried down
completely by evaporation. • Limited number of DNA
Dried-down DNA templates tested
3. SNP reaction mix is added
delivery repeatedly on different SNP
and the DNA disperses in
targets.
the final reaction mix.
• Large number of DNA
samples prepared in plates,
dried down, and stored
before use.
Set up the PCR IMPORTANT! This procedure is optimized for TaqMan® Genotyping Master Mix. See
reactions: wet Table 13 on page 17 for alternative master mixes and plate and cycling conditions.
DNA method
Prepare the reaction mix: wet DNA method
1. Calculate the number of reactions to be performed for each assay, including
recommended controls (see Table 12 on page 17).
Note: Multiple assays can be run on one reaction plate. Controls for each assay
must be included on the same plate.

2 Chapter 2 Methods
Set up the PCR reactions
2. Combine the following components for the number of reactions required, plus
10% overage.
Table 15 Reaction mix for wet DNA method
384-well 96-well Fast[1] 96-well

Component
(5‑µL reaction) (10‑µL reaction) (25‑µL reaction)
2X TaqMan® Master
2.50 µL 5.00 µL 12.50 µL
Mix
20X Assay Working
0.25 µL 0.50 µL 1.25 µL
Stock
Nuclease-Free
— — —
Water
Total volume per
2.75 µL 5.50 µL 13.75 µL
well
[1] When using TaqMan® Genotyping Master Mix, use Fast reaction plates only on the QuantStudio™ 6 and 7
Flex, the QuantStudio™ 12K Flex, ViiA™ 7, or the 7500 Fast Real-Time PCR System and only with
Standard thermal cycling conditions.
3. Vortex briefly to mix.
4. Centrifuge briefly to bring the reaction mix to the bottom of the tube and
eliminate air bubbles.
Prepare the reaction plate: wet DNA method

1. Dilute each DNA sample, including controls, in Nuclease‑Free Water to deliver
1–20 ng per well.
IMPORTANT! All wells using the same assay must contain similar amounts of
sample. A final concentration of at least 0.2 ng/µL is recommended.
2. Add the appropriate volume of reaction mix to each well of the reaction plate
(see Table 15 on page 20).
3. Seal the plate with adhesive film, then centrifuge briefly to bring the reaction mix
to the bottom of the well and eliminate air bubbles.
A non‑optical seal can be used for this step.
4. Remove adhesive film from the plate, then add the appropriate volume of
sample or control to the wells. Be sure to include wells for use as no template
controls.
Reaction plate DNA sample volume

™
MicroAmp Optical 384-Well Reaction Plate 2.25 µL
™
MicroAmp Fast Optical 96-Well Reaction Plate 4.50 µL
™
MicroAmp Optical 96-Well Reaction Plate 11.25 µL
IMPORTANT! Ensure that no cross‑contamination occurs between wells.

Chapter 2 Methods
Set up the PCR reactions 2
5. Seal the plate with adhesive film, then centrifuge briefly to bring the reaction mix
to the bottom of the well and eliminate air bubbles.
IMPORTANT! Use a MicroAmp™ Optical Film Compression Pad when using:
· MicroAmp™ Optical 96‑Well Reaction Plate on the 7900HT Real‑Time PCR
Instrument
· MicroAmp™ Optical 96‑Well Reaction Plate or MicroAmp™ Optical 384‑Well
Reaction Plate on the GeneAmp™ PCR System 9700
Set up the PCR IMPORTANT! This procedure is optimized for TaqMan® Genotyping Master Mix. See
reactions: Table 16 on page 21 for alternative master mixes and plate and cycling conditions.
dried‑down DNA
method
Prepare the reaction mix: dried‑down DNA method
1. Calculate the number of reactions to be performed for each assay, including
recommended controls (see “Prepare assays, DNA samples, and master mix“ on
page 18).
Note: Multiple assays can be run on one reaction plate. Controls for each assay
must be included on the same plate.
2. Combine the following components for the number of reactions required, plus
10% overage.
Table 16 Reaction mix for dried‑down DNA method
384-well 96-well Fast[1] 96-well

Component
(5‑µL reaction) (10‑µL reaction) (25‑µL reaction)
2X TaqMan® Master
2.5 µL 5.00 µL 12.50 µL
Mix
20X Assay Working
0.25 µL 0.50 µL 1.25 µL
Stock
Nuclease-Free
2.25 µL 4.50 µL 11.25 µL
Water
Total volume per
5.0 µL 10.0 µL 25.00 µL
well
[1] When using TaqMan® Genotyping Master Mix, use Fast reaction plates only on the QuantStudio™ 6 and 7
Flex, the QuantStudio™ 12K Flex, ViiA™ 7, or the 7500 Fast Real-Time PCR System and only with
Standard thermal cycling conditions.
3. Vortex briefly to mix.
4. Centrifuge briefly to bring the reaction mix to the bottom of the tube and
eliminate air bubbles.

2 Chapter 2 Methods
(Recommended) Perform a pre‑PCR plate read
Prepare the reaction plate: dried‑down DNA method

1. Add 2–5 µL of sample or control (1–20 ng of purified gDNA or no‑template
control consisting of Nuclease‑Free Water) to each well of the reaction plate.
2. Dry the samples completely by evaporation at room temperature in a dark,

amplicon‑free location. Cover the plate with a lint‑free tissue while drying.
IMPORTANT! Do not accelerate drying by heating the plate. Heating the plate
can cause poor gDNA recovery.
3. Add the appropriate volume of reaction mix to each well of the reaction plate
(see Table 16 on page 21).
4. Seal the plate with optical adhesive film, then centrifuge briefly to bring the
reaction mix to the bottom of the well and eliminate air bubbles.
IMPORTANT! Use a MicroAmp™ Optical Film Compression Pad when using:
· MicroAmp™ Optical 96‑Well Reaction Plate on the 7900HT Real‑Time PCR
Instrument
· MicroAmp™ Optical 96‑Well Reaction Plate or MicroAmp™ Optical 384‑Well
Reaction Plate on the GeneAmp™ PCR System 9700
(Recommended) Perform a pre‑PCR plate read

A pre‑PCR plate read records the background fluorescence of each well of the plate
before PCR. During the post‑PCR plate read, the pre‑read fluorescence is subtracted
from the post‑read fluorescence to account for pre-amplification background
fluorescence, ensuring accurate results. If no pre‑read data is available, no pre‑read
background subtraction is performed.
See the appropriate instrument user guide for instructions on how to use the system
software to perform the pre‑PCR plate read and analysis.
Perform PCR
IMPORTANT! This procedure is optimized for TaqMan® Genotyping Master Mix. See
Table 13 on page 17 for alternative master mixes and plate and cycling conditions.
Note: (Optional) Perform PCR in real‑time experiment mode. Real‑time PCR allows
observation of genotyping data over time to evaluate the accuracy of genotype calls
by observing the location of a given sample relative to others throughout all cycles. If
the quantity of DNA added is too high, resulting in merged genotyping clusters, the
experiment can be reanalyzed at fewer cycles to recover genotyping accuracy.

Chapter 2 Methods
Perform the post-PCR plate read and analysis 2
See the appropriate instrument user guide for more information.
1. Program the instrument using the following conditions:

Predesigned and Custom
TaqMan® DME Genotyping
TaqMan® SNP Genotyping
Step Assays[1]
Assays
Temp. Length Cycles Temp. Length Cycles
Polymerase
95°C 10 minutes HOLD 95°C 10 minutes HOLD
activation
Denaturation 95°C 15 seconds 95°C 15 seconds
Annealing/ 40 50
60°C 1 minute 60°C 90 seconds
extension
[1] TaqMan® DME Genotyping Assays have a longer extension time with additional cycles due to the longer
average amplicon lengths.
2. Select Standard run mode in the plate document or experiment file.

IMPORTANT! Use only Standard Mode thermal cycling conditions with
TaqMan® Genotyping Master Mix.
3. Enter the reaction volume.

Reaction plate Reaction volume
™
MicroAmp Optical 384-Well Reaction Plate 5 µL
™
MicroAmp Fast Optical 96-Well Reaction Plate 10 µL
™
MicroAmp Optical 96-Well Reaction Plate 25 µL
4. Load the reaction plate, then start the run.
Perform the post-PCR plate read and analysis

Fluorescence measurements collected during the post‑PCR plate read are used to plot
the reporter signal normalized to the fluorescence signal of ROX™ (Rn) for each
sample well. This data is used to then determine the genotypes present in the DNA
samples.
To analyze data for allelic discrimination or genotyping:
1. Create a post‑PCR plate document or experiment file.
2. Perform a post‑PCR plate read on a real‑time PCR instrument.
Note: If no pre‑read data is available, no pre‑read background subtraction is
performed.

2 Chapter 2 Methods
3. Analyze the experimental data using the following:
Software Features
Real-time • Instrument software
instrument • View real‑time trace data to aide genotype calling
software
Data analysis varies depending on your real-time PCR system.
See the instrument User Guide for more information.
TaqMan® Genotyper • Desktop software
Software • Create studies
• Overlay data from multiple plates
See “Data analysis with TaqMan® Genotyper Software“ on
page 24.
Thermo Fisher • Cloud software
Cloud Genotyping • Create studies
Application
• Overlay data from multiple plates
• View real-time trace data to aide genotype calling
See “Data analysis with the Thermo Fisher Cloud Genotyping
Application“ on page 25.
Note: TaqMan® Genotyper Software and the Thermo Fisher Cloud Genotyping
Application use algorithms that yield more accurate genotype calls and quality
control functionality than instrument analysis software.
4. Make automatic or manual allele calls. See “Allelic discrimination plots“ on
page 26 for more information.
5. Verify allele types.
Data analysis with TaqMan® Genotyper Software is a standalone software application that can be used to
TaqMan® analyze raw data from genotyping experiments created on an Applied Biosystems™
Real‑Time PCR system.
Genotyper
Software The TaqMan® Genotyper Software can be used to:
• Create a study:
– Import multiple experiments into a single study
– Import assay information files (*.txt or *.xml) to update assay
information
– Define analysis settings
– Import Supplementary Sample Information (SSI) files to update sample
information such as gender or population
– Import reference panel files to add reference samples to a study
• Generate a study template, then use the study template to create new studies. The
software analyzes the data according to the analysis settings that were defined in
the study template.
• Analyze the study data using one of two call methods:
– Autocalling—The software algorithm is used to call the data points.
– Classification Scheme—You define the cluster boundaries that are used to
call the data points.

Chapter 2 Methods
• View the study results; for example, a summary of the Quality Control (QC)
statistics at the study level, assay level, experiment level, and sample level.
• Export the following data:
– Analysis results
– Analysis settings
– Audit trails
• Transfer studies from one TaqMan® Genotyper Software application to another.
• Ensure data security. The security feature allows you to:
– Set security parameters to manage users (set up user accounts and assign
user roles)
– Track changes to the data
For more information on using TaqMan® Genotyper Software, see the TaqMan®
Genotyper Software Getting Started Guide (Pub. No. 4448637).
Data analysis with The Thermo Fisher Cloud Genotyping Application is a stand‑alone software
the Thermo Fisher application that can be used to analyze raw data from genotyping experiments that
are created on an Applied Biosystems™ real‑time PCR system.
Cloud Genotyping
Application Use the Thermo Fisher Cloud Genotyping Application to:
• Create a study:
– Import multiple experiments into a single study.
– Import data from probes that are labeled with FAM™, VIC™, ABY™, or JUN™.
– Import assay information files (*.txt or *.xml).
– Define the analysis settings.
– Import Supplementary Sample Information (SSI) files to update sample
information such as gender or population.
– Import reference panel files to add reference samples to a study.
• Analyze study data using one of two call methods:
– Autocalling—The software algorithm is used to call the data points.
– Classification Scheme—User-defined cluster boundaries are used to call the
data points.
• Analyze a subset of data by creating analysis groups with a subset of data files or
samples. Each analysis group has its own set of analysis settings and analysis
results. The analysis group table displays the analysis status for each group.
• View real‑time data, including amplification plots, multi component plots, and
traces of individual samples for a given assay by cycle in allelic discrimination
plots.
• View the study results. For example, a summary of the Quality Control (QC)
statistics at the study level, assay level, experiment level, and sample level.
• Export the following data:
– Analysis results
– Analysis settings
– Genotype call settings
– Flag settings
– Genotype matrix of samples vs. assays

2 Chapter 2 Methods
– QC by assays showing the assay call rates and flag statistics

– QC by samples showing the sample call rates and flag statistics
• Transfer or share studies from the Thermo Fisher Cloud Genotyping Application
with another user.
See the Thermo Fisher Cloud Genotyping Application Release Notes (https://
apps.thermofisher.com/app-gt-web/src/release-notes.html) for more information.
Allelic TaqMan® Genotyper Software, the Thermo Fisher Cloud Genotyping Application, and
discrimination real‑time PCR instrument software plot the results of the allelic discrimination data as
a plot of Allele 1 (VIC™ dye) versus Allele 2 (FAM™ dye). The allelic discrimination
plots
(AD) plot, also known as a cluster plot or scatter plot, represents each sample well as an
individual point on the plot.
A typical AD plot shows Homozygote clusters, a Heterozygote cluster, and the no‑
template controls. The points in each cluster are grouped closely together and each
cluster is located well away from the other clusters. See Figure 1 and Table 17 on
page 27 for expected cluster locations in an AD plot.
Figure 1 Typical allelic discrimination plot

Chapter 2 Methods
Table 17 Cluster assignments in an allelic discrimination plot
Content of samples Location in AD plot
Allele 1 (homozygote), labeled with VIC™ dye Lower right corner
Allele 2 (homozygote), labeled with FAM™ dye Upper left corner
Approximately midway between Allele

Alleles 1 and 2 (heterozygote)
1 and Allele 2 clusters
No‑template control Bottom left corner
Anywhere outside the regions

Undetermined
described above
With NTC cluster in the bottom left

No amplification
corner
Unexpected distributions in allelic discrimination plots can result from:

• Genetic characteristics of the assay and/or the sample
• Sample preparation errors
• Assay preparation errors
• Instrument setup and maintenance errors
• Data analysis (software) errors
See Appendix A, “Troubleshooting“ for more information.

A Troubleshooting
■ Troubleshooting the AD plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

■ Troubleshooting the AD plot: genetic characteristics of the assay and/or
the samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
■ Troubleshooting the AD plot: sample preparation . . . . . . . . . . . . . . . . . . . . . . . . 45
■ Troubleshooting the AD plot: assay preparation . . . . . . . . . . . . . . . . . . . . . . . . . . 47
■ Troubleshooting the AD plot: instrument set‑up and maintenance . . . . . . . . . . 51
■ Troubleshooting the AD plot: data analysis (software) . . . . . . . . . . . . . . . . . . . . . 53

Appendix A Troubleshooting
Troubleshooting the AD plot A
Troubleshooting the AD plot

See “Allelic discrimination plots“ on page 26 for more information about expected
cluster locations in an AD plot .
Observation Possible cause Recommended action

There is only one cluster present Genetic characteristics of the
assay and/or the samples
The MAF is too low for the See “Low allele frequency“ on
sample size from the tested page 36.
population.
s
his SVG.
uts or arrows
Arrow_Libary. The clusters are cloudy or diffuse Sample preparation errors
ed. Sample DNA is degraded. See “Degraded DNA“ on
page 45.
sed
y. There are PCR inhibitors in See “PCR inhibitors in

the sample. sample“ on page 47.
1
Assay preparation errors
ROX™ dye was not present in See “Using a master mix
the PCR Master Mix. without ROX™ dye“ on
page 48.
There was evaporation from See “Evaporation from the
the reaction well. reaction well“ on page 49.
There were pipetting errors. See “Pipetting errors“ on
page 49.
Instrument set-up and
maintenance errors
ROX™ dye was not designated See “ROX™ dye not
as the reference dye. designated as a passive
reference“ on page 55.
If only one cluster is present, See “Single cluster assay“ on
the allelic discrimination plot page 55.
was incorrectly scaled.

A Appendix A Troubleshooting

There are trailing clusters Sample preparation errors
Sample DNA is degraded. See “Degraded DNA“ on
page 45.
There are PCR inhibitors in See “PCR inhibitors in
Insufficient DNA was added to See “Insufficient DNA added
the well. to the reaction well“ on
page 48.
DNA concentration is highly Use the same concentration
variable. of DNA for each sample. See
“Quantify the sample
gDNA“ on page 18.
The reagents were See “Reagents mishandled or
mishandled or are expired. expired“ on page 47.
ROX™ dye was not present in See “Using a master mix
PCR master mix. without ROX™ dye“ on
page 48.
page 49.
There was inefficient mixing See “Inefficient mixing or
and/or insufficient centrifugation“ on page 50.
centrifugation.
maintenance errors
The thermal cycler is poorly See “Instrument
calibrated. calibration“ on page 52.
Data analysis (software)
errors
ROX™ dye was not designated See “ROX™ dye not
as the reference dye. designated as a passive
Some samples cluster with the NTCs Genetic characteristics of the
There is an extra SNP under See “Additional SNP present
the probe or the primer. under a probe or primer“ on
page 39.
The individual sample has two See “Deletion alleles in an
deletion alleles. individual“ on page 38.
The SNP is triallelic. See “SNP is triallelic“ on
page 42.


Some samples cluster with the NTCs The SNP is on the Y See “Gene on the Y
chromosome and some chromosome“ on page 45.
samples are female.
Note: A Y chromosome SNP
assay will only have just one
or two homozygous clusters,
and no heterozygous cluster.
Sample preparation errors
page 45.
DNA or reagent was not See “DNA or assay reagent
added to the well. not added to the reaction
well“ on page 48.
the well. to the reaction well“ on
page 48.
page 49.
centrifugation.
All samples cluster with the NTC Sample preparation errors
page 45.
the samples. sample“ on page 47.
The reagents were See “Reagents mishandled or
DNA or reagent was not See “DNA or assay reagent
added to the wells. not added to the reaction
well“ on page 48.
the wells. to the reaction well“ on
page 48.
page 49.
maintenance errors


All samples cluster with the NTC Annealing temperature on the Perform several PCR
thermal cycler was too high reactions with decreasing and
or too low. increasing annealing
temperatures, then select the
conditions that amplify the
target of interest.
Annealing temperature on the See “Instrument
thermal cycler was too high calibration“ on page 52.
or too low for the primers or
probes due to poor
calibration.
The polymerase was not See “Perform PCR“ on
efficiently activated due to page 22.
incorrect thermal cycler
conditions.
The polymerase was not See “Instrument
efficiently activated due to calibration“ on page 52.
poor calibration of the
thermal cycler.
NTCs generate high fluorescence signals that Assay preparation errors
cluster with samples instead of close to the origin The reagents were See “Reagents mishandled or
The NTC was contaminated. See “Good laboratory
practices for PCR and RT-
PCR“ on page 63.
maintenance errors
The block was contaminated. See “Routine instrument
maintenance“ on page 51.
errors:
The passive reporter dye was See “ROX™ dye not
assigned incorrectly. designated as a passive
The sample (or samples) did not cluster with the Genetic characteristics of the
specific genotype assay and/or the samples
the primer or the probe. under a probe or primer“ on
page 39.
The SNP is triallelic or tetra- See “SNP is triallelic“ on
allelic. page 42.
There is a copy number See “Gene has a copy number
polymorphism. polymorphism“ on page 41.
page 45.


The sample (or samples) did not cluster with the There was evaporation from See “Evaporation from the
specific genotype the sample well. reaction well“ on page 49.
page 49.
There was more than one See “More than one sample in
sample in the well. the well“ on page 50.
centrifugation.
maintenance errors
The block was contaminated. See “Routine instrument
maintenance“ on page 51.
The samples are not in the Hardy-Weinberg Genetic characteristics of the

equilibrium (the expected ratios of each genotype assay and/or the samples
are not seen) There is a copy number See “Gene has a copy number
The gene is on the X See “Gene on the X
chromosome. chromosome“ on page 44.
It is a non-random No action. Non-random
population. population are not expected
to follow Hardy-Weinberg
equilibrium.
errors
The detectors and markers, See “No assay or marker
or assays, were set up assigned to the well“ on
incorrectly. page 54.
Some (or all) of the data is missing (no data points Data analysis (software)
are shown on the AD plot) errors
No marker was assigned to See “No assay or marker
the sample. assigned to the well“ on
page 54.
Omit was checked for the well See “Omit option checked in
or wells with missing data. Real Time PCR instrument
software“ on page 54.


Some or all the alleles are not called (X is shown Data analysis (software)
on the AD plot) errors
The NTC task was not See “NTCs not assigned“ on
assigned to the NTC wells. page 57.
The autocall option was not See “Autocall is not
selected. selected“ on page 54.
The sample only has two See “Two cluster calling not
clusters, but the two-cluster selected“ on page 54.
calling option was not
selected (the software cannot
assign alleles).
The sample only has one See “Single cluster assay“ on
cluster (the software cannot page 55.
assign alleles).
The sample was an outlier too See “Outlier too far off
far off the scale for alleles to scale“ on page 56.
be called for other samples.
There are more than 3 clusters Assay preparation errors
More than one assay was See “More than one assay in
included in a well. the well“ on page 51.
Genetic characteristics of the
page 39.
The SNP is triallelic or tetra- See “SNP is triallelic“ on
allelic. page 42.
errors
Several assays were run but See “Too many alleles called
only one marker was in AD plot“ on page 58.
assigned.

Troubleshooting the AD plot: genetic characteristics of the assay and/or the samples A

There are vector clusters (the sample data has two Genetic characteristics of the
clusters at the same angle) assay and/or the samples
page 39.
Samples are not in equal See “Degraded DNA“ on
quantity due to degraded page 45.
DNA.
Samples are not in equal See “DNA quantification“ on
quantity due to inaccurate page 61.
DNA quantitation.
Troubleshooting the AD plot: genetic characteristics of the assay

and/or the samples
Unexpected distributions in allelic discrimination plots can result from genetic
characteristics of the assay and/or the samples, including:
• Low allele frequency (page 36)
• Deletion alleles in an individual (page 38)
• Additional SNP present under a probe or primer (page 39)
• Gene has a copy number polymorphism (page 41)
• SNP is triallelic (page 42)
• Gene on the X chromosome (page 44)
• Gene on the Y chromosome (page 45)

Troubleshooting the AD plot: genetic characteristics of the assay and/or the samples
Low allele Only one or two clusters can occur in the AD plot, as shown in Figure 2, when the
frequency minor allele occurs at a low frequency in the population being studied.
ows
bary.
Figure 2 Allelic discrimination plot shows a single cluster (in addition to the NTCs).
What to do
1 To determine if the size of your sample population is sufficient to detect the minor
allele of interest:
1. Find the MAF for your assay on the TaqMan® SNP Genotyping Assays page at
thermofisher.com/taqmansnp, which is frequently updated.
Alternatively, the MAF is in the Assay Information File that is distributed with
the assays. Allele frequency data can also be found using the public SNP
identifier, from public websites such as:
a. The dbSNP index at http://www.ncbi.nlm.nih.gov/SNP/index.html
b. The 1,000 Genomes project at www.1000genomes.org
2. Using the Hardy‑Weinberg Equilibrium equation, determine if the minor allele is
detectable for a sample the size of your test population (see “Example
calculation“ on page 37).
In the Hardy‑Weinberg Equilibrium equation, q2 + 2qp + p2 = 1, the expected
genotype frequencies are q2, 2qp, and p2, where q and p represent the allele
frequencies.
The values for q2, 2qp, and p2 correspond to the fraction of a population that
would be homozygous for the minor allele (qq), heterozygous (qp), and
homozygous for the major allele (pp), respectively.
3. Multiply your sample size by the fraction for each allele to determine the number
of individuals with each genotype that you expect to see.
If your sample size is small, you might not be able to detect rare alleles.

Example calculation
For an SNP with a MAF of 5% (0.05), the predicted frequencies are 0.0025 q:q, 0.095
q:p, and 0.9025 p:p.
If you test 20 genomic DNA samples from this population, you might expect:
• Homozygotes for the minor allele—0.0025 × 20 = 0.05, no individuals
• Heterozygotes—0.095 × 20 = 1.9, about 2 individuals
• Homozygotes for the major allele—0.9025 × 20 = 18.05, about 18 individuals
To detect one homozygote for the minor allele, it would take a sample size of
approximately 400 individuals (Sample Size = 1/MAF2)
Discussion
Many assays in TaqMan® SNP and DME Assays collection show low or 0 for the
minor allele frequency. Many of these SNPs are believed to be functional
polymorphisms that can occur at low frequencies, depending on the population you
are studying. The MAF indicates the frequency of the less‑frequent allele in a
population. Traditionally, only the minor allele frequency is reported. The major allele
frequency is calculated as 1 - MAF. From the MAF, you can calculate how large the
sample population should be to detect a specific allele. The lower the frequency of the
minor allele, the larger the sample size that is required to detect the allele.

Deletion alleles in When an individual does not have the gene or the portion of the gene that contains
an individual the SNP of interest, the data point in the allelic discrimination plot will either:
• Appear as a homozygote of the allele that is present (where there is one deletion
allele)
• Cluster with the NTCs (when there are two deletion alleles)
If an individual sample consistently clusters with the NTCs for a particular assay, it
may indicate the individual has a deletion allele. There are documented occurrences
of copy number variation and deletion alleles in the genes CYP2A6 , GSTM1 , GSTT1 ,
SULT1A1 and CYP2D6 in the TaqMan® DME Genotyping Assays collection.
Uncalled samples
indicated by Xs
NTCs indicated by
squares
Area of plot around
NTC, zoomed in
Figure 3 Allelic discrimination plot showing a deletion allele for the assay
C__8717770_20.
What to do
1. Evaluate the overall assay performance:
• Do the assay results appear in tight clusters?
• Do the clusters have good separation?
2. Repeat the experiment. If the same sample(s) consistently cluster with the NTC
while other samples show fluorescence, a deletion allele may be present.
3. Examine the sample's performance in other assays to rule out problems caused
by this particular sample, such as sample impurity or degradation.

4. Perform a literature search for documentation reporting the presence of deletion

alleles for the gene.
5. Perform analysis using a TaqMan® Copy Number Assay (Cat. No. 4400291) on all
samples to confirm the sample has a deletion allele to rule out assay interference
caused by a SNP present in the individual's DNA, perform comparative
sequencing on the subjects to identify any undocumented SNPs.
To locate the copy number assay of interest, visit thermofisher.com/taqmancnv.
Additional SNP A non‑target SNP under a primer or probe can result in off-cluster data. The location
present under a of the non‑target SNP under the primer or probe, as well as the MAF, influences the
extent to which the cluster pattern is atypical. The number of individuals exhibiting
probe or primer
this pattern depends on the allele frequency of the non‑target SNP. A SNP under a
probe can result in an outlier that falls between the heterozygote and one of the
homozygotes (an angle cluster), due to a lack of amplification of the sample when
there is an additional polymorphism under the primer (Figure 4). The presence of a
polymorphism under a primer generally leads to lower PCR efficiency.
and Arrows
arrow to use in this SVG.
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1
Figure 4 Additional SNP under a primer. The points in black between the green and red
clusters constitute angle clusters.
A SNP under a probe can also result in an outlier that has the same angle as a cluster
but trails behind the main cluster (a vector cluster). You may see points that are in
agreement with a cluster but trail behind the main cluster (a "vector cluster") when

there is an additional SNP under the probe (Figure 5). The presence of a SNP under a
probe leads to lower fluorescence intensity.
SNP under probe circled in red. The points trailing behind the main clusters are vector clusters.
What to do
To confirm the presence of another SNP under the probe or primer:
1. Repeat the experiment, then evaluate overall assay performance.
• Do clusters have good separation?
2. Verify the presence of the outlier.
4. Search the public databases, such as dbSNP, to see if the additional SNP has been
discovered.
5. Perform comparative sequencing on the subjects to identify any undocumented
SNPs present under the primer or probe. The presence of extra SNPs may cause
angle clusters or vector clusters.
Discussion
Our assay design process included many checks to assure that primers and probes
were not designed over significant polymorphisms other than the intended SNP
target. However, the growing number of SNPs discovered in studies of different
ethnic populations make it possible that some of the primers and/or probes in the
TaqMan® SNP and DME Genotyping Assays may overlap currently unknown
polymorphisms in certain populations. In some rare cases, there are some assays
where primers and probes are located over SNPs or other polymorphisms due to the
close proximity of the two SNPs. For these assays, the vector cluster falls in line with
samples of the same genotype, but the reduced PCR efficiency causes a reduction in
signal intensity.

Gene has a copy A copy number polymorphism for a gene may or may not appear as an anomaly in
number the allelic discrimination plot.
polymorphism • If an individual is homozygous with more than three copies of the gene and each
copy has the same genotype, the data will most likely appear in the homozygous
cluster.
• If an individual is heterozygous with an odd number of copies and the copies
have different genotypes, then the data will probably fall between the clusters for
the heterozygote (T:A) and the homozygote (A:A).
Several of the genes included in the TaqMan® Genotyping and DME Assays are
known to have copy number polymorphisms: CYP2D6, GSTM1, GSTT, CYP2E1, and
CYP2A6.
Figure 5 Allelic discrimination plot for assay to CYP2D6*2A -1584C>G (C__32407252_30),

showing samples with a copy number polymorphism.
What to do
1. Evaluate overall assay performance
• Do clusters have good separation?
2. Repeat the experiment to confirm the presence of the off-cluster sample.
4. Perform a literature search for documentation of copy number polymorphisms
for the gene.
5. Perform comparative sequencing on the subjects to identify any undocumented
SNPs present under the primer or probe; extra SNPs may cause angle clusters.

6. Perform analysis using a TaqMan® Copy Number Assay (Cat. No. 4400291) on all
samples to determine the copy number for the gene in which the polymorphism
resides.
• The Copy Number Assay of interest can be found at thermofisher.com/
taqmancnv.
• A Custom TaqMan® Copy Number Assay can be designed if a pre‑designed
TaqMan® Copy Number Assay does not exist for a target of interest.
• A TaqMan® Copy Number Assay must be run in duplex with a reference
assay. Reference assays are commercially available for human and mouse
analysis. If working with other species, you need to identify and use your
own reference assay target.
Discussion
Data points for samples from homozygous individuals with extra copies of a gene
will generally cluster with the homozygous cluster. Data points for heterozygous
individuals with copy number polymorphisms may appear as outliers such as a
fourth or fifth cluster between the heterozygote cluster and one of the homozygous
clusters. Since a copy number variation may not present itself in all individuals, a
gene dosage assay should be performed on all samples to determine which
individuals carry extra copies of the gene.
SNP is triallelic When an SNP is triallelic, you may see outlier samples in the allelic discrimination
plot, although the samples may not be well separated from the main clusters. These
situations are best confirmed by running replicate plates. We provide pairs of
TaqMan® SNP Genotyping Assays to several important DME and CFTR gene variants
that are triallelic (see the Pharmacogenomics Experiments Application Guide (Pub. No.
MAN0009612) and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)
Genotyping Experiments User Guide (Pub. No. MAN0014405).
What to do
1. Evaluate overall assay performance: Are there consistent outlier samples?
2. Examine the sample performance in other assays to rule out problems that are
caused by this particular sample, such as sample impurity or degradation.
3. Perform comparative sequencing on the subjects to verify the presence of more
than two alleles.
4. Repeat the experiment. If the same samples are consistently in the same outlier
space (away from the NTCs, the heterozygotes, and the homozygotes) your gene
may be triallelic.
5. Check the literature for the SNP in question. There may be newly reported
polymorphisms that are described in the literature. Calculate the allele
frequencies for your plate and compare them to the literature to confirm your
results agree with the literature.

Discussion
If an SNP is triallelic, you might see six clusters (three homozygotes and three
heterozygotes) instead of the typical pattern of three clusters (two homozygotes and
one heterozygote). Figure 6 and Table 18 show the possible genotypes that are
associated with SNP rs2032582 in the ABCB1 gene that is known to have three alleles
in several populations. Note that the alleles reported by the ABCB1 SNP assays are
given in the (+) strand genome orientation whereas the ABCB1 gene maps to the (−)
genome strand. Thus, the reported SNP assay alleles and the SNP cDNA annotations
are the reverse complement of one another.
Figure 6 Allelic discrimination plot for triallelic SNP rs2032582. (Left) ABCB1 c.3095G>A assay (C_11711720D_40) to
the plus strand C wild type and T mutant allele. (Right) ABCB1 c.3095G>T assay ( C_11711720C_30 ) to the C wild type
and A mutant allele.
Table 18 Translation table for the ABCB1 c.3095G>T/A triallelic SNP rs2032582
assays
C/T assay C/A assay

ABCB1 c.3095G>T/A
C_11711720D_40 C_11711720C_30
G/G C/C C/C
G/A C/T C/C
A/A T/T noamp
G/T C/C C/A
T/A T/T A/A
T/T noamp A/A

Gene on the X When a gene is on the X chromosome and the population being studied is made up of
chromosome both males and females, the genotype frequencies of the samples do not correspond to
the predicted autosomal Hardy‑Weinberg frequencies. For a sample population
composed of a mixture of males and females, the number of heterozygotes will be
noticeably lower than predicted by the Hardy‑Weinberg equilibrium equation. None
of the males should be heterozygous because males have only one X chromosome.
Figure 7 Allelic discrimination plot with a small number of heterozygotes for a gene on
the X chromosome
What to do
1. Check the AIF included with the assay or the TaqMan® SNP or DME Genotyping
Assays pages (thermofisher.com/taqmansnp or thermofisher.com/taqmandme)
to determine if the assay is for a target located on the X chromosome.
2. Check your results by gender.
Discussion
When a SNP is located on the X chromosome, only the females in the population can
be heterozygous. Males, with only one X chromosome, will always be homozygous.
Depending upon the minor allele frequency, you may see males in only one of the two
homozygous forms.

Troubleshooting the AD plot: sample preparation A
Gene on the Y When a gene is on the Y chromosome and the population is made up of both males
chromosome and females, female samples will appear with the NTC samples. This result does not
reflect assay performance but is due to the fact that female samples will not amplify.
As result, only two clusters will be visible because males have only one Y
chromosome. In Figure 8, female samples are yellow.
Figure 8 Allelic discrimination for a gene on the Y chromosome
Troubleshooting the AD plot: sample preparation

Unexpected distributions in allelic discrimination plots can result from sample
preparation errors, including:
• Degraded DNA (page 45)
• PCR inhibitors in sample (page 47)
• DNA quantification (page 61)
Degraded DNA Degraded DNA can affect PCR efficiency due to the presence of fewer template
copies. Degradation can result from:
• Using very old DNA samples
• Using DNA extracted from formalin-fixed paraffin embedded samples
• Freezing and thawing DNA samples repeatedly
• Leaving DNA samples at room temperature
• Exposing DNA samples to heat, physical shearing, or UV light in ambient
lighting
• Purifying DNA samples inefficiently so residual nucleases remain

Troubleshooting the AD plot: sample preparation
What to do
• Run an agarose gel to determine if your DNA is degraded. Look for a tight band
of high molecular weight. Smearing indicates degraded DNA. Figure 9 illustrates
DNA degraded by heat.
30 seconds
10 minutes
15 minutes
30 minutes
Size stnd.
5 minutes
3 minutes
0 second
1 minute Sample A
Sample B
Figure 9 Agarose gel stained with ethidium bromide showing two samples of human
gDNA subjected to heating at 99°C for 0 to 30 minutes.
• If the DNA is substantially degraded, use more caution in interpreting your
results. If possible, consider repeating the assay using freshly prepared genomic
DNA samples.
• For future experiments, follow the sample storage guidelines in Table 19.
Discussion
Because the average size of the DNA in a degraded sample approaches the size of the
target sequence, the amount of PCR product generated is reduced because there are
fewer intact templates in the size range necessary for amplification.
Table 19 Recommendations for sample storage conditions to minimize DNA

degradation.
Tissue type Storage conditions
Buccal tissue Store between –15°C and –25°C
Immediately place tissue in liquid nitrogen and store at –80°C

Tissue or
Freeze and store between –15° and –25°C
• Whole blood: store between –15°C and –25°C
Blood • Buffy coat: store between –15°C and –25°C and thaw at room
temperature before use

Troubleshooting the AD plot: assay preparation A
PCR inhibitors in Potential PCR inhibitors can originate from the tissue source of the DNA sample or
sample from the purification method. Inhibitors originating from the cell include heparin,
proteins, and heme. Inhibitors originating from DNA preparation include phenol,
proteases, detergents (SDS), and salts (EDTA and citrate).
The presence of polymerase inhibitors can decrease PCR efficiency, leading to:
• Trailing clusters
• Nonamplification such that some (or all) samples cluster with the NTCs
What to do
• Dilute the sample and run the assay with the diluted sample. If the inhibition
decreases, then it is likely there are PCR inhibitors in the sample.
• Repurify the sample and run the assay again.
• Consider using a master mix more tolerant to inhibitors such as TaqPath™
ProAmp™ Master Mix.
Discussion
Inhibition of PCR is always possible when DNA is extracted from tissue and/or blood
samples.
Because DNA purification method that is used to prepare gDNA can affect the
success of PCR, the selected method must minimize degradation and remove
inhibitors. One method for evaluating DNA purity is to calculate the A260 /A280 ratio.
In addition, absorbance at 230 nm can indicate the presence of phenol. See “UV
spectroscopy“ on page 62 for more information.
Troubleshooting the AD plot: assay preparation

Unexpected distributions in allelic discrimination plots can result from assay
preparation errors, including:
• Reagents mishandled or expired (page 47)
• Using a master mix without ROX™ dye (page 48)
• DNA or assay reagent not added to the reaction well (page 48)
• Insufficient DNA added to the reaction well (page 48)
• Evaporation from the reaction well (page 49)
• Pipetting errors (page 49)
• Inefficient mixing or centrifugation (page 50)
• Assay has high background fluorescence (page 50)
• More than one sample in the well (page 50)
Reagents The use of mishandled or expired reagents may result in:

mishandled or • Some or all samples clustering with the NTCs
expired • Trailing clusters
• Weak overall reaction (weak signals)
What to do
Perform the assay again with newly prepared reagents. Follow the Assay and Master
Mix Considerations for reagent storage and handling.

Assay consideration
• Store TaqMan® Assays between –15 and –25°C when they are not in use.
• Minimize freeze‑thaw cycles to no more than ten cycles. Too many freeze thaw
cycles can cause cleavage of the dye from the probe.
• Limit the assay exposure to light. The fluorescent dyes are susceptible to photo‑
bleaching. Photo‑bleaching can result in a lower overall signal for the reaction.
TaqMan® Genotyping Master Mix considerations
• Store TaqMan® Genotyping Master Mix at 2–8°C.
• Make sure the master mix is thoroughly mixed prior to use.
Using a master The use of a PCR Master Mix that does not contain ROX™ dye (or a similar passive
mix without ROX™ reference) can cause:
dye • Trailing clusters
• Undetermined data (an "X" instead of a called allele in the AD plot)
• Diffuse clusters
What to do
Use a TaqMan® Genotyping Master Mix which includes ROX™ dye.
Discussion
ROX™ dye is a passive reference dye that improves the precision of the results by
compensating for small fluorescent fluctuations, such as bubbles and small well‑to‑
well variations.
Our analysis software for allele discrimination and genotyping experiments will not
call the alleles when ROX™ dye (or another passive reference) is not present.
See “ROX™ dye not designated as a passive reference“ on page 55 for more
information.
DNA or assay When gDNA or one of the assay reagents is not added to the reaction well, no PCR
reagent not added amplification takes place and the sample clusters with the NTCs.
to the reaction What to do
well Perform the assay again, making sure to:
• Follow the TaqMan® SNP Genotyping Assay protocol exactly.
• Pipette carefully.
• Mix thoroughly.
Insufficient DNA When insufficient gDNA is added to the reaction well, no PCR amplification takes
added to the place and the sample clusters with the NTCs.
reaction well What to do
Perform the assay again, making sure to:
• Quantitate your DNA accurately (see “Quantify the sample gDNA“ on page 18).
• Follow the TaqMan® SNP Assays protocol as recommended, adding 1–20 ng of
purified genomic DNA (ensuring final concentration no less than 0.2 ng/µL).
• Pipette carefully.
• Mix thoroughly.

Troubleshooting the AD plot: assay preparation A
Evaporation from Evaporation of your reaction can occur if the reaction plates are not properly sealed,
the reaction well leading to:
• Outliers (mild/moderate evaporation)
• Trailing clusters (moderate evaporation)
• Samples clustering at the NTC (extreme evaporation)
What to do
1. Check the location of the wells for the problem calls. Evaporation can most often
occur around the edges of the plate.
2. Check the seals of the optical adhesive cover for leaks.
3. If there are leaks, perform the assay again.
Use a MicroAmp™ Adhesive Film Applicator (Cat. No. 4333183) to thoroughly seal
the cover. Make sure to run the applicator over the edges of the seal.
Discussion
Evaporation can occur if your plate is not properly sealed. As evaporation occurs, the
water in the reaction decreases, causing the signals from the reporter and ROX™ dyes
to increase due to increased concentration of the dyes. The degree of evaporation
influences the assay results:
• Mild — If the PCR reaction is not affected, the ROX dye can compensate for the
increased signals and the assay will work correctly.
• Mild to moderate — You may see outlier samples. Depending on the number of
wells affected, the plot may show only a few outliers or it may show a trailing
cluster.
• Extreme evaporation occurring early in the reaction — The PCR reaction fails and
the samples cluster with the NTC.
Pipetting errors Pipetting errors can cause inconsistent delivery of reagents or sample to the wells,
which can cause:
• Some (or all) samples clustering with the NTCs
• Cloudy or diffuse clusters
• One or more samples that do not cluster with a specific allele (i.e. an outlier)
What to do
• Improve pipetting precision, using the following techniques:
– Calibrate and clean the pipettors regularly.
– Pipette larger volumes (no less than 5 µL) for greater accuracy and precision.
– Reduce the number of pipetting steps whenever possible.
– Increase the consistency of the pipetting method (such as using robotic
pipetting).
– See the manufacturer instructions for the correct method of dispensing liquid
volumes accurately from the pipettor. For example, some pipettors are
designed to deliver the designated volume at the first plunger stop, so
blowing out the remaining volume can cause errors.
• Check your pipetting process by preparing a replicate plate (same assay and
sample over a plate) to be sure that results are reproducible.

Inefficient mixing Insufficient mixing or centrifugation can cause trailing clusters in the AD plot.
or centrifugation What to do
Rerun the assay, mixing the samples well (by pipetting up and down a few times) and
performing the centrifugation steps as described in the protocol. Centrifuging the
samples ensures that the contents of the sample well are pooled at the bottom of the
well, allowing for the most efficient PCR reaction and the most accurate endpoint
read.
Assay has high Some assays have higher levels of background fluorescence than others which can
background cause:
fluorescence • The position of the NTCs moving away from the origin of the allelic
discrimination plot
• The position of the homozygous cluster moving towards the heterozygous
clusters
What to do
If the clusters are well separated from each other, TaqMan® Genotyper Software or the
Thermo Fisher Cloud Genotyping Application can autocall the clusters. You can also
manually call the clusters.
More than one Sometimes samples are inadvertently mixed together due to poor lab technique,
sample in the well resulting in an outlier.
Figure 10 Allelic discrimination plot with different samples in the same well (outlier
sample circled in red)

Troubleshooting the AD plot: instrument set-up and maintenance A
What to do
Perform the assay again making sure that two samples are not combined in the same
wells.
More than one Sometimes assays are inadvertently mixed together due to poor lab technique,
assay in the well resulting in more than 3 clusters in a plot.
ws
n this SVG.
outs or arrows
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ded.
used
ts
ry.
Figure 11 Allelic discrimination plot with different assays in the same well
What to do
Perform the assay again making sure that two assays are not combined in the same
wells.
Troubleshooting the AD plot: instrument set-up and maintenance

For best results, make sure to calibrate and maintain your instrument as
recommended.
Improper instrument set‑up and maintenance can result in unexpected distributions
in allelic discrimination plots, including:
• Some or all samples cluster with the NTCs
• High signal for the NTCs in one or more of the sample wells
Routine To ensure optimal performance of your thermal cycler or real‑time PCR system, we
instrument strongly recommend that you perform routine maintenance. Maintenance schedules
vary by instrument; refer to your relevant instrument manual for details.
maintenance

Troubleshooting the AD plot: instrument set-up and maintenance
Instrument We recommend that you regularly calibrate your thermal cycler or real‑time PCR
calibration system to ensure optimal performance. Calibration varies by instrument. See your
specific instrument user guide for details.

Troubleshooting the AD plot: data analysis (software) A
Types of
calibration ROI calibration
ROI calibration allows the Real Time PCR Instrument Software to map the position of
the wells on the sample block so that, during instrument operation, the software can
associate increases in the fluorescence with specific wells of the reaction plates.
Background calibration
The background calibration measures the ambient fluorescence that is generated from
background electrical signals, sample blocks, water inside consumables, and from the
consumables themselves. This calibration enables the Real Time PCR Instrument
Software to eliminate background signal from the fluorescent samples, thus increasing
the precision of the instrument.
Pure dye spectra calibration

The pure dye spectra calibration enables the instrument to distinguish the fluorescent
dyes being used in the system. The Real Time PCR Instrument Software uses the
spectral data from a set of pure dye standards to process the raw spectral data it
receives after each run.
Instrument verification run

The test verifies that the instrument can generate a standard curve and its ability to
calculate the quantities of two unknowns. This test requires an RNase P Verification
Plate that contains pre‑loaded reagents that create a standard curve with known copy
numbers and two unknowns (also with known copy numbers).
Troubleshooting the AD plot: data analysis (software)

For complete instructions for setting up, running and analyzing TaqMan® SNP
Genotyping Assays experiments, see the user manual for your instrument or software.
Unexpected distributions in allelic discrimination plots can result from data analysis
errors, including:
• “Empty allelic discrimination plots“ on page 53
• “No alleles called in the AD plot“ on page 54
• “Homozygous allele frequencies reversed“ on page 57
• “Too many alleles called in AD plot“ on page 58
Empty allelic Incorrectly creating and/or selecting the detector and marker can result in an empty
discrimination allelic discrimination plot even when the assay chemistry is successful.
plots When the allelic discrimination plot is empty, causes include:
• “No assay or marker assigned to the well“ on page 54
• “Omit option checked in Real Time PCR instrument software“ on page 54
• “ROX™ dye not designated as a passive reference“ on page 55

No assay or marker assigned to the well

An assay or a marker must be assigned to each well before the Real Time PCR
Instrument Software, the TaqMan® Genotyper Software, or the Thermo Fisher Cloud
Genotyping Application can analyze the plate and obtain results.
What to do
If you have a post‑read plate that appears to have no data:
1. Check to see if an assay or a marker is assigned.
2. If no assay or marker is assigned, assign one and reanalyze the data.
Omit option checked in Real Time PCR instrument software

If there is a red X in the plate document for a well, the Omit Well option may have
been checked for this well.
The Omit Well option removes the selected well from the analysis. Consult your
instrument user manual for information on how to check and uncheck Omit well
options.
No alleles called To assist with cluster calling, the Real Time PCR Instrument Software can autocall the
in the AD plot data. When autocalling fails, you will see "X" on the plot rather than the symbol for
called alleles. There are some instances where the software will not autocall the data:
• “Autocall is not selected“ on page 54
• “Two cluster calling not selected“ on page 54
• “Single cluster assay“ on page 55
• “ROX™ dye not designated as a passive reference“ on page 55
• “Outlier too far off scale“ on page 56
• “NTCs not assigned“ on page 57
Autocall is not selected

If the Autocall is not enabled in the software analysis settings, the Allelic
Discrimination Plot displays an X (Undetermined) for each sample. Default analysis
settings vary depending on your real‑time PCR system. See the instrument User
Guide for more information on how to use autocall options.
Two cluster calling not selected

On an instrument other than a QuantStudio™ instrument, the 2 cluster calling option
in the Real Time PCR Instrument Software must be selected if the software is to
successfully autocall plates when only two clusters are detected. Detection of only two
clusters can happen if the MAF is low and/or you ran too few samples to detect all
three genotypes. If this is the case, you must select the two cluster calling option and
re‑analyze the plate.

Single cluster assay

The Real Time PCR Instrument Software cannot autocall single cluster assays. For
many assay/sample combinations, a single cluster assay is the correct result. For
example, a single cluster assay can be correct for a SNP with a very low MAF, such as
many of the TaqMan® DME Genotyping Assays (see “Low allele frequency“ on
page 36).
Use TaqMan® Genotyper Software or theThermo Fisher Cloud Genotyping
Application to assign genotype calls to single clusters. However, if these applications
do not assign a genotype to the cluster and if you believe that the single cluster is
correct, manually call the alleles. You may need to rescale the plot before you can call
the alleles accurately. For complete instructions for plot rescaling, see the user manual
of your instrument or software.
Figure 12 shows an example of a single cluster plot before and after rescaling.
Single cluster before rescaling Single cluster after rescaling
Figure 12 Allelic discrimination plot before and after rescaling.
ROX™ dye not designated as a passive reference

When ROX™ dye is not designated as a passive reference, some of the Real Time PCR
Instruments will not autocall the alleles while others may exhibit trailing clusters in
the allelic discrimination plots. ROX™ dye is a passive reference that improves the
precision of the results by compensating for small fluorescent fluctuations, such as
bubbles and small well‑to‑well variations that occur in the plate. We recommend
TaqMan® Genotyper Software or the Thermo Fisher Cloud Genotyping Application to
assign genotype calls when no passive reference is used. We also recommend using
TaqMan® Genotyping Master Mix that contains ROX™ as a passive reference dye to

normalize for well‑to‑well variations in volume. Consult your specific instrument

manual for more information on ROX™ designation for genotyping applications.
Outlier too far off scale

If you have an assay that shows clustering around the NTCs, you may want to look
for data from an outlier sample. In some cases, the software scales to include the
outlier giving the other samples the appearance of clustering around the NTC. If you
remove the outlier from the analysis, the software rescales the data and the analysis
can proceed.
For more details on how to remove outlier, refer to the user manual of your
instrument or software.
Outlier circled in red Data reanalyzed with outlier omitted
Figure 13 Data analyzed with and without outlier included.

What to do
Remove the outlier(s) using the omit well function in your Real Time PCR Instrument
software and re‑analyze. The program will adjust the scaling.

NTCs not assigned

If the wells containing the NTCs are not assigned with the NTC task in the software,
the software may not call the alleles.
Without NTCs labeled With NTCs assigned
Figure 14 Data analyzed with and without the NTCs assigned.

What to do
In some instances where the data is diffuse and software does not autocall, labeling
the NTC wells with the NTC task provides a point of reference for the software,
improving clustering and autocalling.
Assign the NTC task to the NTC wells in the plate and re‑analyze the data. Refer to
the instruction manual appropriate for your software.
Note: NTCs are not required for autocalling.
Homozygous Your observed major and minor allele frequencies for homozygotes are reversed from
allele frequencies those predicted by the Hardy‑Weinberg Equilibrium equation. For example, for a SNP
with a MAF of 5% (0.05), the predicted frequencies are 0.0025 q:q, 0.095 q:p, and
reversed
0.9025 p:p. If the allele frequencies are reversed, you see 0.9025 q:q, 0.095 q:p, and
0.0025 p:p.
Reporter dye assigned incorrectly to the allele in the detector
In the software, you must set up two detectors and one marker in order for the alleles
to be called. The detector defines which dye is assigned to the allele. If you
inadvertently assigned the dyes to the wrong alleles when you created the detectors,
the observed frequencies will be the reverse of those predicted from the Hardy‑
Weinberg equation.
Note: The AIF included with your order contains the correct allele‑dye assignments.

Too many alleles In the software, you must set up one marker for each assay run on the plate. Running
called in AD plot more than one assay per marker can result in more than three clusters in the allelic
discrimination plot.
The same markers and detectors are assigned to two The same markers and detectors are assigned to one of
assays the two assays
Figure 15 Allelic discrimination plots showing two assays assigned to one detector and
marker.
What to do
1. Create markers and detectors for each assay on the plate. Refer to the instructions
in your instrument manual appropriate for your software.
2. Assign each marker to the correct assays.
3. Re‑analyze your data.
Multiple assays may be run on a single plate, however it is essential that each assay is
assigned its own marker. Each assay has its own unique run characteristics. Running
two assays with the same marker name may result in genotyping miscalls and the
appearance of assay failure.

B Supplemental information
TaqMan® SNP Genotyping Assays chemistry overview

TaqMan® MGB and TaqMan® MGB probes consist of target-specific oligonucleotides with:
QSY™ probes • A reporter dye at the 5´ end of the probe:
– VIC™ dye for Allele 1 probe
– FAM™ dye for Allele 2 probe
• A non-fluorescent quencher (NFQ) dye at the 3´ end of the probe.
• A minor groove binder (MGB) at the 3´ end of the probe that:
– Increases the melting temperature (Tm) without increasing probe length.
– Allows for the design of shorter probes.
TaqMan® QSY™ probes consist of target-specific oligonucleotides with:
• A reporter dye at the 5´ end of the probe.
• A QSY™ quencher at the 3´ end of the probe
Two TaqMan® QSY™ probes can be used in multiplex with two MGB probes to
analyze two SNPs per well or more than two alleles per well.

ements
wository.
to use in this SVG.
anced callouts or arrows

B
e_Callout&Arrow_Libary.
1
Appendix B Supplemental information
TaqMan® SNP Genotyping Assays chemistry overview
th, as needed. 1
About the 5' Note: The following figures are general representations of PCR with TaqMan® MGB
e, and unused
nuclease assay probes and TaqMan® SNP Genotyping Assays. The sequence regions are not
G elements necessarily drawn to scale. The principals of the assay are the same for TaqMan®
e repository.
QSY™ probes.
The 5' nuclease assay process takes place during PCR amplification. It occurs in every
1 cycle and does not interfere with the exponential accumulation of product.
1 For the PCR amplification, genomic DNA is introduced into a reaction mixture
consisting of master mix, forward and reverse primers, and two TaqMan® probes
Figure 17.
Minor Groove
V VIC™ dye MGB Binder Probe
AmpliTaq Gold™ Primer

F FAM™ dye DNA Polymerase
Quencher
Q (NFQ or QSY™) DNA Template Extended primer
Figure 16 Legend
B B
MG MG
Forward primer Probe 1 Probe 2 Reverse primer
V Q F Q
A G
and Arrows DNA template 3'

5' [G/A]
rrow to use in this SVG.
3' [C/T] 5'
dvanced callouts or arrows
cape_Callout&Arrow_Libary.
Figure 17 TaqMan® SNP Assay components and DNA template

ength, as needed.
During the PCR, the forward and reverse primers anneal to complementary sequences
along the denatured cDNA template strands (Figure 18). The TaqMan® probe anneals
ngle, and unused
specifically to a complementary sequence between the forward and reverse primer
SVG elements
o the repository. sites. When the probe is intact, the proximity of the reporter dye to the quencher dye
results in suppression of the reporter fluorescence primarily by Förster‑type energy
transfer.
1
1 5' 3'
B B
MG Probe 2 MG Reverse primer
Probe 1
V Q F Q
Forward primer A G
[C/T]
3' 5'
Figure 18 Primers and probes anneal to the denatured DNA template

During polymerization, the DNA polymerase only cleaves probes that hybridize to
the target sequence. Cleavage separates the reporter dye from the probe. The
separation of the reporter dye from the quencher dye results in increased fluorescence
by the reporter (Figure 19). The increase in fluorescence occurs only if the target
sequence is complementary to the probe and amplified during PCR. Because of these

her SVG elements
G to the repository.
1 1
DNA quantification B
1
requirements, nonspecific amplification is not detected, and the fluorescence signal
indicates which alleles are in the sample (Table 20).
B 5' 3'
Probe 1 MG
F Q
Reverse primer
A
Forward primer V Probe 2

G MGB
Q
C
Match
Figure 19 Polymerization and signal generation
Table 20 Correlation between fluorescence signals and sample sequence.
Fluorescence signals Sample genotype
VIC™ signal Homozygosity for Allele 1
FAM™ signal Homozygosity for Allele 2
VIC™ and FAM™ signals Heterozygosity Allele 1-Allele 2
Mismatches between probes and target sequences

The probes in SNP Genotyping Assays are designed so their hybridization to off-
target template is highly reduced by even single nucleotide mismatches between a
probe and the target sequence. Poor probe hybridization reduces the amount of
reporter dye cleaved from the quenched probe. Furthermore, AmpliTaq Gold™ DNA
Polymerase is more likely to displace a mismatched probe without cleaving it. Each of
these factors minimizes the production of nonspecific fluorescence signals from off
target templates.
DNA quantification
In an assay and/or study, gDNA concentration uniformity leads to accurate, robust,
and reproducible results and ensures efficient use of valuable samples. Precise
handling and quantitative measurements before running an assay can prevent errors
without waste of reagents and samples.
Variability in gDNA concentrations in TaqMan® SNP Genotyping Assays can lead to
experimental anomalies that can affect interpretation of genotyping results, including:
• Some (or all) samples clustering with the NTCs
• A sample (or samples) does not cluster with a specific allele (i.e. is an outlier)

B Appendix B Supplemental information
DNA quantification
Figure 20 shows how variability in gDNA can affect interpretation of genotyping

results.
127 ng
10 ng
5 ng
135 ng
5 ng
NTC 0.5 ng
All samples have 3 ng gDNA Samples with gDNA ranging from 0.5 to 127 ng
®
Figure 20 Allelic discrimination plots for TaqMan DME Genotyping Assay
C__1204092_20.
To ensure accurate results:
• Always perform your own concentration measurements before using any
genomic DNA (gDNA), even commercially prepared DNA.
• Use the recommended amount of gDNA, 1–20 ng per sample per assay.
• Always use the same quantity of gDNA for all samples of an assay on a plate.
There are numerous methods for quantitating genomic DNA, including:
• UV spectroscopy
• Absolute quantification
– TaqMan® RNase P method
– SYBR™ Green assay
• Fluorometric analysis
We recommend quantifying gDNA using UV spectroscopy or absolute quantification
using the TaqMan® RNase P method.
UV spectroscopy UV spectroscopy can be used to quantitate gDNA by reading sample absorbance at

260 nm (A260). The A260 is most accurate when using pure nucleic acid and is most
useful for DNA in microgram to nanogram quantities. Proteins, particles in the
solution, and aromatic chemicals can affect the reading. Samples are usually
concurrently read at 280 nm, to determine the concentration of contaminating
proteins. The A260 /A280 ratio is used to determine purity of a DNA sample (see “PCR
inhibitors in sample“ on page 47).
The effective read range of UV spectroscopy is 0.1 to 0.999, which corresponds
approximately to 4 ng/µL to 50 ng/µL of gDNA. Values above or below are outside
the linear range for concentration determination. To ensure accurate quantitative
results, gDNA samples should be diluted so that the A260 reading is between 0.1 and
0.999.

Good laboratory practices for PCR and RT-PCR B
Absolute Absolute quantification measures the total amount of amplifiable gDNA. This
quantification technique requires the creation of a standard curve using gDNA samples of known
quantities. The standard samples must be pre-quantified and confirmed using an
independent method such as spectrophotometry or fluorometry. The unknown
samples are compared to the known samples for quantification.
Absolute
quantification Description
method
TaqMan® RNase P TaqMan® RNase P is a highly accurate 5' nuclease assay that
detects and quantifies genomic copies of the human RNase P
gene. TaqMan® DNA Template Reagents (Cat. No. 401970) and
TaqMan® RNase P Detection Reagents Kit (Cat. No. 4316831) allow
for convenient means to quantify gDNA. See RNase P
Quantification for Genotyping Experiments
(Pub. No. MAN0014349 ) for more information.
SYBR™ Green assay SYBR™ Green dye binds to double-stranded DNA (dsDNA).
Quantification with theSYBR™ Green assay is less specific than
TaqMan® RNase P because the dye binds to any dsDNA whereas
TaqMan® reagent chemistry targets a specific DNA sequence. The
SYBR™ Green method requires melt curve analysis to ensure the
specificity of the assay.
For either technique, run the standard curve and unknown samples on the same
plates in the real‑time PCR instrument.
Fluorometric You can quantify DNA by fluorometric analysis using the Qubit™ 3.0 Fluorometer or
analysis various intercalating dyes.
Good laboratory practices for PCR and RT-PCR

When preparing samples for PCR or RT‑PCR amplification:
• Wear clean gloves and a clean lab coat.
– Do not wear the same gloves and lab coat that you have previously used
when handling amplified products or preparing samples.
• Change gloves if you suspect that they are contaminated.
• Maintain separate areas and dedicated equipment and supplies for:
– Sample preparation and reaction setup.
– Amplification and analysis of products.
• Do not bring amplified products into the reaction setup area.
• Open and close all sample tubes carefully. Avoid splashing or spraying samples.
• Keep reactions and components capped as much as possible.
• Use a positive‑displacement pipettor or aerosol‑resistant barrier pipette tips.
• Clean lab benches and equipment periodically with 10% bleach solution or
DNAZap™ Solutions (Cat. No. AM9890).

C Safety
WARNING! GENERAL SAFETY. Using this product in a manner not specified

in the user documentation may result in personal injury or damage to the
instrument or device. Ensure that anyone using this product has received
instructions in general safety practices for laboratories and the safety
information provided in this document.
· Before using an instrument or device, read and understand the safety
information provided in the user documentation provided by the
manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data
Sheets (SDSs) and use appropriate personal protective equipment (gloves,
gowns, eye protection, etc). To obtain SDSs, see the “Documentation and
Support” section in this document.

Appendix C Safety
Chemical safety C
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,
ensure laboratory personnel read and practice the general safety guidelines for
chemical usage, storage, and waste provided below. Consult the relevant SDS
for specific precautions and instructions:
· Read and understand the Safety Data Sheets (SDSs) provided by the
chemical manufacturer before you store, handle, or work with any chemicals
or hazardous materials. To obtain SDSs, see the “Documentation and
Support” section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing).
· Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).
· Check regularly for chemical leaks or spills. If a leak or spill occurs, follow
the manufacturer's cleanup procedures as recommended in the SDS.
· Handle chemical wastes in a fume hood.
· Ensure use of primary and secondary waste containers. (A primary waste
container holds the immediate waste. A secondary container contains spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container storage.)
· After emptying a waste container, seal it with the cap provided.
· Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
· Ensure that the waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.
· IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.

C Appendix C Safety
Biological hazard safety
Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Conduct all work in properly equipped facilities
with the appropriate safety equipment (for example, physical containment
devices). Safety equipment can also include items for personal protection, such
as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety
glasses, or goggles. Individuals should be trained according to applicable
regulatory and company/ institution requirements before working with
potentially biohazardous materials. Follow all applicable local, state/provincial,
and/or national regulations. The following references provide general
guidelines when handling biological samples in laboratory environment.
· U.S. Department of Health and Human Services, Biosafety in Microbiological
and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)
21‑1112, Revised December 2009; found at:
www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,
WHO/CDS/CSR/LYO/2004.11; found at:
www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

D Documentation and support
Related documentation
PDF versions of this guide and the documents listed in this section are available at
thermofisher.com/support.
Note: To open the user documentation, use the Adobe™ Reader™ software available
from www.adobe.com.
Table 21 Product web pages
Product Web page
TaqMan® SNP Genotyping Assays thermofisher.com/taqmansnp
TaqMan® Drug Metabolism Genotyping

thermofisher.com/taqmandme
Assays
Custom TaqMan® SNP Genotyping Assays thermofisher.com/taqmancustomsnp
Table 22 Product documentation
Document Pub. No.
TaqMan® Genotyping Master Mix Protocol 4371131
TaqMan® Genotyping Master Mix Quick Reference 4371130
Custom TaqMan® Assays Design and Ordering Guide 4367671
Pharmacogenomics Experiments Application Guide MAN0009612
Cystic Fibrosis Transmembrane Conductance Regulator

MAN0014405
(CFTR) Genotyping Experiments User Guide
TaqMan® Genotyper Software Getting Started Guide 4448637
TaqMan® Genotyper Software Quick Reference 4448638
Custom Primers and TaqMan® Probes shipped at ambient

temperature reduce environment impact and retain their 090071
quality and stability (White Paper)

D Appendix D Documentation and support
TaqMan® Assays qPCR Guarantee
Table 23 Instrument documentation
Document Pub. No.
Applied Biosystems™ 7300/7500/7500 Fast Real‐Time PCR

System Getting Started Guide: Allelic Discrimination 4347822
Assays
Applied Biosystems™ ViiA™ 7 Real-Time PCR System

4441434
Getting Started Guide
Applied Biosystems™ ViiA™ 7 Real-Time PCR System User

4442661
Guide: Calibration, Maintenance, Networking, and Security
Applied Biosystems™ StepOne™ and StepOnePlus™ Real-

Time PCR Systems Genotyping Experiments Getting 4376786
Started Guide
QuantStudio™ 12K Flex Real-Time PCR System:

4470935
OpenArray™ Experiments User Guide
QuantStudio™ 12K Flex Real-Time PCR System: Multi‐Well

4470050
Plates and Array Card Experiments User Guide
QuantStudio™ 6 and 7 Flex Real-Time PCR System

4489822
Software Getting Started Guide
Applied Biosystems™ 7900HT Fast Real-Time PCR System

4364015
Allelic Discrimination Getting Started Guide
QuantStudio™ 3 and 5 Real-Time PCR Systems Installation,

MAN0010407
Use, and Maintenance Guide
TaqMan® Assays qPCR Guarantee

Only the following assays are covered by the TaqMan® Assay qPCR Guarantee:
predesigned TaqMan® SNP Genotyping Assays and TaqMan® Drug Metabolism
Genotyping Assays. Without limiting the foregoing, custom SNP Assays are expressly
excluded from the TaqMan® Assay qPCR Guarantee. Visit thermofisher.com/
taqmanguarantee for detailed information about the TaqMan Assay qPCR Guarantee.
Customer and technical support

Visit thermofisher.com/support for the latest in services and support, including:
• Worldwide contact telephone numbers
• Product support, including:
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support

Appendix D Documentation and support
Limited product warranty D
• Product documentation, including:

– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.
Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies' website at www.thermofisher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please contact Life
Technologies at www.thermofisher.com/support.

Index
5' nuclease assay 6 F

FAM dye 6, 25, 59
A Fluorometric analysis 45
Functionally Tested assay 8
Absolute quantitation 45
ABY dye 9, 25
Allele G
Automatic call 23–25 Genotyper Software 23, 24
Hetrozygote 26
Homozygote 26
Manual call 23–25 H
Allelic discrimination Hardy‑Weinberg, Equilibrium equation 53
Plate read 23
Plot 23, 26, 29
Assay database 10 J
Assay Information File (AIF) 7, 12, 24 JUN dye 9, 25
Autocalling 53
L
B
limited product warranty 69
biohazard safety 66
M
C
Minor‑groove binder (MGB), Probe 6
Chromosome Minor‑groove binding (MGB), Probe 59
X 35
Y 35
Cluster N
Diffuse 47, 51 Nonfluorescent quencher (NFQ) 6
Trailing 47, 51
Context sequence 7
Custom Assay Design Tool (CADT) 9, 11 O
Outliers 47, 53
D
DNA P
Degradation 45 PCR inhibition 45
Quantitation 45 Pre‑PCR plate read 22
DNA delivery Probe hybridization 59
Dried‑down DNA 19
Wet DNA 19
documentation, related 67 Q
Drug Metabolism (DME) Index 11 QSY quencher 9
Qualified assay 8

Index
R U
related documentation 67 UV spectroscopy 45
Rescaling 53
V
S Validated assay 8
safety, biohazard 66 VIC dye 6, 25, 59
Single nucleotide polymorphism (SNP) 6
Supplementary Sample Information (SSI) 24
support, customer and technical 68 W
warranty 69
T
terms and conditions 69
Thermo Fisher Cloud Genotyping Application 23,
25

thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com
29 September 2017

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For Research Use Only. Not for use in diagnostic procedures.

The information in this guide is subject to change without notice.

Revision history: Revision history of Pub. No. MAN0009593

A.0 30 January 2014 New document

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

TaqMan® SNP Genotyping Assays User Guide 3

■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

4 TaqMan® SNP Genotyping Assays User Guide

■ APPENDIX D Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

TaqMan® SNP Genotyping Assays User Guide 5

TaqMan® SNP Genotyping Assay overview

6 TaqMan® SNP Genotyping Assays User Guide

Table 2 Example allele-to-dye associations

TaqMan® SNP Genotyping Assays User Guide 7

Master mix Guidelines for use

Predesigned TaqMan® SNP Genotyping Assays

8 TaqMan® SNP Genotyping Assays User Guide

Predesigned TaqMan® SNP Genotyping Assays are made‑to‑order, available in

Custom TaqMan® SNP Genotyping Assays

TaqMan® SNP Genotyping Assays User Guide 9

Custom TaqMan® SNP Genotyping Assays allow you to:

For maximal efficiency in a multiplex format, Custom TaqMan® SNP Genotyping

Assay format, size, and storage

Table 4 Predesigned TaqMan® SNP Genotyping Assays: single‑tube

Number of reactions Cat. No.

Small 1,500 750 300 40X 4351379 4351384

Medium 5,000 2,500 1,000 40X 4351376 4351382

Large 12,000 6,000 2,400 80X 4351374 4351380

10 TaqMan® SNP Genotyping Assays User Guide

Table 5 TaqMan® DME Genotyping Assays: single‑tube

Number of reactions Cat. No.

Small 750 150 20X 4362691 —

Table 6 Custom TaqMan® SNP Genotyping Assays: single‑tube

Number of reactions Cat. No.

Small 1,500 750 300 40X 4331349 4332077

Medium 5,000 2,500 1,000 40X 4332072 4332075

Large 12,000 6,000 2,400 80X 4332073 4332076

TaqMan® SNP Genotyping Assays User Guide 11

Table 7 TaqMan® SNP Genotyping Assays plate products and formats

OpenArray™ plates[1] thermofisher.com/openarray

96- or 384-well plates (fast or standard) thermofisher.com/customplating

Shipment contents Single‑tube shipments include:

12 TaqMan® SNP Genotyping Assays User Guide

• Do not perform more than 10 freeze‑thaw cycles. If you expect to freeze‑thaw

Dilute, then dispense aliquots of TaqMan® SNP Genotyping Assays

2. Vortex, then centrifuge the mixture.

TaqMan® SNP Genotyping Assays User Guide 13

Required materials not supplied

Table 8 Kits for genomic DNA extraction

Item Sample type Source

Select the appropriate kit for your

Table 9 Supported instruments

Item Block modules Source

96-well, 96-well Fast, 384-well, and 8-strip

96-well, 2 × 96-well, 2 × 384-well, and 8-strip

3 × 32-well, 96-well, 2 × 96-well, 2 × 384-well,

SimpliAmp™ Thermal Cycler[1] 96-well and 8-strip tubes