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TaqMan SNP Genotyping

Assays

Protocol

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June 9, 2004 11:08 am, 4332856_Title.fm
Copyright 2004, Applied Biosystems. All rights reserved.
For Research Use Only. Not for use in diagnostic procedures.
Information in this document is subject to change without notice. Applied Biosystems assumes no responsibility for any errors that
may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall
Applied Biosystems be liable for incidental, special, multiple, or consequential damages in connection with or arising from the use
of this document.
NOTICE TO PURCHASER: LIMITED LICENSE
TaqMan probes are covered by U.S. Patent 5,723,591 and foreign counterparts and patents pending owned by Applera
Corporation, and are covered by U.S. Patents 5,801,155 and 6,084,102 and foreign counterparts licensed to Applied Biosystems.
NOTICE TO PURCHASER: DISCLAIMER OF LICENSE
This product is optimized for use in the Polymerase Chain Reaction (PCR) and 5 nuclease detection methods covered by patents
owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No license under these patents to use the PCR process
is conveyed expressly or by implication to the purchaser by the purchase of this product. A license to use the PCR process for
certain research and development activities accompanies the purchase of certain Applied Biosystems reagents when used in
conjunction with an authorized thermal cycler, or is available from Applied Biosystems. Further information on purchasing
licenses to practice the PCR process may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln
Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, California
94501 USA.
NOTICE TO PURCHASER: DISCLAIMER OF LICENSE FOR CUSTOM SEQUENCE DETECTION PRIMERS
This product is optimized for use in the Polymerase Chain Reaction (PCR) and 5 nuclease detection methods covered by patents
owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd. No license under these patents to use the PCR process
or 5 nuclease detection methods is conveyed expressly or by implication to the purchaser by the purchase of this product. A
license to use the PCR process for certain research and development activities accompanies the purchase of certain Applied
Biosystems reagents when used in conjunction with an authorized thermal cycler, or is available from Applied Biosystems. Further
information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing, Applied
Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404 or at Roche Molecular Systems, Inc., 1145 Atlantic Avenue,
Alameda, California 94501 USA.
ABI PRISM, Applied Biosystems, MicroAmp, and VIC are registered trademarks and AB (Design), ABI PRISM, Applera, and FAM
are trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries.
AmpErase, AmpliTaq Gold, GeneAmp, and TaqMan are registered trademarks of Roche Molecular Systems, Inc.
All other trademarks are the sole property of their respective owners.

Part Number 4332856 Rev. B


06/2004

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Contents

Product Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Product Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Available Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Product Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Assay Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
About SNP Genotyping Assay Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
About the Assay Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocol Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
About This Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Procedure Flowchart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
About TaqMan SNP Genotyping Assays . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Assay Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
About the Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
5 Nuclease Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Materials and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Available Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Assay Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Ordering Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Storage and Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Materials and Equipment Not Included . . . . . . . . . . . . . . . . . . . . . . . 9
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Documentation User Attention Words . . . . . . . . . . . . . . . . . . . . . . . 12
Chemical Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Chemical Waste Hazard Warning . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Site Preparation and Safety Guide . . . . . . . . . . . . . . . . . . . . . . . . . . 13
About MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Ordering MSDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Preventing Contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
General PCR Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Assay Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
About the Assay Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

TaqMan SNP Genotyping Assays Protocol 3


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Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Determining Tube Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
General Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Reagent Preparation Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
Recommended Template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Quantifying Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
Methods for Adding DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Preparing the Reaction Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Preparing the Reaction Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Selecting a Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Performing PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
Allelic Discrimination Plate Read and Analysis . . . . . . . . . . . . . . . . . . . . .29
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
General Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
Obtaining Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32
Applied Biosystems Web Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

4 TaqMan SNP Genotyping Assays Protocol


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Product Overview
Product TaqMan SNP Genotyping Assays provide the largest collection of
Description ready-to-use SNP assays available. All assays were designed using
our powerful bioinformatics pipeline and software, as well as
genomic information from Celera Genomics and public databases:
TaqMan Validated SNP Genotyping Assays: > 150,000
gene-centric assays. These are our highest performing SNP
genotyping assays, which have undergone minor allele frequency
(MAF) validation on 2 or 4 ethnic group populations (45
individual samples per ethnic group). Inventoried for fast
availability.
TaqMan Coding SNP Genotyping Assays: ~ 30,000 assays for
the detection of informative and putative functional SNPs in
gene-coding regions. Inventoried assays, which are functionally
tested to assure quality performance.
TaqMan Pre-Designed SNP Genotyping Assays: ~ 1.7 million
genome-wide and genome-unique in silico assays. Manufactured
and functionally tested upon ordering.
TaqMan SNP Genotyping Assays provide optimized assays for
genotyping single nucleotide polymorphisms (SNPs). The products
use the 5 nuclease assay for amplifying and detecting specific SNP
alleles in purified genomic DNA samples. Each assay allows
researchers to genotype individuals for a specific SNP.

Available Visit our Web site to view the available TaqMan SNP Genotyping
Products Assays (PNs 4331183, 4351374, 4351376, 4351379):
http://www.appliedbiosystems.com
For more information about ordering TaqMan SNP Genotyping
Assays, see Ordering Assays on page 8.
If a particular SNP of interest is not available as a product on our
Web site, you can use our Custom TaqMan SNP Genotyping Assays
Service.

TaqMan SNP Genotyping Assays Protocol 1


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The Custom TaqMan SNP Genotyping Assays Service is an assay
development service that designs, synthesizes, formulates, and
delivers analytically quality-controlled primer and probe sets for SNP
genotyping assays based on sequence information submitted by the
customer. To place an order, contact your Applied Biosystems
representative.
TaqMan SNP Genotyping Assay Products

Concen- Part
Type Scale Number of Reactions
tration Number

25-L 5-mL
Reaction Reaction
96-Well 384-Well

Inventoried Small 20X 150 750 4331183

Non-inventoried Small 20X 150 750 4351379

Non-inventoried* Medium 40X 600 3,000 4351376

Non-inventoried* Large 80X 2,400 12,000 4351374

*Available by the latter half of 2004.

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Product Properties of TaqMan SNP Genotyping Assays:
Properties All TaqMan SNP Genotyping Assays are designed and optimized
to work with the TaqMan Universal PCR Master Mix, No
AmpErase UNG, using the same universal thermal cycling
conditions. This facilitates the workflow for all throughput
requirements and in any SNP genotyping study.
The assays require only three components:
1 to 20 ng of purified genomic DNA sample
20X or 40X or 80X SNP Genotyping Assay Mix (specific for
each polymorphism)
2X TaqMan Universal PCR Master Mix (with or without
AmpErase UNG)
The assays require only one amplification step and an endpoint
reading to obtain results.

Assay Contents Each SNP genotyping assay consists of:


One tube containing 20X or 40X or 80X SNP Genotyping Assay
Mix. Concentration depends on product and scale ordered.
CD-ROM containing assay information and PDFs of the protocol
and product insert

About SNP The SNP Genotyping Assay Mix contains:


Genotyping Sequence-specific forward and reverse primers to amplify the SNP
Assay Mix of interest
Two TaqMan MGB probes:
One probe labeled with VIC dye detects the Allele 1 sequence
One probe labeled with FAM dye detects the Allele 2
sequence

About the Assay The assay information consists of:


Information Genomic information about the SNP, including chromosomal
location, allele frequency (for validated assays), and context
sequence
Information about the packaging of each assay tube, including the
location in the plate rack and the 2-D bar code

TaqMan SNP Genotyping Assays Protocol 3


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Protocol Overview
About This This protocol provides:
Protocol Background information about SNP genotyping assays
A list of materials and equipment required to perform SNP
genotyping assays using TaqMan SNP Genotyping Assays
Instructions for preparing reaction plates and performing PCR
Overview of procedures for performing endpoint plate reads and
analyzing results

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Procedure The following diagram provides a simplified overview of the
Flowchart procedure for using TaqMan SNP Genotyping Assays.

Prepare reaction mix


PCR Amplification

1 2 3 4 5 6 7 8 9 10 11 12
A

Prepare reaction plate D

384-well plate 96-well plate

Perform PCR or GeneAm


PCR Systemp
9700

SDS instrumentation Thermal cycler


Allelic Discrimination

Set up a new
plate read document
Plate Read

Perform post-PCR
plate read
I

SDS instrumentation
Allelic Discrimination

Analyze the
plate read document
Analysis

Call allele types

TaqMan SNP Genotyping Assays Protocol 5


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About TaqMan SNP Genotyping Assays
Assay Each SNP genotyping assay consists of a single tube containing:
Components Two primers for amplifying the sequence of interest
Two TaqMan MGB probes for detecting alleles

About the Probes Each TaqMan MGB probe contains:


A reporter dye at the 5 end of each probe
VIC dye is linked to the 5 end of the Allele 1 probe.
FAM dye is linked to the 5 end of the Allele 2 probe.
A minor groove binder (MGB)
This modification increases the melting temperature (Tm) without
increasing probe length (Afonina et al., 1997; Kutyavin et al.,
1997), which allows the design of shorter probes. This results in
greater differences in Tm values between matched and mismatched
probes, which produces more accurate allelic discrimination.
A nonfluorescent quencher (NFQ) at the 3 end of the probe
Because the quencher does not fluoresce, Applied Biosystems
sequence detection systems can measure reporter dye
contributions more accurately.

5 Nuclease During PCR, the following events occur:


Assay 1. Each TaqMan MGB probe anneals specifically to a
complementary sequence between the forward and reverse
primer 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 Frster-type energy transfer (Frster, 1948;
Lakowicz, 1983).
2. AmpliTaq Gold DNA polymerase cleaves only probes that are
hybridized to the target.
3. Cleavage separates the reporter dye from the quencher dye,
which results in increased fluorescence by the reporter.
The increase in fluorescence signal occurs only if the amplified
target sequence is complementary to the probe. Thus, the
fluorescence signal generated by PCR amplification indicates
which alleles are present in the sample.

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Mismatches Between Probe and Target Sequences
Mismatches between a probe and target reduce the efficiency of
probe hybridization. Furthermore, AmpliTaq Gold DNA polymerase
is more likely to displace a mismatched probe without cleaving it,
which does not produce a fluorescent signal.

SNP Genotyping Assay Results


The next figure illustrates results from matches and mismatches
between target and probe sequences in TaqMan SNP Genotyping
Assays (Livak et al., 1995).

F
Allele
1 V NFQ MGB NFQ
MGB

Match Mismatch

V
Allele F NFQ
2 NFQ MGB MGB

Match Mismatch

Legend
Nonfluorescent
V VIC dye NFQ quencher AmpliTaq
Gold DNA
Minor groove Polymerase
F 6-FAM dye MGB binder

GR2180

The table below shows the correlation between fluorescence signals


and sequences present in the sample.

A substantial increase in Indicates


VIC dye fluorescence only homozygosity for Allele 1.
FAM dye fluorescence only homozygosity for Allele 2.
both fluorescent signals Allele 1-Allele 2 heterozygosity.

TaqMan SNP Genotyping Assays Protocol 7


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Materials and Equipment
Available The TaqMan SNP Genotyping Assays are available as shown below:
Products Part Numbers 4331183, 4351374, 4351376, 4351379
*Contents 20X or 40X or 80X SNP Genotyping Assay Mixes
*Volumes 187.5 L 375 L, or 750 L (setup for 5-L
reaction format)
Description One tube containing sequence-specific primers,
one probe for Allele 1 labeled with VIC dye, and one probe for
Allele 2 labeled with FAM dye
*Contents and volumes are dependent on scale ordered.
For a list of available products, visit our Web site:
http://www.appliedbiosystems.com/

Assay Naming SNP genotyping assays from the TaqMan SNP Genotyping Assays
are named using a unique assay identifier containing up to 16 digits.

Ordering Assays Visit our Web site to order the available assays:
www.appliedbiosystems.com
Use the part numbers (PN 4331183, 4351374, 4351376, 4351379)
and the assay identifier to locate and order the assay you need.

Storage and Follow the guidelines below for storage:


Stability Store the assay mixes at 15 to 25 C.
Minimize freeze-thaw cycles.
Keep all TaqMan SNP Genotyping Assays protected from direct
exposure to light. Excessive exposure to light may affect the
fluorescent probes.

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Materials and The following tables include required and optional equipment and
Equipment Not materials for using the TaqMan SNP Genotyping Assays. Unless
Included otherwise noted, the items listed are available from major laboratory
suppliers (MLSs).
Instruments from Applied Biosystems

Instruments Source
ABI PRISM 7900HT Sequence Detection Contact your local
System Applied Biosystems
sales office.
ABI PRISM 7700 Sequence Detection System
GeneAmp PCR System 9700 thermal cycler
Applied Biosystems 7300 Real-Time PCR
System
Applied Biosystems 7500 Real-Time PCR
System

User-Supplied Materials

Materials Source

ABI PRISM 96-Well Optical Reaction Plate With Applied Biosystems
Barcode (code 128) (PN 4326659)
ABI PRISM 384-Well Clear Optical Reaction Applied Biosystems
Plate With Barcode (code 128) (PN 4309849)
ABI PRISM Optical Adhesive Covers Applied Biosystems
(PN 4311971)
ABI PRISM Optical Adhesive Cover Starter Kit Applied Biosystems
(PN 4313663)
ABI PRISM Optical Caps, 8 caps/strip Applied Biosystems
(PN 4323032)
ABI PRISM Cap Installing Tool Applied Biosystems
(PN 4330015)
Control DNA CEPH Individual 1347-02, 180 L Applied Biosystems
(PN 403062)
MicroAmp Multi Removal Tool Applied Biosystems
(PN 4313950)
TaqMan DNA Template Reagents Applied Biosystems
(PN 401970)
TaqMan RNase P Detection Reagents Kit Applied Biosystems
(PN 4316831)
TaqMan Universal PCR Master Mix, No Applied Biosystems
AmpErase UNG, 200 reactions (PN 4324018)

TaqMan SNP Genotyping Assays Protocol 9


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User-Supplied Materials (continued)

Materials Source
TaqMan Universal PCR Master Mix, 200 Applied Biosystems
reactions (PN 4304437)
TaqMan Universal PCR Master Mix, No Applied Biosystems
AmpErase UNG, 2000 reactions (PN 4326614)
TaqMan Universal PCR Master Mix, 2000 Applied Biosystems
reactions (PN 4326708)
10-Pack, TaqMan Universal PCR Master Mix, Applied Biosystems
No AmpErase UNG (PN 4324020)
10-Pack, TaqMan Universal PCR Master Mix Applied Biosystems
(PN 4305719)
Accessories for tubes of assay mixes:
Decapper for single caps (PN 54000) Micronic BV *
Decapper for eight caps (PN 54001) PO Box 604
8200 AP Lelystad
Netherlands
Telephone:
+31(0)320.277077
Fax:
+31(0)320.277088
traxis.micronic.com
Solid caps for 0.7-mL/1.4-mL tubes, Apogent Technologies, Inc.*
10 capmats (for 96-wells) per case (PN 4463) 30 Penhallow St.
Portsmouth NH 03801
USA
Telephone:
+1.603.433.6131
Fax:
+1.603.431.0860
www.Apogent.com
Centrifuge with plate adapter MLS
DNase-free, sterile-filtered water MLS
Disposable gloves MLS
Microcentrifuge MLS
Microsoft Excel
or equivalent spreadsheet and Software suppliers
analysis software
Pipet tips, aerosol-resistant MLS

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User-Supplied Materials (continued)

Materials Source
Pipettors: MLS
Positive-displacement
Air-displacement
Multichannel
Polypropylene tubes MLS
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0, MLS
made using DNase-free, sterile-filtered water)
*Other vendors supply similar products

TaqMan SNP Genotyping Assays Protocol 11


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Safety
Documentation Five user attention words appear in the text of all
User Attention Applied Biosystems user documentation. Each word implies a
Words particular level of observation or action as described below.
Note: Calls attention to useful information.
IMPORTANT! Indicates information that is necessary for proper
instrument operation, accurate chemistry kit use, or safe use of a
chemical.
Indicates a potentially hazardous situation which, if
not avoided, may result in minor or moderate injury. It may also be
used to alert against unsafe practices.
Indicates a potentially hazardous situation which, if
not avoided, could result in death or serious injury.
Indicates an imminently hazardous situation which, if
not avoided, will result in death or serious injury. This signal word is
to be limited to the most extreme situations.

Chemical Hazard CHEMICAL HAZARD. Some of the chemicals


Warning used with Applied Biosystems instruments and protocols are
potentially hazardous and can cause injury, illness, or death.
Read and understand the material safety data sheets (MSDSs)
provided by the chemical manufacturer before you store, handle,
or work with any chemicals or hazardous materials.
Minimize contact with chemicals. Wear appropriate personal
protective equipment when handling chemicals (e.g., safety
glasses, gloves, or protective clothing). For additional safety
guidelines, consult the MSDS.
Minimize the inhalation of chemicals. Do not leave chemical
containers open. Use only with adequate ventilation (e.g., fume
hood). For additional safety guidelines, consult the MSDS.
Check regularly for chemical leaks or spills. If a leak or spill
occurs, follow the manufacturers cleanup procedures as
recommended on the MSDS.
Comply with all local, state/provincial, or national laws and
regulations related to chemical storage, handling, and disposal.

12 TaqMan SNP Genotyping Assays Protocol


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Chemical Waste CHEMICAL WASTE HAZARD. Wastes produced
Hazard Warning by Applied Biosystems instruments are potentially hazardous and
can cause injury, illness, or death.
Read and understand the material safety data sheets (MSDSs)
provided by the manufacturers of the chemicals in the waste
container before you store, handle, or dispose of chemical waste.
Handle chemical wastes in a fume hood.
Minimize contact with chemicals. Wear appropriate personal
protective equipment when handling chemicals (e.g., safety
glasses, gloves, or protective clothing). For additional safety
guidelines, consult the MSDS.
Minimize the inhalation of chemicals. Do not leave chemical
containers open. Use only with adequate ventilation (e.g., fume
hood). For additional safety guidelines, consult the MSDS.
After emptying the waste container, seal it with the cap provided.
Dispose of the contents of the waste tray and waste bottle in
accordance with good laboratory practices and local,
state/provincial, or national environmental and health regulations.

Site Preparation A site preparation and safety guide is a separate document sent to all
and Safety Guide customers who have purchased an Applied Biosystems instrument.
Refer to the guide written for your instrument for information on site
preparation, instrument safety, chemical safety, and waste profiles.

About MSDSs Some of the chemicals used with this instrument may be listed as
hazardous by their manufacturer. When hazards exist, warnings are
prominently displayed on the labels of all chemicals.
Chemical manufacturers supply a current material safety data sheet
(MSDS) before or with shipments of hazardous chemicals to new
customers and with the first shipment of a hazardous chemical after
an MSDS update. MSDSs provide you with the safety information
you need to store, handle, transport and dispose of the chemicals
safely.
We strongly recommend that you replace the appropriate MSDS in
your files each time you receive a new MSDS packaged with a
hazardous chemical.
CHEMICAL HAZARD. Be sure to familiarize
yourself with the MSDSs before using reagents or solvents.

TaqMan SNP Genotyping Assays Protocol 13


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Ordering MSDSs You can order free additional copies of MSDSs for chemicals
manufactured or distributed by Applied Biosystems using the contact
information below.

To order documents by automated telephone service:

1. From the U.S. or Canada, dial 1.800.487.6809.

2. Follow the voice instructions to order documents (for


delivery by fax).
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To order documents by telephone:

In the U.S. Dial 1.800.345.5224, and press 1.

In Canada Dial 1.800.668.6913, and press 1 for English or 2


for French.

To view, download, or order documents through the


Applied Biosystems Web site:

1. Go to
http://docs.appliedbiosystems.com/msdssearch.html

2. In the SEARCH field, type in the chemical name, part


number, or information that will appear in the MSDS and
click SEARCH.
Note: You may also select the language of your choice from
the drop-down list.

3. When the Search Results page opens, find the document


you want and click on it to open a PDF of the document.

For chemicals not manufactured or distributed by


Applied Biosystems, call the chemical manufacturer.

14 TaqMan SNP Genotyping Assays Protocol


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Preventing Contamination
Introduction PCR assays require special laboratory practices to avoid false
positive amplifications (Kwok and Higuchi, 1989). The high
throughput and repetition of these assays can lead to amplification of
a single DNA molecule (Saiki et al., 1985; Mullis and Faloona,
1987).

General PCR Follow these recommended procedures:


Practices Wear a clean lab coat (not previously worn while handling
amplified PCR products or used during sample preparation) and
clean gloves when preparing samples for PCR amplification.
Change gloves whenever you suspect that they are contaminated.
Maintain separate areas, dedicated equipment, and supplies for:
Sample preparation
PCR setup
PCR amplification
Analysis of PCR products
Never bring amplified PCR products into the PCR setup area.
Open and close all sample tubes and reaction plates carefully. Try
not to splash or spray PCR samples.
Keep reactions and components capped as much as possible.
Use positive-displacement pipet or aerosol-resistant pipet tips.
Clean lab benches and equipment periodically with freshly diluted
10% bleach solution.

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Assay Information
Overview This section provides a description of the assay information that you
received with the shipment of SNP genotyping assays.

About the Assay You receive assay information for each order on a CD-ROM.
Information The file name includes the number from the bar code on the plate
The information on the file is provided in tab-delimited format.

Uses You can use the assay information to determine:


Which assay is included in each tube that you received
The location of each tube in the assay rack
Assay IDs
Chromosome location of the SNP
Minor allele frequencies (calculated for validated assays)
Context sequence

Determining Tube To determine the contents of each assay tube, match the Assay ID
Contents and the plate rack position on the tube label with the values in the
Assay ID and Well Loc columns. View assay information in the
corresponding row of the file.

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Columns The table below lists and describes some of the assay information
columns.

Column Example Description

Order ID 185024159 Assigned by Applied Biosystems, and appears on the plate


rack in the 1-D bar code.
Note: This is the unique identifier you should use if
contacting Applied Biosystems about your order.

Item No 1 Row number in the file.

Part Number 4331183 The part number ordered and shipped.

Assay ID C_8395642_11 Assigned by Applied Biosystems. Unique assay identifier


used to name each SNP genotyping assay. Contains up to
16 digits.
Note: This is the unique identifier you should use to locate
and order the assay you need on our Web site.

Qty 1 Number of copies of a particular SNP genotyping assay


ordered in this order.

Plate ID 50286240_E Assigned by Applied Biosystems. Includes the Order ID


value and appears on the plate rack as the 1-D bar code.

Well Loc A1 Location of assay tube in the plate rack.

Vial ID 0005885579 2-D bar code number on the bottom of each tube.
Note: There are leading zeros in this number. If you open
the file in a spreadsheet, the leading zeros may be dropped.

Reporter 1 VIC Dye used to label the TaqMan probe that detects the Allele 1
sequence.

Reporter 2 FAM Dye used to label the TaqMan probe that detects the Allele 2
sequence.

Quencher NFQ Quencher used at the 3 end of the TaqMan probes.

Context NNN[A1/A2]NNN 25 nucleotides on both sides of SNP site (in brackets).


Sequence A1= Allele 1, A2 = Allele 2.

Design Strand Forward Indicates if the probe contains the sequence of the context
strand or of its reverse complement.

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PCR Amplification
Overview In the first step in performing SNP genotyping assays, AmpliTaq
Gold DNA polymerase from the TaqMan Universal PCR Master
Mix, No AmpErase UNG*, amplifies target DNA, using
sequence-specific primers and TaqMan MGB probes from the SNP
Genotyping Assay Mix.
*Alternatively, TaqMan Universal PCR Master Mix, containing
AmpErase UNG, may be used. Henceforth, only Master Mix without
UNG is referred to in this protocol.

General Process PCR amplification involves the following procedures:


1. Preparing the reaction mix
2. Preparing an optical reaction plate containing the following for
each assay:
No Template Controls (NTCs)
Known genomic DNA controls (optional)
Unknown genomic DNA samples
3. Performing PCR

Reagent The following guidelines will ensure optimal PCR performance:


Preparation Keep all TaqMan SNP Genotyping Assays protected from light, in
Guidelines the freezer, until you are ready to use them. Excessive exposure to
light may affect the fluorescent probes.
Minimize freeze-thaw cycles.
Prior to use:
Thoroughly mix the TaqMan Universal PCR Master Mix, No
AmpErase UNG, by swirling the bottle.
Resuspend the assay mix by vortexing and then centrifuge the
tube briefly.
After thawing frozen genomic DNA samples, resuspend the
samples by vortexing and then centrifuge the tubes briefly.
Prepare the reaction mix for each assay before transferring it to the
optical reaction plate for thermal cycling and fluorescence
analysis.

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Recommended The recommended template for TaqMan SNP Genotyping Assays is
Template purified genomic DNA (1 to 20 ng). Quantify genomic DNA using
the TaqMan RNase P Detection Reagents Kit (PN 4316831) and the
TaqMan DNA Template Reagents Kit (PN 401970).

Quantifying Applied Biosystems recommends quantifying the amount of genomic


Genomic DNA DNA in samples before using TaqMan SNP Genotyping Assays.
Generate a standard curve using the DNA template standards
provided in the TaqMan DNA Template Reagents Kit (PN 401970)
and the RNase P gene primers and probe provided in the TaqMan
RNase P Detection Reagents Kit (PN 4316831).
Note: Refer to the appropriate instrument user guide for detailed
instructions on performing and analyzing runs.
! CAUTION CHEMICAL HAZARD. TaqMan Universal PCR
Master Mix may cause eye and skin irritation. Exposure may cause
discomfort if swallowed or inhaled. Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.

To quantify genomic DNA:

1. Create and set up a sequence detector plate document.

2. Prepare the reaction plate using the following components:


2X TaqMan Universal PCR Master Mix, No AmpErase
UNG
20X Primer and TaqMan Probe (FAM) dye mix
DNA standard template or genomic DNA sample
DNase-free, sterile-filtered water
Note: Use at least 3 replicates of each standard or sample,
and use all five DNA standards to ensure an accurate
standard curve is generated. The range of known copy
number should bracket anticipated copy numbers of the
unknown samples on the same plate.

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To quantify genomic DNA: (continued)

3. Run the plate on an ABI PRISM Sequence Detection System


or Real-Time PCR System using the following thermal
cycling conditions:
AmpliTaq
Gold
PCR
Enzyme
Activation
HOLD CYCLE (40 cycles)
Denature Anneal/
Extend
Time 10 min 15 sec 1 min
Temp 95 C 92 C 60 C

4. Generate a standard curve to quantify the amount of DNA in


each sample.

Methods for There are two methods for adding genomic DNA to the reaction.
Adding DNA Decide which method to use based on the experimental design.
The two methods are described in the table below.

Method Method Description Experimental Uses

Wet DNA 1. SNP reaction mix is aliquoted to an Many different DNA templates tested on
delivery optical reaction plate. a limited number of SNP targets
2. Genomic DNA is delivered to the final
reaction mix.
Note: In this method, the liquid used to
resuspend the DNA is used as a
component of the final reaction.

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Method Method Description Experimental Uses

DNA 1. Genomic DNA is delivered to the DNA concentration requires large


predelivery bottom surface of an optical reaction sample volumes (2 to 5 L) to run the
and dry-down plate. assay
2. The DNA sample is dried down or
completely by evaporation.
Limited number of DNA templates
3. The SNP reaction mix is added, and tested repeatedly on different SNP
the DNA disperses in the final targets
reaction mix.
or
A large number of DNA samples are
prepared in plates, dried down, and
stored before use

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Preparing the The reaction mix contains TaqMan SNP Genotyping Assay Mix,
Reaction Mix TaqMan Universal PCR Master Mix, No AmpErase UNG, and
DNase-free water. The recommended reaction size is 5 L for a
384-well setup and 25 L for a 96-well setup.
CHEMICAL HAZARD. SNP Genotyping Assay
Mix contains formamide. Exposure causes eye, skin, and respiratory
tract irritation. It is a possible developmental and birth defect hazard.
Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
! CAUTION CHEMICAL HAZARD. TaqMan Universal PCR
Master Mix may cause eye and skin irritation. Exposure may cause
discomfort if swallowed or inhaled. Read the MSDS, and follow the
handling instructions. Wear appropriate protective eyewear, clothing,
and gloves.

To prepare the reaction mix:

1. Calculate the number of reactions to be performed for each


assay.
Note: Include at least two NTCs and known genomic DNA
controls on each plate for optimal performance of TaqMan
SNP Genotyping Assays.

2.
If using the method Then go to

Wet DNA delivery step 3 below

DNA predelivery and step 4 on page 24


dry-down

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To prepare the reaction mix: (continued)

3. If using the wet DNA delivery method:


a. Calculate the volume of components needed for each
assay, using the table below.

Volume(L)/Reaction

Component
5-L 25-L
Reaction Reaction

2X TaqMan Universal PCR Master 2.50 12.50


Mix, No AmpErase UNG

20X SNP Genotyping Assay Mix* 0.25 1.25

Total 2.75 13.75

Note: Add extra reactions to provide excess volume for the


loss that occurs during reagent transfers.
*40X and 80X Assay Mixes should be diluted to 20X working
solutions with 1X TE. TE should be 10 mM Tris-HCL, 1mM
EDTA, pH 8.0 and use DNase-free, and RNase-free water.

b. Go to step 5 on page 24.

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To prepare the reaction mix: (continued)

4. If using the DNA predelivery and dry-down method:


a. Calculate the volume of components needed for each
assay, using the table below.

Volume(L)/Reaction

Component
5-L 25-L
Reaction Reaction

2X TaqMan Universal PCR Master 2.50 12.50


Mix, No AmpErase UNG

20X SNP Genotyping Assay Mix* 0.25 1.25

DNase-free water 2.25 11.25

Total 5.00 25.00

Note: Add extra reactions to provide excess volume for the


loss that occurs during reagent transfers.
*40X and 80X Assay Mixes should be diluted to 20X working
solutions with 1X TE. TE should be 10 mM Tris-HCL, 1mM
EDTA, pH 8.0 and use DNase-free, and RNase-free water.

b. Continue with step 5 below.

5. Swirl the bottle of 2X TaqMan Universal PCR Master Mix,


No AmpErase UNG, gently to resuspend.

6. Vortex and centrifuge the 20X SNP Genotyping Assay Mix


briefly.

7. Pipet the required volumes of 2X TaqMan Universal PCR


Master Mix, No AmpErase UNG, and 20X SNP Genotyping
Assay Mix into a sterile tube.

8. Invert the tube(s) to mix.

9. Centrifuge the tube(s) briefly to spin down the contents and


to eliminate any air bubbles from the solution.

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Preparing the To ensure optimal performance of TaqMan SNP Genotyping Assays,
Reaction Plate run controls for each assay and on each reaction plate:
Two NTCs
Known genomic DNA controls (optional)

To prepare the reaction plate:

1. If using the DNA predelivery and dry-down method:


a. Pipet one control or sample (1 to 20 ng of purified
genomic DNA) into each well of a 96-well or 384-well
optical reaction plate.
b. Dry down the samples completely by evaporation at room
temperature.
Note: Multiple SNP genotyping assays may be run on one
reaction plate.
Note: Use a calibrated, positive-displacement pipettor to
minimize contamination and error.

2. Invert the reaction mix tube(s) prepared in Preparing the


Reaction Mix on page 22.

3. Centrifuge the tube(s) briefly to spin down the contents and


to eliminate air bubbles.

4. Pipet reaction mix into each well.

Volume of Reaction Mix (L)

Plate Format DNA Predelivery


Wet DNA Delivery
and Dry-Down
Method
Method

384-well 2.75 5

96-well 13.75 25

IMPORTANT! Be sure that no cross-contamination occurs


from well to well.

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To prepare the reaction plate: (continued)

5. If using the wet DNA delivery method:


a. Dilute 1 to 20 ng of each purified genomic DNA sample
into DNase-free water.

Then the volume of


DNA sample and
If you are preparing a
DNase-free water
per reaction should be

384-well reaction plate 2.25 L

96-well reaction plate 11.25 L

b. Pipet one control or sample, using the volumes in step 5a


above, into each well of the 96-well or 384-well optical
reaction plate.
Note: Multiple SNP genotyping assays may be run on one
reaction plate.
Note: Use a calibrated, positive-displacement pipettor to
minimize contamination and error.

6. Look at each well to check pipetting accuracy, and note


which wells do not appear to contain the proper volume.

7. Cover the plate with an optical adhesive cover or with


optical caps.

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Selecting a Because the data acquired during PCR amplification is not necessary
Thermal Cycler for analysis, any of the following instruments can be used for PCR
amplification:
GeneAmp PCR System 9700 thermal cycler
IMPORTANT! Because of differences in ramp rates and thermal
accuracy, you may need to adjust the settings if you choose to use
other thermal cyclers.
ABI PRISM 7900HT SDS
ABI PRISM 7700 SDS
Applied Biosystems 7500 Real-Time PCR System
Applied Biosystems 7300 Real-Time PCR System
Note: Use of the 7900HT or 7700 Sequence Detection Systems or
the 7500 or 7300 Real-Time PCR Systems allows for real-time
analysis of PCR, which is helpful for troubleshooting. If using a
sequence detection system for PCR amplification, perform the
endpoint plate read separately.

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Performing PCR To perform PCR:

1. Program the thermal cycling conditions.


IMPORTANT! These conditions are optimized for use only
with TaqMan SNP Genotyping Assays on the instruments
specified on page 27.

AmpliTaq
Gold
PCR
Enzyme
Activation
HOLD CYCLE (40 cycles)
Denature Anneal/
Extend
Time 10 min 15 sec 1 min
Temp 95 C 92 C 60 C

Note: Refer to the appropriate instrument user guide for


help with programming your thermal cycler.

2. Set the reaction volume according to the table below.

Plate Format Reaction Volume (L)

384-well 5

96-well 25

3. Load the reaction plate into the thermal cycler, and start the
run.

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Allelic Discrimination Plate Read and Analysis
Overview After PCR amplification, you perform an endpoint plate read. The
SDS software calculates the fluorescence measurements made during
the plate read and plots Rn values based on the signals from each
well. Using the software, you can determine which alleles are present
in each sample.
Note: Refer to the allelic discrimination section of the appropriate
instrument user guide for instructions on how to use the system
software to perform the plate read and analysis.

General Process The general process for analyzing data for SNP genotyping involves
the following procedures:
1. Creating and setting up an allelic discrimination plate read
document
2. Performing an allelic discrimination plate read on an SDS
instrument
3. Analyzing the plate read document
4. Making automatic or manual allele calls
5. Confirming allele types

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Troubleshooting
Observation Possible Cause Recommended Action
NTCs generated PCR contamination Test your reagents for the presence of nucleic
fluorescence signals. may have occurred. acid.
Distinct clusters were Reporter dyes were not 1. Confirm the Dye settings.
not observed or a appropriately assigned.
2. Reanalyze the plate read.
sample did not cluster
Quencher was
with one specific allele
improperly selected.
type.
A sample may: 1. Recheck the DNA concentrations of the
samples, using the procedure in
Contain more or less
Quantifying Genomic DNA on page 19.
DNA than other
samples 2. Retest the sample to confirm.
Contain a rare allelic 3. Test the sample using a different SNP
variation or genotyping assay.
sequence
duplication
Contain mixtures of
multiple alleles or
contamination
Inaccurate reagent Check each well for a variation in volume, note
delivery or evaporation which wells do not appear to contain the
occurred. proper volume, and redo any reactions that did
not contain the proper volume.
Bubbles are present in Check each well for bubbles, and redo any
the wells. reactions that contained bubbles.
Unknown genomic The sample may: 1. Recheck the DNA concentrations of the
samples did not samples using the procedure in Quantifying
Contain no DNA
generate fluorescence Genomic DNA on page 19.
signals. Contain PCR
inhibitors 2. Test a positive control: Control DNA CEPH
Individual 1347-02 (PN 403062).
Be homozygous for
a rare allelic variation 3. Retest the sample to confirm.
4. Test the sample using a different SNP
genotyping assay.

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Bibliography
Afonina, I., Zivarts, M., Kutyavin, I., et al. 1997. Efficient priming of
PCR with short oligonucleotides conjugated to a minor groove
binder. Nucleic Acids Res. 25:26572660.
Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B.
1997. Oligonucleotides with conjugated dihydropyrroloindole
tripeptides: base composition and backbone effects on hybridization.
Nucleic Acids Res. 25:37183723.
Kwok, S. and Higuchi, R. 1989. Avoiding false positives with PCR.
Nature 339:237238.
Livak, K.J., Marmaro, J., and Todd, J.A. 1995. Towards fully
automated genome-wide polymorphism screening [letter]. Nat.
Genet. 9:341342.
Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil
DNA glycosylase to control carry-over contamination in polymerase
chain reactions. Gene 93:125128.
Mullis, K.B. and Faloona, F.A. 1987. Specific synthesis of DNA in
vitro via a polymerase-catalyzed chain reaction. Methods Enzymol.
155:335350.
Saiki, R.K., Scharf, S., Faloona, F., et al. 1985. Enzymatic
amplification of -globin genomic sequences and restriction site
analysis for diagnosis of sickle cell anemia. Science 230:13501354.

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Obtaining Technical Support

Services and Support

Applied A services and support page is available on the Applied Biosystems


Biosystems Web site. To access this, go to:
Web Site http://www.appliedbiosystems.com
and click the link for services and support.
At the services and support page, you can:
Search through frequently asked questions (FAQs)
Submit a question directly to Technical Support
Order Applied Biosystems user documents, MSDSs, certificates of
analysis, and other related documents
Download PDF documents
Obtain information about customer training
Download software updates and patches
In addition, the services and support page provides worldwide
telephone and fax numbers to contact Applied Biosystems Technical
Support and Sales facilities.

32 TaqMan SNP Genotyping Assays Protocol


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Notes

TaqMan SNP Genotyping Assays Protocol 33


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34 TaqMan SNP Genotyping Assays Protocol
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Worldwide Sales and Support

Applied Biosystems vast distribution and


service network, composed of highly trained
support and applications personnel,
reaches 150 countries on six continents.
For sales office locations and technical support,
please call our local office or refer to our
Web site at www.appliedbiosystems.com.

Applera is committed to providing the


worlds leading technology and information
for life scientists. Applera Corporation
consists of the Applied Biosystems and
Celera Genomics businesses.

Headquarters
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Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884

06/2004

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