TaqMan® Allelic Discrimination Demonstration Kit
Contents
1. T T Sa 1 00 0 00 1 00 2 00 3 00 400 5 00 600 7 00 800 9 00 10 00 ALZ2Rn 1 TAMBA NTEM Tim m 60 1 1 00 0 001 0 008 2 NTC 44755 0 1305 2 3665 7 4 01 0 003 NTC 0 458 0 3865 2 7165 1 0E 00 0 006 0 014 0 000 Jo 000 Amp 4 NTC 2292 042339 26758 1 0 00 0 004 10 009 0 000 jo 000 1 2 3 E 5 6 7 8 9 10 11 12 No Amp No Amp No Amp No Amp No Amp No Amp No Amp No Amp 1 1 1 T 1 1 1 1 2 2 2 2 yA 2 2 2 1 and 2 and 2 1 and 2 1 and 2 i and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 i and 2 1 and 2 1 and 2 and 2 i and 2 1 and 2 i and 2 1 and 2 1 and 2 1 and 2 and 2 i and 2 and 2 1 and 2 1 and 2 and 2 1 and 2 1 and 2 i and 2 1 and 2 1 and 2 1 and 2 and 2 1 and 2 and 2 1 and 2 1 and 2 and 2 1 and 2 1 and 2 i and 2 1 and 2 1 and 2 and 2 and 2 1 and 2 i and 2 1 and 2 1 and 2 and 2 i and 2 1 and 2 i and 2 i and 2 1 and 2 1 and 2 and 2 1 and 2 and 2 1 and 2 1 and 2 and 2 1 and 2 i and 2 l and 2 i and 2 1 and 2 i and 2 and 2 1 and 2 1 and 2 1 and 2 Tommoom 4 If the populations do not show good discrimination choose Raw Spectra 88 Y from the Analysis menu Check whether the signal for at least one replicate of each of the two allele standards is within the dynamic range of t2. Step Action 1 Place the MicroAmp Optical 96 Well Reaction Plate in the GeneAmp PCR System 9600 or GeneAmp PCR System 9700 in 9600 Emulation Mode 2 Program the thermal cycler with the parameters shown in Table 5 on page 17 3 Perform PCR amplification 4 Store the PCR products at 2 6 C until you are ready to analyze them in the ABI PRISM 7200 Sequence Detector or TaqMan LS 50B PCR Detection System Allelic Discrimination on the ABI PRISM 7700 and 7200 Overview Analysis on the ABI PRISM 7700 or 7200 The TaqMan Allelic Discrimination Demonstration Kit is designed for Plate Read end point detection Plate Read detection collects one fluorescence scan per tube after PCR is completed and can be used to perform the allelic discrimination assay on either the ABI PRISM 7700 or ABI PRISM 7200 Sequence Detector The ABI PRISM 7700 or ABI PRISM 7200 Sequence Detector performs the Plate Read and generates multicomponented columns for No DNA Allele 1 and Allele 2 The data is then normalized for each allele and a genotype call is made for Allele 1 homozygote 1 Allele 2 homozygote 2 or Allele 1 2 heterozygote Samples run from the Genomic Control DNA included in this kit should all receive Allele 1 2 calls Refer to your instrument user s manual for more information To perform allelic discrimination Step Action 1 Launch the Sequence Detection Systems software 2 If the untitled pla
3. 62 Emulation Mode ABI PRISM 7700 HOLD HOLD CYCLE Sequence 2 min 10 min 15 sec 1 min Detector 50 95 C 95 62 IMPORTANT The two minute 50 C step is required for optimal AmpErase UNG activity The 10 minute 95 C step is required to activate AmpliTaq Gold DNA Polymerase Use one of the allelic discrimination probes with its target at a concentration of 100 nM Run atleast four replicates of each of the nine conditions defined by the 3 x 3 matrix below as well as four No Template Control NTC and four No Amplification Control NAC replicates The NTC and NAC replicates should be run at 900 nM forward and reverse primer concentrations Forward Primer nM Reverse Primer nM 50 300 900 50 50 50 300 50 900 50 300 50 300 300 300 900 300 900 50 900 300 900 900 900 25 Universal FAM 20 1M 20 1M Total PCR 10 1M Template Forward Reverse Volume Master FAM Target Primer Primer Deionized Well Wells uL Probe pL uL pL uL Water uL A1 A4 25 0 5 5 0 0 125 0 125 19 25 50 A5 A8 25 0 5 5 0 0 125 0 75 18 625 50 9 12 25 0 5 5 0 0 125 2 25 17 125 50 B1 B4 25 0 5 5 0 0 75 0 125 18 625 50 B5 B8 25 0 5 5 0 0 75 0 75 18 0 50 B9 B12 25 0 5 5 0 0 75 2 25 16 5 50 C1 C4 25 0 5 5 0 2 25 0 125 17 125 50 C5 C8 25 0 5 5 0 2 25 0 75 16 5 50 C9 C12 25 0 5 5 0 2 25 2 25 15 0 50 D1 D4 25 0 5 0 2 25 2 25 20 0 50 NTC D5 D8 25 0 5 5 0 2
4. Weak PCR amplifications for AL1 or AL2 Observation Outcome Probable Cause Solution Diffuse distribution of Incorrect Weak PCR amplifications Repeat reactions paying heterozygote allele calls particular attention to pipetting normalized results in technique and pipet calibration plotted data Use fresh reagents and prepare a master mix You can also try a larger reaction volume Distorted distribution Incorrect Poor reproducibility of Allow spreadsheet to recalculate vertically or allele calls NTC ALI or AL2 distributions and calls in the horizontally reactions absence of the replicate value s elongated of farthest from the mean heterozygote Repeat reactions paying particular attention to pipetting technique and pipet calibration Use fresh reagents and prepare a Master Mix You can also try a larger reaction volume 22 Appendix A Guidelines for Custom Applications Nine Step We recommend the following steps for the development of custom 5 Program nuclease assays for allelic discrimination applications Step Action See page 1 Install and use Primer Express software 2 Identify target seguence 23 3 Design TagMan probe 23 4 Design primers 24 5 Order reagents 24 6 Ouantitate probe and primers 24 7 Optimize primer concentrations 25 8 Optimize probe concentrations 28 9 Set up and run an Allelic Discrimination plate 33 Identify Target A target is a nucleoti
5. 3 4 5 6 7 8 3 10 11 12 A No Amp No Amp No Amp Mo Amp No Amp No Amp No Amp 1 1 1 1 B 1 1 1 2 2 2 2 2 2 2 2 C and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 D and2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 t and 2 1 and 2 E i and 2 i and 2 1 and 2 i and 2 i and 2 1 and 2 1 and 2 i and 2 i and 2 1 and 2 i and 2 i and 2 F fi and2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 G fi and2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 1 and 2 H 1 and 2 1 and 2 1 and 2 1 and 2 i and 2 i and 2 1 and 2 1 and 2 1 and 2 2 Bring all samples on scale Confirm that the standards and samples in the plot under Graph are distributed into up to four populations 3 If the populations are well distributed use the lasso tool and the Call pop up menu in the upper right corner of the Allelic Discrimination window see below to call them manually as No Amp Allele 1 Allele 2 and Allele 1 2 heterozygote Note Manual calling allows this application to be run without NTCs or standards with up to 96 individual samples per plate Troubleshooting Custom Allelic Discrimination Applications continued Step Action Allelic Discrimination Dye Components hai Allele 1 Y
6. A9 A12 and B1 B4 AL2 5 uL of Allele 2 FAM standard to wells B5 B12 UNKN 5 uL of each unknown sample to wells C1 B12 5 Close the plate with MicroAmp Optical Caps 6 Centrifuge the plate to collect the liquid at the bottom of the tubes and remove the air bubbles Thermal Cycling Use the thermal cycler conditions in Optimize Primer Concentrations on page 25 Run Allelic Discrimination Plate Step Action 1 Launch the Sequence Detection Systems software 2 If the untitled plate that opens is not the correct Allelic Discrimination plate for your instrument a Close the untitled plate b From the File menu choose New Plate 88 N c Inthe New Plate dialog box choose Allelic Discrimination from the Plate Type pop up menu The Run pop up menu will disappear d Choose the correct instrument from the Instrument pop up menu Note The correct plate type and instrument can be set in Preferences under the Edit menu Define the plate wells as shown in Set Up and Run an Allelic Discrimination Plate on page 33 Click the Show Analysis button Click the Post PCR Read button The software will perform the Plate Read From the File menu choose Save as to save the plate From the Analysis menu choose Analyze L The computer analyzes the data IMPORTANT Spectral Compensation for Endpoint must be on for the ABI PRISM 7700 Sequence Detector and off for the ABI PRISM 72
7. Appl 4 357 362 Longo N Berninger N S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Lyamichev V Brow M A D and Dahlberg J E 1993 Structure specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases Science 260 778 783 Mullis K B and Faloona F A 1987 Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction In Wu R ed Methods in Enzymology Vol 155 Academic Press Inc San Diego CA Pp 335 350 Orrego C 1990 Organizing a laboratory for PCR work In Innis M A Gelfand D H Sninsky J J and White T J eds PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA pp 447 454 PCR Technical Information 1996 In Perkin Elmer Systems Reagents amp Consumables 1996 1997 Foster City CA Saiki R K Scharf S J Faloona F A Mullis K B Horn G T Erlich H A and Amheim N 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Saiki R K Gelfand D H Stoffel S Scharf S J Higuchi R Horn G T Mullis K B and Erlich H A 1988 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase Science 239 487 491 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A La
8. Plasmid Allele 2 probe 5 FAM CCA GCA AGC ACT GAT GCC TC TAMRA 3 Plasmid Allele 1 standard 250 uL One tube 10 fg uL sufficient for 100 reactions Plasmid Allele 2 standard 250 uL One tube 10 fg uL sufficient for 100 reactions Genomic Control DNA human 1 0 mL Two tubes 10 ng uL sufficient for 200 reactions Storage and Stability Store the TaqMan Allelic Discrimination Demonstration Kit or its components at 2 6 C If stored under the recommended conditions the product will maintain performance through the control date printed on the label continued on next page Instruments One of the following instrument systems in Table 2 is required Required User Supplied Materials Table 2 Instrument Platforms for Allelic Discrimination Equipment Item Source ABI PRISM 7700 Sequence Detector ABI PRISM 7200 Sequence Detector and GeneAmp PCR System 9600 or GeneAmp PCR System 9700 in 9600 Emulation Mode TagMan LS 50B PCR Detection System and GeneAmp PCR System 9600 or GeneAmp PCR System 9700 in 9600 Emulation Mode Applied Biosystems call your regional sales office for the instrument best suited your needs The following items in Table 3 may be required in addition to the reagents supplied in the TaqMan Allelic Discrimination Demonstration Kit Table 3 User supplied Materials Reagent Equipment Item Source Deionized
9. Set Up and Run an Allelic Discrimination Plate This procedure involves PCR amplification of the target DNA followed by fluorescence analysis When performing allelic discrimination using your Allele 1 and Allele 2 standards the analysis requires that the controls and samples be run Figure 3 Eight No Template Control wells NTC Eight Allele 1 standard wells AL1 Eight Allele 2 standard wells AL2 Seventy two Genomic Control DNA wells UNKN IMPORTANT Eight replicates of No Template Control Allele 1 standard and Allele 2 standard must be run to make allele calls at a 99 796 confidence level using the automated allele calling routine Manual allele calling with less than eight replicates is possible Refer to Chapter 4 of the ABI PRISM 7200 Sequence Detector User s Manual Figure3 Plate diagram showing placement of control and sample reactions 33 34 Prepare Controls and Unknowns Step Action 1 Prepare 575 uL of a solution that contains your optimized primers and probes in concentrations 10X the optimal values you determined 2 Combine the following TaqMan Universal PCR Master Mix for 115 reactions 2 875 mL in 1 725 mL of water 575 uL of 10X Primer and Probe Solution 3 Deliver 45 uL of this mixture to each of the 96 wells in the plate 4 If preparing Then add NTC 5 uL of TE buffer to wells A1 A8 AL1 5 uL of Allele 1 TET standard to wells
10. Sninsky J J and White T J eds PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA pp 129 141 Holland P M Abramson R D Watson R and Gelfand D H 1991 Detection of specific polymerase chain reaction product by utilizing the 5 to exonuclease activity of Thermus aquaticus DNA polymerase PNAS USA 88 7276 7280 Kwok S 1990 Procedures to minimize PCR product carry over In Innis M A Gelfand D H Sninsky J J and White T J eds PCR Protocols A Guide to Methods and Applications Academic Press Inc San Diego CA pp 142 145 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Lakowicz J R 1983 Energy Transfer In Principles of Fluorescent Spectroscopy Plenum Press N Y pp 303 339 Lawyer F C Stoffel S Saiki R K Myambo K B Drummond R and Gelfand D H 1989 Isolation characterization and expression in Escherichia coli of the DNA polymerase gene from the extreme thermophile Thermus aquaticus J Biol Chem 264 6427 6437 Lee L G Connell C R and Bloch W 1993 Allelic discrimination by nick translation PCR with fluorogenic probes Nucleic Acids Res 21 3761 3766 Livak K J Flood S J A Marmaro J Giusti W and Deetz K 1995 Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization PCR Meth
11. 00 Sequence Detector From the Analysis menu choose Allelic Discrimination 88 K The Allelic Discrimination window appears Check the Allelic Discrimination window and confirm that the No Amp NTC 1 Allele 1 standard 2 Allele 2 standard and 1 and 2 heterozygote calls have been made If these calls are correct the custom application is running under optimal conditions 35 36 Troubleshooting Custom Allelic Discrimination Applications Step Action 1 If the Allelic Discrimination window does not show autocalls select Dye Components from the pop up menu to the right of the toolbar as shown below Dye Components zm Allele Components ic Discrimination 2 7 Graph I I a 110 lI 1 4 I 1 IL T ae ji H e 0304 t 2 T T T 0 10 0 00 0 10 0 20 030 O40 0 50 O60 0 70 O80 090 100 1 10 1 20 Allele 1 v Plate well Sample ALZ ALI TAM Rn NTCm Tim T2m Tin T2n Call eo 1 00 0 001 0 008 Amp A2 NTC 44755 D1305 2 3665 7 4 01 0 005 0 261 0 000 0 000 No NTC 0 458 0 3865 2 7165 1 0 00 0 006 0 014 0 000 Jo 000 Amp 4 NTC 1 2392 0 4339 26758 1 0 00 0 004 10 009 0 000 0 000 No Amp 7 Tray 1 2
12. 1 Protein Sequencing Press FAX 32 650 638 5981 Chemiluminescence Telephone FAX 1 800 542 2369 781 275 8581 U S only or Tropix 1 781 271 0045 9 00 a m to Tropix 5 00 p m ET For Support On This Product Dial 1 800 831 6844 and LC MS Telephone FAX 1 800 952 4716 650 638 6223 9 00 a m to 5 00 p m PT Documents on Free 24 hour access to Applied Biosystems technical documents including MSDSs is available by fax or e mail Demand You can access Documents on Demand through the internet or by telephone If you want to order Then through the Use http www appliedbiosystems com techsupport internet You can search for documents to order using keywords Up to five documents can be faxed or e mailed to you by title by phone fromthe a Call 1 800 487 6809 from a touch tone phone Have United States or Canada your fax number ready b Press 1 to order an index of available documents and have it faxed to you Each document in the index has an ID number Use this as your order number in step d below c Call 1 800 487 6809 from a touch tone phone a second time d Press 2 to order up to five documents and have them faxed to you by phone from outside the United States or Canada a Dial your international access code then 1 858 712 0317 from a touch tone phone Have your complete fax number and co
13. 25 2 25 14 50a Add 1 uL of 0 5 sodium dodecyl sulfate SDS to each of the four NAC wells to inhibit any enzyme activity in those wells To optimize primer concentrations Step Action 1 Launch the Sequence Detection Systems software 2 If the untitled plate that opens is not a Single Reporter Plate Read document for your instrument a Close the untitled plate b From the File menu choose New Plate 88 N c Inthe New Plate dialog box choose Single Reporter from the Plate Type pop up menu and Plate Read from the Run pop up menu d Choose the correct instrument from the Instrument pop up menu 3 Select wells as follows 1 12 unknowns UNKN 01 04 No Template Controls NTC 05 08 No Amplification Controls NAC To optimize primer concentrations continued Step Action 4 Click the Show Analysis button 5 Click the Post PCR Read button The software will perform the Plate Read From the File menu choose Save as to save the plate From the Diagnostics submenu under the Instrument menu choose Advanced Options Under Miscellaneous Options deselect the Use Spectral Compensation for Endpoint checkbox Advanced Options Viewer Display mse Multicomponent View EJ Display best fit in Raw Spectra View Analysis Spectra Components Use background in Spectra Components folder Ll Use pure s
14. 5 for a list of required reagents and equipment Quantitate Probes Use a spectrophotometric method to determine the concentrations of and Primers the probes and primers received Measure the absorbance at 260 nm of a 1 100 dilution of each oligonucleotide in TE buffer Calculate the oligonucleotide concentration C in uM using the method shown in the table below Extinction Extinction Coefficient Chromophore Coefficient Number Contribution A 15 200 1 15 200 C 7 050 6 42 300 G 12 010 5 60 050 T 8 400 6 50 400 FAM 20 958 1 20 958 TAMRA 31 980 1 31 980 TET 16 255 0 Total 220 888 Absorbance 260 nm sum of extinction coefficient contributions x cuvette pathlength x oligonucleotide concentration 100 0 13 220 888 1 x 0 3 cm x C 100 C 196 uM Optimize Primer The purpose of this procedure is to determine the minimum primer Concentrations concentrations that give the maximum The ABI PRISM 7700 Sequence Detector can provide additional data for optimization using the minimum threshold cycle C7 See Appendix B on page 39 for more information regarding Cr Use the TaqMan Universal PCR Master Mix Use the thermal cycler conditions in the table below Times and Temperatures Thermal Cycler m Each of 40 Cycles Initial Steps Melt Anneal Extend GeneAmp PCR HOLD HOLD CYCLE System 9600 or 2 min 10 min 15 sec 1 min 9700 in 9600 50 C 95 95
15. TagMan Allelic Discrimination Demonstration Kit Protocol BSS BioSystems Copyright 2008 2010 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 APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF Notice to Purchaser Limited License Use of this TaqMan Allelic Discrimination Demonstration Kit P N 4303263 is covered by US patent claims and patent claims outside the US The purchase of this product includes a limited nontransferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as apparatus or system claims for real time PCR and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fe
16. Use this probe ratio in your allelic discrimination assay 14 If the probes are not well balanced at any ratio use the TET probe at 350 nM ABI PRISM 7700 Sequence Detector Prepare the plate shown in the table below Use 50 uL of the indicated solution in each well Final Universal 1 uM 1 uM Final FAM TET PCR FAM TET Total Probe Probe Master Probe Probe Deionized Volume Conc Conc Wells Mix uL uL uL Water uL uL nM nM A1 A4 125 12 5 50 62 5 250 50 200 A5 A8 125 25 50 50 250 100 200 A9 A12 125 25 25 75 250 100 100 B1 B4 125 50 25 50 250 200 100 B5 B8 125 50 12 5 62 5 250 200 50 To optimize probe concentrations on the 7700 Step Action 1 Launch the Sequence Detection Systems software 2 If the untitled plate that opens is not the correct Allelic Discrimination plate for your instrument a Close the untitled plate b From the File menu choose New Plate 88 c Inthe New Plate dialog box choose Allelic Discrimination from the Plate Type pop up menu The Run pop up menu will disappear d Choose the correct instrument from the Instrument pop up menu Note correct plate type and instrument can be set in Preferences under the Edit menu 3 Select wells A1 B8 as unknowns UNKN 4 Click the Show Analysis button Click the Post PCR Read button The software will perform the Plate Read 6 From th
17. al Offices Sales and Service section below for how to contact local service representatives outside of the United States and Canada Call Technical Support at 1 800 831 6844 and select the appropriate option below for support on the product of your choice at any time during the call To open a service call for other support needs or in case of an emergency press 1 after dialing 1 800 831 6844 For Support On This Product Dial 1 800 831 6844 and ABI PRISM9 3700 DNA Press FAX Analyzer 8 650 638 5981 ABI PRISM 3100 Genetic Analyzer Press FAX 26 650 638 5891 DNA Synthesis Press FAX 21 650 638 5981 For Support On This Product Dial 1 800 831 6844 and Fluorescent DNA Press FAX Sequencing 22 650 638 5891 Fluorescent Fragment Press FAX Analysis includes GeneScan applications 23 650 638 5891 Integrated Thermal Cyclers Press FAX 24 650 638 5891 Biolnformatics includes BioLIMS BioMerge and Press CHA SQL GT applications 25 505 982 7690 PCR and Sequence Press FAX Detection 5 or call 240 453 4613 1 800 762 4001 and press 1 for PCR or 2 for Sequence Detection EMAT Telephone FAX 1 800 899 5858 and press 1 then press 6 508 383 7855 Peptide and Organic Press FAX Synthesis 31 650 638 598
18. boratory Manual Cold Spring Harbor Press 41 42 Worldwide Sales Offices Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches into 150 countries on six continents For international office locations please call our local office or refer to our web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free 1 800 345 5224 Fax 1 650 638 5884 Technical Support For technical support Toll Free 1 800 831 6844 ext 23 Fax 1 650 638 5891 www appliedbiosystems com D Applied AS Biosystems Printed in the USA 09 2010 Part Number 4303267E
19. d involves substituting dUTP for dTTP in the Reagent Master Mix and adding AmpErase UNG to the mix prior to amplification Kwok and Higuchi 1989 Longo et al 1990 PCR products from previous amplifications are not reamplified Misprimed nonspecific PCR products created before thermal cycling are degraded but native DNA template is not affected When dUTP replaces dTTP as a dNTP substrate in PCR AmpErase UNG treatment can remove up to 100 000 copies of contaminating amplicon per 25 uL reaction The 2 minute hold cycle at 50 C is necessary for optimum AmpErase UNG cleavage of the uracil deoxyribose linkage The 10 minute hold cycle at 95 C necessary to activate AmpliTaq Gold DNA Polymerase also cleaves the phosphate ester backbone of the PCR products that contained uracil nucleotides and reduces the AmpErase UNG activity substantially Because UNG is not completely deactivated during the 95 C incubation it is important to keep the reaction temperatures greater than 55 C to prevent amplicon degradation Do not use AmpErase UNG in subsequent amplification of dU containing PCR template such as in nested PCR protocols The UNG will degrade the dU containing PCR product preventing further amplification continued on next page 13 General PCR Although the protocol and reagents described above are capable of Practices degrading or eliminating large numbers of carried over PCR products we encourage users to use the following pr
20. de sequence two primers and a probe Sequence 4 For allelic discrimination each allele associated with a target has probe labeled with its own fluorescent reporter dye The shortest amplicons work the best Consistent results are obtained for amplicon ranges from 50 150 bp Primers are common and have complete homology for both alleles Design TaqMan Use the following guidelines Probe 4 Keep the G C content in the 20 80 range if possible Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more Gs should be avoided Donot put a on the 5 end Using Primer Express software the melting temperature Tm should be 65 67 C Select the strand that gives the probe with more Cs than Gs Position the polymorphic site approximately in the middle third of the sequence Adjust the probe lengths so that both probes have the same T 23 Design Primers Use the following guidelines Keep the G C content in 30 8096 range Avoid runs of an identical nucleotide This is especially true for guanine where runs of four or more Gs should be avoided Using Primer Express software the Tm should be 58 60 C The five nucleotides at the 3 end should have no more than two G and or C bases Place the forward and reverse primers as close as possible to the probe without overlapping the probe Order Reagents Refer to User Supplied Materials on page
21. e File menu choose Save as to save the plate 31 32 To optimize probe concentrations on the 7700 continued Step Action 7 From the Diagnostics submenu under the Instrument menu choose Advanced Options Under Miscellaneous Options select the Use Spectral Compensation for Endpoint checkbox as shown below Advanced Options Viewer CI Display mse in Multicomponent View x Display best fit in Raw Spectra View Analysis Spectra Components Use background in Spectra Components folder Ll Use pure spectra in Spectra Componentz folder Miscellaneous Options Set 7700 Exposure Time Use Spectral Compensation for Real Time EJ Use Spectral Compensation for Endpoint Note If you change the option a dialog box will appear telling you to quit the application and restart it to use the changes From the Analysis menu choose Analyze 88 L From the Analysis menu choose Allelic Discrimination 88 From the Export submenu under the File menu choose Multicomponent Export the Multicomponent file 11 Quit the Sequence Detection Systems software 12 Open the Multicomponent file exported from the Sequence Detection Systems software 13 Identify the probe ratio at which the FAM and TET multicomponent values are closest to each other 14 Use this probe ratio in your allelic discrimination assay continued on next page
22. e and Stability ice bb Rx EE RS 4 Instruments Required 5 User Supplied Materials gt Technical Support ci sss rinpa IA GA REC E 6 To Reach Us On the Web 6 Hours for Telephone Technical Support 6 To Reach Us by Telephone or Fax in North America 6 JDocumentsonDemand 9 To Reach Us by E Mail II EIA EA 6 Regional Offices Sales and Service 7 Preventing Contamination 13 OV rVIEW REPERI 13 Prevention of PCR Product Carryover 13 General PCR Practi es tecie wee ta ee EHE ee ws 14 Fluorescent Contaminants 14 Preparing Control Reactions and Control DNA Samples 15 OVELVIEW 2 eoa De Iv e e Mae NW eh ras 15 Prepare Controls and Unknowns 16 PCR Amplification Pe cece eens 18 Thermal Cycling Parameters 18 Real Time Run on the ABI PRISM 7700 19 Performing PCR on the GeneAmp 9600 and 9700 19 Allelic Discrimination on the ABI PRISM 7700 and 7200 20 OVerVleW remo RENE ett ea ARS 20 Analysis on the ABI PRISM 7700 or 7200 20 Alle
23. e or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Probes labeled at the 5 and 3 end and their use in the 5 nuclease assay are covered by patents issued and pending owned by Applied Biosystems TRADEMARKS Applied Biosystems AB Design ABI PRISM and MicroAmp are registered trademarks and ABI and Primer Express are trademarks of Applied Biosystems 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 Contents IntrOdUCHON van SANA A AEE BOREL 1 DNS SN EROR NORD AR ah AAA PARA CHU OR C R 1 5 Nuclease ASSAY se IAA A PRU bee ees 1 sequence Detection oisi RSE a ns 3 Allelic Discrimination 3 System Performance Guarantee 3 Demonstrated Performance 3 Materials and Eguipment 4 Kit Contents cs oec ere haces WE EC M MEER ae 4 Storag
24. ecautions and those referenced in Appendix C on page 40 to minimize sample cross contamination and PCR product carryover Fluorescent Contaminants 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 and dedicated equipment and supplies for sample preparation for PCR setup and for PCR amplification and analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes carefully Try not to splash or spray PCR samples Use positive displacement or air displacement pipettors with filter plugged tips Change tips after each use Keep reactions and components capped as much as possible Clean lab benches and equipment periodically with 1096 bleach solution Because sample protein and fluorescent contaminants may interfere with this assay and give false positive results it may be necessary to include a No Amplification Control tube that contains the sample and no enzyme If the absolute fluorescence of the No Amplification Control is greater than that of the No Template Control after PCR fluorescent contaminants may be present in the sample Preparing Control Reactions and Control DNA Samples Overview This procedure involves PCR amplification of the target DNA f
25. el 39 0 39 83891 Fax 39 0 39 838 9492 Czech Republic and Slovakia Praha Tel 420 2 61 222 164 Fax 420 2 61 222 168 The Netherlands Nieuwerkerk a d IJssel Tel 31 0 180 331400 Fax 31 0 180 331409 Denmark Naerum Tel 45 45 58 60 00 Fax 45 45 58 60 01 Norway Oslo Tel 47 23 12 06 05 Fax 47 23 12 05 75 Europe Finland Espoo Tel 358 0 9 251 24 250 Fax 358 0 9 251 24 243 Poland Lithuania Latvia and Estonia Warszawa Tel 48 22 866 40 10 Fax 48 22 866 40 20 France Paris Tel 33 0 1 69 59 85 85 Fax 33 0 1 69 59 85 00 Portugal Lisboa Tel 351 0 22 605 33 14 Fax 351 0 22 605 33 15 Germany Weiterstadt Tel 49 0 6150 101 0 Fax 49 0 6150 101 101 Russia Moskva Tel 7 095 935 8888 Fax 7 095 564 8787 Spain Tres Cantos Tel 34 0 91 806 1210 Fax 34 0 91 806 1206 South Africa Johannesburg Tel 27 11 478 0411 Fax 27 11 478 0349 Sweden Stockholm Tel 46 0 8 619 4400 Fax 46 0 8 619 4401 United Kingdom Warrington Cheshire Tel 44 0 1925 825650 Fax 44 0 1925 282502 Switzerland Rotkreuz Tel 41 0 41 799 7777 Fax 41 0 41 790 0676 South East Europe Zagreb Croatia Tel 385 1 34 91 927 Fax 385 1 34 91 840 Middle Eastern Countries and North Africa Monza Italia Tel 39 0 39 8389 481 Fax 39 0 39 8389 493 Africa English Speaking and West A
26. emplates See Appendix A Guidelines for Custom Applications on page 23 Using the Genomic Control DNA and protocol for the TaqMan Allelic Discrimination Demonstration Kit automated allele calls will be reported by the Sequence Detection System with a 99 7 confidence level The minimum and maximum detection range is from 10 100 ng of Genomic Control DNA which is approximately 104 105 copies of a single copy gene Materials and Equipment Kit Contents The TaqMan Allelic Discrimination Demonstration Kit P N 4303263 has been designed to provide 200 reactions of 50 uL each Experiments have been performed with the ABI PRISMO 7700 and ABI PRISM 7200 Sequence Detectors showing that a 25 uL final reaction volume will provide the same precision for TaqMan allelic discrimination assays We do not recommend final reaction volumes lower than 25 uL The contents of the TagMan Allelic Discrimination Demonstration Kit are listed in Table 1 Table 1 Kit Components Component Volume Description TagMan Universal PCR Master Mix 5 75 mL One bottle sufficient for 200 reactions of 50 uL each Probe and Primer Mix 3 45 mL Two tubes sufficient for 200 reactions of 50 uL each containing the following Forward primer 5 TGC CAG CTC AGC A 3 Reverse primer 5 GAG GTG GAA 3 TagMan Plasmid Allele 1 probe 5 GCA ACC GAT GCC CGT T TAMRA 3 TaqMan
27. es This method permits the analysis of thousands of samples per day with high sample to sample reproducibility The TaqMan Allelic Discrimination Demonstration Kit employs a probe technology that exploits the 57 3 nuclease activity of AmpliTaq Gold DNA Polymerase to allow direct detection of the PCR product by the release of a fluorescent reporter as a result of PCR This PCR system is optimized for yield AmpErase UNG is required for the prevention of PCR product carryover Longo et al 1990 For more information on the 5 nuclease assay refer to Lawyer et al 1989 Holland et al 1991 and Lyamichev et al 1993 Two TagMan probes are used in this allelic discrimination assay one probe for each allele in a two allele system Each probe consists of an oligonucleotide with a 5 reporter dye and a 3 quencher dye TET 6 carboxy 4 7 2 7 tetrachlorofluorescein is covalently linked to the 5 end of the probe for the detection of Allele 1 FAM 6 carboxyfluorescein is covalently linked to the 5 end of the probe for the detection of Allele 2 Each of the reporters is quenched by TAMRA 6 carboxy N N N N tetramethylrhodamine attached via a linker arm located at the 3 end of each probe When the probe is intact the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer F rster 1948 Lakowicz 1983 During PCR forward and reverse pr
28. he detector a Ifthe signal exceeds the dynamic range rerun the plate with a proportional reduction in the concentrations of both probes b If the signal is within the dynamic range proceed to step 5 5 From the Diagnostics submenu under the Instrument menu choose Advanced Options Under Viewer select the Display best fit in Raw Spectra View checkbox a Ifthe Raw Spectra View shows a poor fit for the Pure Dye files rerun the Pure Dye standards b Ifthe Raw Spectra View shows a good fit for the raw data proceed to the next step Note Updating your Pure Dye files every 90 days is a good practice for running allelic discrimination assays To do this use the Sequence Detection Systems Spectral Calibration Kit P N 4305822 Refer to ABI PRISM 7700 User Bulletin 4 Generating New Spectra Components P N 4306234 for instructions on creating new Pure Dye standards 37 38 Troubleshooting Custom Allelic Discrimination Applications continued Step Action 6 Rerun the reaction using an extension temperature of 64 C to improve the separation between populations 7 If the separation between populations still does not allow them to be called manually reinspect the probe sequences and samples to confirm that they have been labeled and run correctly Appendix B Real Time Detection on the ABI PRISM 7700 Threshold Cycle Real Time detection on the ABI PRism 7700 Sequence Detector monit
29. he following and deliver 50 pL of B1 B4 the mixture to each of the 8 wells 220 uL of 2X Master 132 uL of Probe and Primer Mix 441 of Plasmid Allele 1 standard 44 uL of TE buffer B5 B12 AL2 Combine the following and deliver 50 pL of the mixture to each of the 8 wells 220 uL of 2X Master 132 uL of Probe and Primer Mix 44 uL of Plasmid Allele 2 standard 44 uL of TE buffer C1 H12 Unknowns Combine the following and deliver 50 pL of UNKN the mixture to each of the 72 wells 2000 uL of 2X Master Mix 1200 uL of Probe and Primer Mix 400 uL of Genomic Control DNA 400 uL of TE buffer a TE buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 IMPORTANT With the ABI PRISM 7700 and ABI PRISM 7200 Sequence Detectors you must use MicroAmp Optical disposables Do not use MicroAmp Optical Tubes with the ABI PRISM 7200 PCR Amplification Thermal Cycling The thermal cycling parameters in Table 5 are used for the TaqMan Parameters Allelic Discrimination Demonstration Kit control reactions on the GeneAmp PCR Systems 9600 and 9700 and the ABI PRISM 7700 Sequence Detector The GeneAmp PCR System 9600 or GeneAmp PCR System 9700 in 9600 Emulation Mode is used to perform PCR amplification when the TaqMan LS 50B PCR Detection System or ABI PRISM 7200 Sequence Detector is used for fluorescence analysis IMPORTANT All reaction volumes are 50 uL The 2 minute 50 C step is requ
30. imers hybridize to a specific sequence of the target DNA The TagMan probe hybridizes to a target sequence within the PCR product The AmpliTaq Gold enzyme cleaves the TaqMan probe with its 5 3 nuclease activity The reporter dye and quencher dye are separated upon cleavage resulting in increased fluorescence of the reporter Figure 1 on page 2 The 3 end of the TaqMan probe is blocked to prevent extension of the probe during PCR Polymerization R Reporter Q Quencher Forward R Q Probe 5 Primer 5 3 5 5 3 Reverse Strand displacement Primer 5 3 3 5 5 ii Cleavage 5 gt gt 3 3 5 Polymerization completed LA 3 3 5 5 3 lt 0 Figure 1 The fork like structure dependent polymerization associated 5 3 nuclease activity of AmpliTaq Gold DNA Polymerase during one extension phase of PCR Lyamichev et al 1993 This process occurs in every cycle and does not interfere with the exponential accumulation of product The separation of the reporter dyes from the quencher dye results in increase in fluorescence for each of the FAM and TET reporters The increase in fluorescence is measured and is a direct consequence of target amplification during PCR Both primer and probe must hybridize to their targets for amplification and cleavage to occur The fluorescence signals are generated only if the target sequences for the probes are amplified du
31. ired for optimal AmpErase UNG activity The 10 minute 95 C step is required to activate AmpliTaq Gold DNA Polymerase Table 5 Thermal Cycling Conditions Thermal Cycler Times and Temperatures Initial Steps Each of 40 Cycles Melt Anneal Extend GeneAmp PCR HOLD HOLD CYCLE System 9600 or 2 min 10 min 15 sec 1 min 9700 50 95 95 62 ABI PRISM 7700 HOLD HOLD CYCLE Sequence 2 min 10 min 15 sec 1 min Detector 50 95 95 62 continued on next page 17 Real Time Run Use the following procedure to perform a Real Time run on the on the ABI PRISM 7700 Sequence Detector Refer to the ABI PRISM 7700 ABI PRISM 7700 Sequence Detector User s Manual for more information Performing PCR on the GeneAmp 9600 and 9700 Step Action 1 Create a Real Time plate document Refer to the ABI PRISM 7700 Sequence Detector User s Manual for details 2 Place the MicroAmp Optical 96 Well Reaction Plate in the ABI PRISM 7700 Sequence Detector 3 Perform a Real Time run using the thermal cycling conditions shown in Table 5 4 Save the Real Time plate results 5 Close the Sequence Detection Systems software 6 Leave the MicroAmp Optical 96 Well Reaction Plate in the ABI PRISM 7700 Sequence Detector Use the following procedure to amplify samples in the GeneAmp PCR System 9600 or GeneAmp PCR System 9700 in 9600 Emulation Mode
32. lic Discrimination on the LS 50B 22 LS 50B 5ettDgs 5iresv5vex acd KARAK e 22 Measure Fluorescence 22 Appendix A Guidelines for Custom Applications 24 Nine Step Program 1 0 2 a cece eee eee 24 Identify Target Sequence 24 Design TaqMan Probe 0 0 cece cece eee ene 24 Design Primers who e e Gad 25 Order Re gentsc ceres cet Ets KORO HERES 25 Quantitate Probes and Primers 25 Optimize Primer Concentrations 26 Optimize Probe Concentrations 29 Set Up and Run an Allelic Discrimination Plate 34 Appendix B Real Time Detection on the ABI PRISM 7700 40 Threshold Cycle San ee Revi E ase eR 40 Appendix C References eh An SA en SAMAN 41 Introduction Overview 5 Nuclease Assay The TaqMan Allelic Discrimination Demonstration Kit is a model assay to show the allelic discrimination capabilities of the Applied Biosystems Sequence Detection Systems It has been optimized for use with TaqMan Universal PCR Master Mix P N 4304437 Direct detection of polymerase chain reaction PCR product with no downstream processing is accomplished within minutes of PCR completion by measuring the increase in fluorescence of dye labeled DNA prob
33. luorescence is measured directly No thermal cycling is required The procedure is instrument dependent reflecting the optical differences between the ABI PRISM 7200 Sequence Detector page 28 and the ABI PRISM 7700 Sequence Detector page 31 ABI PRISM 7200 Sequence Detector Prepare the plate shown in the table below Use 50 uL of the indicated solution in each well Universal 1 pM 1 pM FinalFAM Final TET PCR FAM TET Total Probe Probe Master Probe Probe Deionized Volume Conc Conc Wells Mix pL uL uL Water uL Well pL nM nM 1 4 25 2 5 2 5 20 50 50 50 A5 A8 25 2 5 5 0 17 5 50 50 100 A9 A12 25 2 5 7 5 15 50 50 150 B1 B4 25 2 5 10 12 5 50 50 200 B5 B8 25 2 5 12 5 10 50 50 250 B9 B12 25 2 5 15 7 5 50 50 300 C1 C4 25 2 5 17 5 5 50 50 350 To optimize probe concentrations on the 7200 Step Action 1 Launch the Sequence Detection Systems software 2 If the untitled plate that opens is not the correct Allelic Discrimination plate for your instrument a Close the untitled plate b From the File menu choose New Plate 88 N c Inthe New Plate dialog box choose Allelic Discrimination from the Plate Type pop up menu The Run pop up menu will disappear d Choose the correct instrument from the Instrument pop up menu Note correct plate type and instrument can be set in Preferences under the Edit menu Selec
34. nm Slit nm nm Slit nm Filter nm FAM 488 4 518 8 515 TET 488 4 538 8 515 TAMRA 488 4 582 8 515 To perform allelic discrimination use the following procedure Refer to the LS 50B Luminescence Spectrometer User s Manual for details The macro receives data from your output file and generates multicomponented data The data is then normalized for each allele and a genotype call is made for Homo 1 homozygote 1 Homo 2 homozygote 2 or Hetero 1 2 heterozygote Samples run from the Genomic Control DNA included in this kit should all receive Hetero 1 2 calls Step Action 1 Transfer the contents of each optical tube from the PCR amplification reactions into the corresponding well of a 96 Well Microplate Portvair Be sure to follow the allelic discrimination plate configuration shown in Figure 2 on page 15 2 Under the Setup Instrument tab configure the TaqMan LS 50B PCR Detection System as shown in Table 6 Run the plate read Name and store the output file Double click on the Standard WPR Multicomponenting Macro When prompted for the spreadsheet name type gtypewpr xls Select the location of your output file macro analyzes the data and makes genotype calls 7 Name and save your spreadsheet continued on next page 21 Troubleshooting on the TaqMan LS 50B PCR Detection System normalized results in plotted data
35. ollowed by fluorescence analysis When performing allelic discrimination using the Plasmid Allele 1 and Plasmid Allele 2 standards the analysis requires that the controls and samples shown below in Figure 2 be run For custom applications see Appendix A on page 23 Note TaqMan LS 50B PCR Detection System uses a Buffer well which must be placed in position A1 The ABI PRISM 7700 and 7200 Sequence Detectors do not use Buffer wells Eight No Template Control wells NTC Eight Plasmid Allele 1 wells AL1 Eight Plasmid Allele 2 wells AL2 Seventy two Genomic Control DNA wells UNKN IMPORTANT Eight replicates of No Template Control Plasmid Allele 1 and Plasmid Allele 2 must be run to make allele calls at a 99 7 confidence level using the automated allele calling routine Manual allele calling with less than eight replicates is possible Refer to Chapter 4 of the ABI PRISM 7200 Sequence Detector User s Manual Figure 2 Plate diagram showing placement of control and sample reactions continued on next page 15 Prepare Controls Prepare reactions in a MicroAmp Optical 96 Well Reaction Plate The and Unknowns plate wells should contain the following Table 4 Plate Well Setup Well If preparing Then A1 A8 NTC Combine the following and deliver 50 pL of the mixture to each of the 8 wells 220 uL of 2X Master Mix 132 uL of Probe and Primer Mix 88 uL of TEA buffer 9 12 AL1 Combine t
36. ors fluorescence and calculates R during each PCR cycle The threshold cycle or C7 value is the cycle at which a statistically significant increase in AR the difference between reporter fluorescence in the sample and that in the No Template Control is first detected Figure 4 On the graph of R versus cycle number the threshold cycle occurs when the Sequence Detection Application begins to detect the increase in signal associated with an exponential growth of PCR product For example in a series of similar reactions where primer concentrations are varied the optimum conditions are those that give the lowest value Rnt Sample Rn Threshold Baseline Cycle Number Figure 4 Amplification plot R versus cycle number 39 Appendix C References Ausubel F M Brent R Kingstin R E Moore D D Seidman J G Smith J A and Struhl K eds 1987 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience John Wiley and Sons New York Delort A M Duplaa A M Molko D and Teoule R 1985 Excision of uracil residues in DNA mechanism of action of Escherichia coli and Micrococcus luteus uracil DNA glycosylases Nucleic Acids Res 13 319 335 F rster V Th 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Ann Phys Leipzig 2 55 75 Gelfand D H and White T J 1990 Thermostable DNA Polymerases In Innis M A Gelfand D H
37. pectra in Spectra Components folder Miscellaneous Options L Set 7700 Exposure Time Use Spectral Compensation for Real Time Use Spectral Compensation for Endpoint If you change the option a dialog box will appear telling you to quit the application and restart it to use the changes From the Analysis menu choose Analyze 88 L From the Export submenu under the File menu choose Results Export the Results file 10 Quit the Sequence Detection Systems software 11 Open the Results file exported from the Sequence Detection Systems software 27 To optimize primer concentrations continued Step Action 12 Tabulate the results for Ry and if using the ABI PRISM 7700 instrument Choose the minimum forward and reverse primer concentrations that yield the maximum R and minimum C7 13 Use these primer concentrations in your allelic discrimination assay 14 If wells 01 04 are different from wells 05 08 check for sources of contamination If a run with fresh reagents still shows significant differences between these wells remove possible interactions between primers and probes by redesigning one of the primers Optimize Probe The purpose of this procedure is to determine the probe concentrations Concentrations that give the most reliable autocalls The initial fluorescence signals from the two probes are matched approximately F
38. ring PCR Because of these requirements non specific amplification is not detected For information on release of a fluorescent reporter during the PCR refer to Lee et al 1993 and Livak et al 1995 continued on next page Sequence Detection Allelic Discrimination System Performance Guarantee Demonstrated Performance The Sequence Detection Systems from Applied Biosystems are used to measure the increase of reporter fluorescence following PCR Reporter signals are normalized to the emission of a passive reference Rn Emission Intensity of Allele 1 Reporter AL1 Emission Intensity of Passive Reference Rn Emission Intensity of Allele 2 Reporter AL2 Emission Intensity of Passive Reference These parameters are used in the Allelic Discrimination analysis software described on pages 19 21 The TaqMan Allelic Discrimination Demonstration Kit illustrates discrimination between the alleles of a two allele system It contains enough PCR reagents for up to 200 reactions of 50 uL each During amplification the Plasmid Allele 1 and Plasmid Allele 2 standards supplied with the kit generate reporter fluorescent signals such that allele calls may be made on unknown samples Allele 1 and Allele 2 probes supplied in the Probe and Primer Mix with the TaqMan Allelic Discrimination Demonstration Kit can be used with the specific Genomic Control DNA included in the kit Custom probes must be designed for detection of any other t
39. sia Fairlands South Africa Tel 27 11 478 0411 Fax 27 11 478 0349 All Other Countries Not Listed Warrington UK Tel 44 0 1925 282481 Fax 44 0 1925 282509 Japan Japan Hatchobori Chuo Ku Tokyo Tel 81 3 5566 6100 Fax 81 3 5566 6501 11 Eastern Asia China Oceania Australia Scoresby Victoria Malaysia Petaling Jaya Tel 61 3 9730 8600 Tel 60 3 758 8268 Fax 61 3 9730 8799 Fax 60 3 754 9043 China Beijing Singapore Tel 86 10 6238 1156 Tel 65 896 2168 Fax 86 10 6238 1162 Fax 65 896 2147 Hong Kong Taiwan Taipei Hsien Tel 852 2756 6928 Tel 886 2 2698 3505 Fax 852 2756 6968 Fax 886 2 2698 3405 Korea Seoul Tel 82 2 593 6470 6471 Fax 82 2 593 6472 Thailand Bangkok Tel 66 2 719 6405 Fax 66 2 319 9788 Preventing Contamination Overview Prevention of PCR Product Carryover The DNA amplification capability of the PCR process makes special laboratory practices necessary Small levels of DNA carryover from samples with high DNA concentrations from the Genomic Control DNA or from previous PCR amplifications can result in product even in the absence of added template DNA See the references in Appendix C on page 40 for more information on PCR and laboratory practices for preventing contamination Treatment with uracil N glycosylase UNG EC 3 2 2 can prevent the reamplification of carryover PCR products This metho
40. t wells A1 C12 as unknowns UNKN Click the Show Analysis button Click the Post PCR Read button The software will perform the Plate Read From the File menu choose Save as to save the plate 29 30 To optimize probe concentrations on the 7200 continued Step Action 7 From the Diagnostics submenu under the Instrument menu choose Advanced Options Under Miscellaneous Options deselect the Use Spectral Compensation for Endpoint checkbox Advanced Options Viewer CI Display mse in Multicomponent View x Display best fit in Raw Spectra View Analysis Spectra Components Use background in Spectra Components folder Ll Use pure spectra in Spectra Componentz folder Miscellaneous Options Set 7700 Exposure Time Use Spectral Compensation for Real Time Use Spectral Compensation for Endpoint If you change the option a dialog box will appear telling you to quit the application and restart it to use the changes From the Analysis menu choose Analyze 88 L From the Analysis menu choose Allelic Discrimination 88 K From the Export submenu under the File menu choose Multicomponent Export the Multicomponent file 11 Quit the SDS software 12 Open the Multicomponent file exported from the SDS software 13 Identify the probe ratio at which the FAM and TET multicomponent values are closest to each other
41. te that opens is not the correct Allelic Discrimination plate for your instrument a Close the untitled plate b From the File menu choose New Plate 88 N c Inthe New Plate dialog box choose Allelic Discrimination from the Plate Type pop up menu The Run pop up menu will disappear d Choose the correct instrument from the Instrument pop up menu Note correct plate type and instrument be set in Preferences under the Edit menu Set up the plate as shown in Figure 2 on page 15 Note See your instrument user s manual for more information Click the Show Analysis button Click the Post PCR Read button The software will perform the Plate Read From the File menu choose Save as to save the plate Click the Show Analysis button 19 To perform allelic discrimination continued Step Action 8 From the Analysis menu choose Analyze 88 L The computer analyzes the data 9 From the Analysis menu choose Allelic Discrimination 88 The Allelic Discrimination Viewer appears 10 Examine data to confirm that allele calls have been made Allelic Discrimination on the LS 50B LS 50B Settings The excitation and emission settings for the TaqMan LS 50B PCR Measure Fluorescence Detection System are summarized in Table 6 Table 6 TaqMan LS 50B PCR Detection System Settings Excitation Excitation Emission Emission Emission Dye
42. untry code ready 011 precedes the country code b Press 1 to order an index of available documents and have it faxed to you Each document in the index has an ID number Use this as your order number in step d below Call 1 858 712 0317 from a touch tone phone a second time d Press 2 to order up to five documents and have them faxed to you To Reach Us by Contact technical support by e mail for help in the following product E Mail areas For this product area Use this e mail address Chemiluminescence info appliedbiosystems com Genetic Analysis galab appliedbiosystems com LC MS apisupport sciex com PCR and Sequence Detection pcrlab appliedbiosystems com Protein Sequencing Peptide and DNA Synthesis corelab appliedbiosystems com Regional Offices Sales and Service your local Applied Biosystems service representative If you are outside the United States and Canada you should contact The Americas United States Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 Tel 650 570 6667 800 345 5224 Fax 650 572 2743 Latin America Del A Obregon Mexico Tel 305 670 4350 Fax 305 670 4349 Europe Austria Wien Hungary Budapest Tel 43 0 1 867 35 750 Tel 36 0 1 270 8398 Fax 43 0 1 867 35 75 11 Fax 36 0 1 270 8288 Belgium Tel 32 0 2712 5555 Fax 32 0 2 712 5516 Italy Milano T
43. water or TE buffer Major laboratory suppliers MLS 10 mM Tris HCl 1 mM EDTA pH 8 0 TagMan Universal PCR Master Applied Biosystems P N 4304437 Mix MicroAmp Optical 96 Well Applied Biosystems P N 403012 Reaction Plate and Optical Caps 96 Well Microplate Portvair Applied Biosystems P N L225 1692 Primer Express software Applied Biosystems P N 402089 Note The ABI PRISM 7700 and ABI PRISM 7200 Sequence Detectors use the MicroAmp Optical 96 Well Reaction Plate and MicroAmp Optical Caps The LS 50B PCR Detection System uses the 96 Well Microplate Portvair Technical Support To Reach Us on the Web Hours for Telephone Technical Support To Reach Us by Telephone or Fax in North America Applied Biosystems web site address is http www appliedbiosystems com techsupport We strongly encourage you to visit our web site for answers to frequently asked questions and to learn more about our products You can also order technical documents and or an index of available documents and have them faxed or e mailed to you through our site see the Documents on Demand section below In the United States and Canada technical support is available at the following times Product Hours Chemiluminescence 9 00 a m to 5 00 p m Eastern Time LC MS 9 00 a m to 5 00 p m Pacific Time All Other Products 5 30 a m to 5 00 p m Pacific Time See the Region
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