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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.
DRAFT
June 9, 2004 11:08 am, 4332856_Title.fm
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
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.
Concen- Part
Type Scale Number of Reactions
tration Number
25-L 5-mL
Reaction Reaction
96-Well 384-Well
1 2 3 4 5 6 7 8 9 10 11 12
A
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
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
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.
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)
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
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
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.
1. Go to
http://docs.appliedbiosystems.com/msdssearch.html
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.
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.
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.
Design Strand Forward Indicates if the probe contains the sequence of the context
strand or of its reverse complement.
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.
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.
2.
If using the method Then go to
Volume(L)/Reaction
Component
5-L 25-L
Reaction Reaction
Volume(L)/Reaction
Component
5-L 25-L
Reaction Reaction
384-well 2.75 5
96-well 13.75 25
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
384-well 5
96-well 25
3. Load the reaction plate into the thermal cycler, and start the
run.
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
Headquarters
850 Lincoln Centre Drive
Foster City, CA 94404 USA
Phone: +1 650.638.5800
Toll Free (In North America): +1 800.345.5224
Fax: +1 650.638.5884
06/2004