MSI Analysis System, Version 1.2 Technical Manual TM255
Contents
1. Ld bi eee ee j AY A i ANd a re B Penta D 80 100 120 140 160 180 Penta CO i 80 100 140 120 160 180 1200 800 400 O beenmerg tas Figure 1 The MSI Analysis System Version 1 2 A single normal genomic DNA template 1 2ng was amplified using the MSI Analysis System and the PCR products were analyzed using an ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary Panel A An electropherogram showing the five mononucleotide loci two pentanucleotide loci and Internal Lane Standard 600 peaks Panel B An electropherogram showing peaks of the fluorescein labeled loci BAT 26 and Penta D Panel C An electropherogram showing the peaks of JOE labeled loci NR 21 BAT 25 and MONO 27 Panel D An electropherogram showing the peaks of the TMR labeled loci NR 24 and Penta C 10070TA The mononucleotide repeat markers included in the MSI Analysis System were selected for high sensitivity and specificity to alterations in samples containing mismatch repair defects These mononucleotide repeat markers are quasi monomorphic that is almost all individuals are homozygous for the same common allele for a given marker see Section 10 C for allele frequencies Use of monomorphic markers simplifies data interpretation The pentanucleotide repeat markers were selected for their high level of polymorphism and low degree of MSI to uniquely identify sampl2. 2004 2007 2009 2011 2012 2014 Promega Corporation All Rights Reserved MagneSil Maxwell PowerPlex and QuantiFluor are registered trademarks of Promega Corporation ReliaPrep is a trademark of Promega Corporation ABI PRISM GeneMapper MicroAmp and POP 4 are registered trademarks of Applera Corporation AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc Applied Biosystems is a registered trademark of Applied Biosystems GenBank is a registered trademark of U S Department of Health and Human Services Hi Di is a trademark of Applera Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com
3. microcentrifuge tubes MicroAmp reaction tube strips or MicroAmp optical 96 well reaction plates Applied Biosystems 1 5ml microcentrifuge tubes aerosol resistant pipette tips o AmpliTaq Gold DNA polymerase Life Technologies Cat N8080242 The MSI Analysis System is optimized to amplify 1 2ng of genomic DNA in a 10ul reaction volume using the protocols detailed below However optimization of input DNA amounts should be performed to adjust for variations in DNA yield and quality due to differences in samples DNA isolation methods or both Using excessive amounts of DNA template may result in peak heights exceeding the linear detection range of the CE instruments Use of insufficient DNA template can result in low PCR yields and peak heights may fall below detection limits 50RFU Accurate quantitation of template DNA is highly recommended We recommend quantitating the FFPE template DNA using the Promega QuantiFluor ONE dsDNA System Cat E4870 or E4871 or QuantiFluor dsDNA System Cat E2670 Testing at Promega has shown that DNA quantitation using the QuantiFlour ONE dsDNA System with the Quantus Fluorometer provided results more similar to those of qPCR from DNA samples extracted from three FFPE tissue types as compared to the NanoDrop 2000 instrument 1 The MSI Analysis System is optimized for use with the GeneAmp PCR System 9600 and 9700 thermal cyclers 4 A Amplification Setup Note We strongly recom
4. software The proper panels and bins files for use with GeneMapper software can be obtained from the Promega web site at www promega com resources tools msi panels and bins for genemapper software Note For recommendations on other polymers or capillaries contact Promega Technical Services at genetic promega com Table 1 The MSI Analysis System Locus Information Major Repeat Size Range K562 Alleles Primer Marker Name GenBank Number Sequence bp bp Dye NR 21 XM_033393 A 94 101 101 JOE BAT 26 U41210 A 103 115 113 FL BAT 25 L04143 A 114 124 122 JOE NR 24 X60152 A 130 133 130 TMR MONO 27 AC007684 A 142 154 150 JOE Penta C AL138752 AAAAG 143 194 164 174 TMR Penta D AC000014 AAAAG _ 135 201 168 187 FL Allele sizes were determined using the ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary Rare alleles outside of these size ranges may exist Allele sizes may vary when using different polymers or instrument configurations TMR carboxy tetramethylrhodamine FL fluorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxyfluorescein 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Plot Setting Cell ID Allelic Ladder vE Panes 4 JFM TME EA i faa mE thi A ai Aa de Me M oh Yk g
5. Revised 8 14 1 Description 1 A The MSI Analysis System The MSI Analysis System Version 1 26 is a fluorescent PCR based assay to detect microsatellite instability MSI in human cells Typically MSI analysis involves comparing allelic profiles of microsatellite markers generated by amplification of DNA from matching normal and test samples which may be mismatch repair MMR deficient Alleles that are present in the test sample but not in corresponding normal samples indicate MSI The MSI Analysis System includes fluorescently labeled primers for co amplification of seven markers including five mononucleotide repeat markers BAT 25 BAT 26 NR 21 NR 24 and MONO 27 and two pentanucleotide repeat markers Penta C and Penta D Figure 1 and Table 1 The mononucleotide markers are used for MSI determination and the pentanucleotide markers are used to detect potential sample mixups or contamination An internal lane size standard Figure 2 is added to the amplified samples to assure accurate sizing of alleles and adjust for run to run variation The PCR products are separated by capillary electrophoresis CE using an ABI PRISM 310 or 3100 or Applied Biosystems 3130 or 3130x Genetic Analyzer and the output data may be analyzed with GeneMapper software Applied Biosystems to determine MSI status of test samples To simplify data analysis we created panels and bins text files to allow automatic assignment of genotypes using GeneMapper
6. X txt then Import 9 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 20 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com 8 C Creating an Analysis Method Using GeneMapper Software Versions 4 0 and 4 1 Select Tools then GeneMapper Manager Select the Analysis Methods tab Select New and a new analysis method dialog box will open Select Microsatellite Select OK Enter a descriptive name for the analysis method such as Promega MSI Select the Allele tab Figure 8 Select the bin set corresponding to the Promega MSI System Promega_MSI Ensure that the Use marker specific stutter ratio if available box is checked n a Enter the values shown in Figure 8 for proper filtering of peaks when using the MSI Analysis System These filter settings will allow the user to display plotted data with the highest peak labeled in each set of amplified fragments within each mononucleotide marker range These settings can be changed if an alternative labeling strategy is desired For an explanation of the proper usage and effect of these settings refer to your GeneMapper software versions 4 0 and 4 1 documentation Analysis Method Editor Microsatellite General Allele Peak Detector Peak
7. capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 Open the ABI PRISM 3100 Data Collection Software 2 Open a new plate record Name the plate and select GeneScan Select the plate size 96 well or 384 well Select Finish 3 Complete the plate record spreadsheet for the well you have loaded Enter appropriate information into the Sample Name column Figure 3 12 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega Plate Editor x File Edit Plate Name MSI_Analysis_System_V1 2 Well Sample Name Color Info Color Comment Project Name Dye Set Run Module 1 A1 1_Normal_Sample 3100_Projectt iF GeneScan36_POP4DefaultModule m oe m 5 e L 1_Tumor_Sample 3100_Project1 GeneScan36_POP4DefaultModule ji T 2_Normal_Sample 3100_Project1 GeneScan36_POP4DefaultModule t T i T ji eee aka 2_Tumor_Sample E 00_Projectt GeneScan36_POP4DefaultModule 3100_Projectt_ 4769TA Figure 3 The Plate Editor 4 5 6 fi 10 In the Project Name column select 3100_Project1 from the drop down menu In the Dye Set colum
8. com O Oo Promega 8 Monitor the electrophoresis by observing the raw data and status windows Each sample will take approximately 30 minutes for syringe pumping sample injection and sample electrophoresis 6 Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 Materials to Be Supplied by the User dry heating block water bath or thermal cycler crushed ice or an ice water bath aerosol resistant pipette tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 Applied Biosystems o 10X genetic analyzer buffer with EDTA Applied Biosystems sample tubes and septa for the 3100 Applied Biosystems Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 Cat DG4650 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamid
9. o 2 2 co 2 foCcCcooeoNMN co OO we Oo co oo oO fo fF o NR 24 Size Caucasian African Asian MONO 27 bp American American American Size Caucasian African Asian bp American American American NR 21 Size Caucasian African Asian bp American American American Allele sizes were determined using the ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary Note that allele sizes may vary when using different polymer or instrument configurations Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 27 www promega com TM255 Revised 8 14 Promega 10 D Summary of Changes The following change was made to the 8 14 revision of this document The module selected in Section 5 C Step 4 was corrected to GS STR POP4 1ml F Module Section 8 G was updated to reflect the fact that the panels and bins text files download package no longer includes an informational document and an example plot settings file U S Pat Nos 7 749 706 and 7 902 343 U S Pat No 6 238 863 Chinese Pat No ZL99802696 4 European Pat No 1058727 Japanese Pat No 4494630 and other patents pending Licensed under U S Pat Nos 6 566 053 STR loci are the subject of U S Pat No RE 37 984 German Pat No DE 38 34 636 C2 and other patents issued to the Max Planck Gesellschaft zur Forderung der Wissenschaften e V Germany
10. over 99 of the time For data analysis the GeneMapper analysis software is required The software allows manual and automated analysis of the raw data and generates electropherograms with accompanying data tables displaying PCR fragment lengths in base pairs and peak heights in RFU Results should be reviewed to eliminate artifact peaks caused by bleedthrough stutter or CE spikes Figures 6 and 7 Refer to the GeneMapper software user s manual for detailed instructions on fragment analysis and genotyping To simplify data analysis we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper software The proper panels and bins text files for use with GeneMapper software can be obtained from the Promega web site at www promega com resources tools msi panels and bins for genemapper software Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 19 www promega com TM255 Revised 8 14 4634TA Figure 7 Bleedthrough or pull up peaks Small peaks indicated by arrows that overlap much higher peaks in another color channel are called bleedthrough or pull up peaks Bleedthrough can occur when peak heights are excessive or if a poor or incorrect matrix was applied to the samples 8 B Importing Panels and Bins Text Files for GeneMapper Software Versions 4 0 and 4 1 To facilitate analysis of data gene
11. was degraded into smaller fragments with the larger loci showing diminished yield Insufficient template DNA Use the recommended amount of template DNA Excess amounts of DNA We recommend 1 2ng of DNA Amplification of gt 2ng DNA template may result in an imbalance in yields with the smaller loci showing more amplification product than the larger loci Use less DNA template or reduce the number of cycles in the amplification program by 2 or more cycles 10 Appendix 10 A Reference 1 Quantitating DNA from FFPE samples using the QuantiFluor ONE dsDNA System and the Quantus Fluorometer Application Note AN268 Promega Corporation 10 B Related Products Product Size Cat Maxwell 16 MDx Instrument each AS3000 Maxwell 16 FFPE Tissue LEV DNA Purification Kit 48 preps AS1130 ReliaPrep FFPE gDNA Miniprep System 10 reactions A2351 100 reactions A2352 MagneSil Genomic Fixed Tissue System 100 samples MD1490 QuantiFluor ONE dsDNA System 100 reactions E4871 500 reactions E4870 QuantiFluor dsDNA System Iml E2670 26 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com 10 C Allele Frequencies for Mononucleotide Repeat Loci BAT 25 BAT 26 Size Caucasian African Asian Size Caucasian African Asian bp American American American bp American American American N vo O
12. 99 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega 8 E Processing Data 1 Inthe File menu select New Project then Microsatellite 2 Import sample files into a new project using the Add Samples to Project selection under the File menu 3 Inthe Sample Type column use the drop down menu to select Sample Positive Control or Negative Control as appropriate 4 Inthe Analysis Method column select the analysis method created in Section 8 C 5 Inthe Panel column select Promega MSI from the Promega MSI folder This is the panels file that was imported in Section 8 B 6 Inthe Size Standard column select the size standard that was created in Section 8 D 7 If analyzing data from an ABI PRISM 310 Genetic Analyzer ensure that the appropriate matrix file is selected in the Matrix column 8 Select Analyze green arrow button to start data analysis 8 F Reviewing the Size Standard When correctly labeled the ILS 600 should give a Sizing Quality score near 1 0 Problems with ILS analysis extra peaks mislabeling etc will generate a Sizing Quality score of between 0 0 and 1 0 and may result in sample analysis failure If analysis fails but the fragments were assigned correctly the Sizing Quality can be overridden by selecting Override SQ The samples then can be re analyzed For more information refer to yo
13. Genetic Analyzer For answers to questions about matrix generation contact Promega Technical Services at genetic promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 9 www promega com TM255 Revised 8 14 O 5 B Sample Preparation The Internal Lane Standard 600 is included in each CE injection to standardize analysis of amplified samples and uses a fourth color 1 Prepare a loading cocktail by combining and mixing the Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 1 0ul ILS 600 24ul Hi Di formamide x injections 2 Combine 25ul of prepared loading cocktail and 1ul of amplified sample Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation section below 3 Denature samples by heating at 95 C for 3 minutes and immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading 4 Place tubes in the appropriate autosampler tray 5 Place the autosampler tray in the instrument and close the instrument doors 5 C Instrument Preparation Refer to the ABI PRISM Genetic Analyzer user s manual
14. ILS 600 and 9 0ul of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 25ul of ILS 600 and 9 75ul of Hi Di formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10ul of formamide internal lane standard mix into each well 4 Add 1ul of amplified sample Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation section below 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 7 C Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze the samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and Applied Biosystems 3130 or 3130x Genetic Analyzer with the following exceptions 1 Inthe Module Manager select New Select Regular in the Type drop
15. Quality Quality Flags Bin Set Promega msi a Marker Repeat Type IV Use marker specific stutter ratio if available Values for dinucleotide repeats are calculated automatically Mono Tr Tetra Penta Hexa Cut off value 0 0 bo o po 0 0 Plus ratio Joss foo foo foo foo Plus distance bs foo foo foo foo Stutter ratio bo po o po po oo oo oo oo oo ToS ies less s 75 ies Range Fitter Factory Defaults x Cancel Figure 8 The Analysis Method Editor The PlusA ratio setting for Mono repeats is 0 9999 6975TA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 21 www promega com TM255 Revised 8 14 Promega 8 C Creating an Analysis Method Using GeneMapper Software Versions 4 0 and 4 1 continued 10 Select the Peak Detector tab We recommend the settings shown in Figure 9 Peak amplitude thresholds should be determined by the individual laboratory based on desired sample peak intensity range dynamic range of the instrument and noise or background signal that is observed in analyzed data Peak amplitude thresholds between 50RFU and 200RFU are commonly used 11 Select OK to save your settings Analysis Method Editor Microsatellite General Allele Peak Detector Peak Quality Quality Flags Peak Detection Algorithm advanced he Ranges Peak Detection Analysis siz
16. TECHNICAL MANUAL MSI Analysis System Version 1 2 Instructions for Use of Product MD1641 For Research Use Only Not for use in diagnostic procedures Q Revised 8 14 TM255 All technical literature is available at www promega com protocols Visit the web site to verify that you are using the most current version of this Technical Manual E mail Promega Technical Services if you have questions on use of this system techserv promega com TD DDPO cca wens scsi cavess voces cauiesces esa cnius sabes cesasaws decease OEE E OE EEE NOE E ATEEN 2 Pi The MSI Analysis 99 SUC UN cacao saceuceuneauaseuencsvunsst cen conesereuescevessnuuieuneauaeudaxstuners NEE NN EEEE 2 1 B Microsatellite Instability Overview sccssccsccssscssccesccescesccesccesccsscesscnsccnscesccssccesccescosccsccescoess 4 1 C The Internal Lane Standard 600 ssessessesseososeoseoseosesseosessoseosessesecseesecseoseseesecseoseoseoseseoseoseoseose 4 2 Product Components and Storage Conditions esseseeseeseeseoseosesseseosecseosessesecsecseoscsecsecsecseoseoseseoseoseoseos 5 3 DNA TE on MeMo iir aie a aN E E EEEE AEE NENS 5 4 DNA Amplification Using the MSI Analysis System ssccsscsssccsccssccnsccsscesscesscnscesscesecesccescesccsceescees 6 4 A Amplification Setup eesseoseoseessesseosecsecoseoseesscsseoseosecoseosecosscsecoseosecoseosecsscseeoseosecsseosecseoseesseoseese 6 4 B Amplification Thermal Cycling eses
17. croll to the right In the Results Group 1 column select the desired results group In the Instrument Protocol 1 column select the protocol you created in Step 2 Be sure this information is present for each row that contains a sample name Select OK Notes 1 To create a new results group select New in the drop down menu in the Results Group column Select the General tab and enter a name Select the Analysis tab and select GeneMapper Generic in the Analysis type drop down list 2 Itis helpful to use names with similar initial characters when naming matched samples so that these samples remain together when analysis results are sorted 5 Place samples in the instrument and close the instrument doors 6 Inthe spectral viewer confirm that dye set F is active and set the correct active calibration for dye set F 7 Inthe run scheduler locate the plate record that you just created in Steps 3 and 4 and click once on the name to highlight it 8 Once the plate record is highlighted click the plate graphic that corresponds to the plate on the autosampler that contains your amplified samples 9 When the plate record is linked to the plate the plate graphic will change from yellow to green and the green Run Instrument arrow becomes enabled 10 Click on the green Run Instrument arrow on the toolbar to start the sample run 11 Monitor electrophoresis by observing the run view array or capillaries viewer wi
18. ctions Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If the peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0ul of ILS 600 and 9 0ul of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 25ul of ILS 600 and 9 75ul of Hi Di formamide 2 Vortex for 10 15 seconds 3 Pipet 10ul of formamide internal lane standard mix into each well 4 Add 1ul of amplified sample Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time injection voltage or the amount of product mixed with loading cocktail may need to be increased or decreased Use the Module Manager in the data collection software to modify the injection time or voltage in the run module see Instrument Preparation section below 5 Centrifuge plate briefly to remove air bubbles from the wells if necessary 6 Denature samples at 95 C for 3 minutes then immediately chill on crushed ice or in an ice water bath for 3 minutes Denature samples just prior to loading the instrument 6 C Instrument Preparation Refer to the ABI PRISM 3100 Genetic Analyzer user s manual for instructions on cleaning the pump blocks installing the
19. d plate records table and the Run Instrument button becomes enabled Figure 4 13 Select Run Instrument on the tool bar to start the sample run 14 Monitor electrophoresis by observing the run status array and capillary views windows in the collection software Each run 16 samples will take approximately 45 minutes 4 3100 Data Collection Software Version 1 1 Fie Yiew Instrument Tools Service Help Plate View Run view Status View Array View Capillary View Pending Plate Records Status pending pending Wells 96 96 Linked Plate Records A Plate Name Application Wells Status RBH_A GS 96 loading BIRBH_B GS 96 pending Processed Plate Records Plate Name Application Wells Status 96 DK310_D13_co GS processed a DK310_ac los 96 processed DK310_ C 02 les E processed DK310_Seq_ Taq GS 96 processed LF_2 19 04 GS 96 processed WMA _2_12_04 GS 96 processed New Uplink pelete Import Waiting for Oven to Stabilize at Run Temperature Figure 4 The Plate View tab 14 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega 7 Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection So
20. ding Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary Amplification reaction components were not added to the bottom of the tube or well Gently tap the tube or plate to move the liquid to the bottom or centrifuge briefly 24 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega S toms Causes and Comments Excessive fluorescent signal for allele peaks Too much template DNA e Make sure DNA was accurately quantitated and use 1 2ng in PCR Dilute PCR product 1 5 to 1 10 in sterile deionized water or 1X Gold ST R Buffer prior to preparing loading solution Extra peaks visible in one or all dye colors Microsatellite artifacts e g stutter Amplification of microsat ellite loci generated artifacts that appear as smaller peaks 1bp above or below the prominent mononucleotide repeat allele or 5bp below a pentanucleotide repeat allele Stutter product peak heights will be higher if too much template DNA was used Excess amount of DNA We recommend 1 2ng of DNA Amplification of gt 2ng of DNA template may result in a higher number of stutter products Use less DNA template or reduce the number of cycles in the amplification program by 2 or more cycles Pull up or bleedthrough Pull up can occur when p
21. down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Confirm that the injection time is 5 seconds and the injection voltage is 3kV Lengthen the run time to 2 000 seconds Give a descriptive name to your run module and select OK Note Sensitivities of instruments may vary The injection time and voltage may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds and for the injection voltage is 1 3kV 2 Inthe Protocol Manager select New Type a name for your protocol Select Regular in the Type drop down list and select the run module you created in the previous step in the Run Module drop down list Lastly select F in the Dye Set drop down list Select OK 16 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega 3 Inthe Plate Manager create a new plate record as described in the instrument user s manual In the dialog box that appears select GeneMapper Generic in the Application drop down list and select the appropriate plate type 96 well Add entries in the owner and operator windows and select OK Note If autoanalysis of sample data is desired refer to the instrument user s manual for instructions 4 Inthe GeneMapper plate record enter sample names in the appropriate cells S
22. e 6 A Spectral Calibration The PowerPlex Matrix Standards 3100 3130 Cat 4 DG4650 is required for spectral calibration on the ABI PRISM 3100 Genetic Analyzer The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to generate a matrix on the ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary For protocols and additional information about spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBDO22 which is available online at www promega com protocols Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary Spectral calibration must be performed for each ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary For answers to questions about spectral calibration contact Promega Technical Services at genetic promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 11 www promega com TM255 Revised 8 14 6 B Sample Preparation The Internal Lane Standard 600 is included in each CE injection to standardize analysis of amplified samples and uses a fourth color 1 Prepare a loading cocktail by combining and mixing the Internal Lane Standard 600 ILS 600 and Hi Di formamide as follows 0 5ul ILS 600 9 5ul Hi Di formamide x inje
23. e MSI Analysis System Protocols to prepare and use this internal lane standard are provided in Sections 5 B 6 B and 7 B 1 200 1 000 800 600 60 80 120 140 160 180 225 250 275 325 350 375 425 450 475 400 200 5751TA 0 Figure 2 Internal Lane Standard 600 4 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega 2 Product Components and Storage Conditions PRODUCT SIZE CAT MSI Analysis System Version 1 2 100 reactions 50 reaction pairs MD1641 For Research Use Only Not For Use In Diagnostic Procedures Each system contains sufficient reagents to perform 100 reactions 50 reaction pairs Includes Pre amplification Components Box e 1x 100ul MSI 10X Primer Pair Mix e 1x 300ul Gold ST R 10X Buffer e 1x1 25ml Nuclease Free Water e 1 x 3ug K562 High Molecular Weight DNA 10ng ul Post amplification Components Box e 1x150ul Internal Lane Standard 600 Storage Conditions Store all components at 30 C to 10 C The MSI 10X Primer Pair Mix and Internal Lane Standard 600 are light sensitive and must be stored in the dark Available Separately PRODUCT SIZE CAT PowerPlex Matrix Standards 310 50ul each dye DG4640 PowerPlex Matrix Standards 3100 3130 25ul each dye DG4650 Not for Medical Diagnostic Use 3 DNA Extraction Methods Obtaining sufficient high qualit
24. eak heights are excessive or if a poor or incorrect matrix was applied to the samples Increase the peak amplitude threshold i e 150 200RFU in the analysis method and re analyze If problems persist generate a new matrix or perform a new spectral calibration and apply the new matrix to samples Samples were not properly denatured prior to loading Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to loading the capillary CE related artifacts contaminants Contaminants in the water used both on the ABI PRISM 310 Genetic Analyzer and to dilute the 10X genetic analyzer buffer may generate peaks in the blue and green dye colors Use autoclaved deionized water change vials and wash the buffer reservoir Contamination with another template DNA or previously amplified DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 25 www promega com TM255 Revised 8 14 9 Troubleshooting continued S toms Causes and Comments Preferential amplification of smaller loci DNA was cross linked in sample DNA prepared from paraffin embedded samples often is cross linked with other DNA or protein molecules preventing amplification of longer DNA fragments Degraded DNA DNA template
25. es helping to confirm that the test sample and the paired normal samples are from the same individual Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 3 www promega com TM255 Revised 8 14 1 B Microsatellite Instability Overview Microsatellites are short tandem repeated DNA sequences 1 6 base pairs in length These repeats are distributed throughout the genome and often vary in length from one individual to another due to differences in the number of tandem repeats at each locus Microsatellite markers can be used to detect a form of genomic instability called microsatellite instability MSI is a change in length of a microsatellite allele due to insertion or deletion of repeat units during DNA replication and failure of the DNA mismatch repair system to correct these errors 1 C The Internal Lane Standard 600 The Internal Lane Standard 600 ILS 600 contains 22 DNA fragments of 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases in length Figure 2 Each fragment is labeled with carboxy X rhodamine CXR and is detected separately as a fourth color in the presence of MSI Analysis System amplified material using the ABI PRISM 310 or 3100 or Applied Biosystems 3130 or 3130xl Genetic Analyzer The ILS 600 is used in each CE injection to increase precision in analysis when using th
26. es and respective fluorescent dyes for loci contained in the MSI Analysis System are provided in Table 1 Commonly samples in which gt 40 of microsatellite markers are altered gt 2 altered markers out of 5 are classified as MSI H Samples with less than 40 mononucleotide repeat markers altered may be classified as nonMSI H However the occurrence of even one altered mononucleotide marker in mismatch repair proficient cells is uncommon and repeating the MSI assay or performing additional methods of analysis may be necessary to accurately classify these samples Pentanucleotide markers are not intended for use in MSI classification Amplification of microsatellite markers will yield one or two major allele peaks depending on whether the individual is homozygous one peak or heterozygous two peaks for that marker In addition amplification of microsatellite markers often generates artifact peaks due to stutter Electropherograms of mononucleotide markers show a number of less intense stutter peaks at 1bp intervals from the most prominent or true allele peak Figure 6 MSI positive for a marker is recognized as alterations in the lengths of the microsatellite due to deletion or insertion of a repeating unit which produces a novel length allele in test DNA compared to matching normal DNA The mononucleotide markers included in the MSI Analysis System are nearly monomorphic so that most individuals are homozygous for the same common allele for a gi
27. eshooting GeneMapper software versions 4 0 and 4 1 refer to GeneMapper Software Versions 4 0 and 4 1 Reference and Troubleshooting Guide S toms Causes and Comments Weak fluorescent signal for allele peaks Insufficient template DNA Make sure DNA is accurately quantitated and use the recommended amount of template DNA Degraded DNA Prepare new genomic DNA Impure DNA template Impurities in DNA preparation may inhibit PCR Clean up DNA or prepare new genomic DNA Poor quality DNA Improper or prolonged fixation of paraffin embedded samples can result in low DNA yields and poor quality DNA Repeat DNA preparation See Section 3 for DNA purification product recommendations Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject sample Check the syringe pump system for leakage Check the laser power Poor quality formamide was used Use only Hi Di formamide when analyzing samples S High salt concentration or altered pH If the DNA template is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the DNA volume should not exceed 20 of the total reaction volume Carryover from DNA sample of K Na Mg or EDTA can negatively affect PCR Changes in pH also may affect PCR Thermal cycler or tube problems Confirm PCR program is correct Use recommended thermal cycler and tubes Calibration of heat block may be required Samples were not properly denatured before loa
28. f a different template volume is required the water volume should be adjusted accordingly Amplification Mix for the MSI Analysis System Component Volume Per Reaction Nuclease Free Water 5 85 ul Gold ST R 10X Buffer 1 00ul MSI 10X Primer Pair Mix 1 00ul AmpliTaq Gold DNA polymerase 5u tl 0 15ul Total reaction volume 8 001 Note Pipetting volumes smaller than 1 ul is inaccurate We recommend preparing enough Amplification Mix to avoid pipetting such small volumes 6 Combine the final volumes of Nuclease Free Water Gold STXR 10X Buffer MSI 10X Primer Pair Mix and AmpliTag Gold DNA polymerase in a sterile 1 5ml tube Mix gently 7 Transfer 8ul of Amplification Mix to the bottom of each reaction tube or well 8 Pipet 2ul of template DNA 1 2ng for each sample into the bottom of the appropriate tube or well containing Amplification Mix Mix by pipetting several times Mix Note Store DNA templates in nuclease free water or TE buffer 10mM Tris HCI pH 8 0 0 1mM EDTA If the template DNA is stored in TE buffer that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA sample added should not exceed 20 of the final reaction volume PCR amplification efficiency and quality can be greatly altered by changes in pH due to added Tris HCl available magnesium concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of template DNA and
29. for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe 1 Open the ABI PRISM 310 Data Collection Software 2 Prepare a GeneMapper sample sheet as described in the ABI PRISM 310 Genetic Analyzer user s manual Enter the appropriate sample information in the Sample Info column 3 Create anew GeneMapper injection list Select the appropriate sample sheet by using the drop down menu 4 Select the GS STR POP4 1ml F Module using the drop down menu Change the injection time to 2 seconds and keep the settings for the remaining parameters as shown below Inj Secs 1 5 Inj kV 15 0 Run kV 15 0 Run C 60 Run Time 30 Note You may need to optimize injection time for individual instruments Injection times of 1 5 seconds are recommended for use with 1 2ng of template DNA 5 Select the appropriate matrix file Section 5 A 6 To analyze the data automatically select the auto analyze check box and the appropriate analysis parameters and size standard Refer to the ABI PRISM 310 Genetic Analyzer user s manual for specific information about these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 10 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega
30. ftware Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or an ice water bath aerosol resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 Applied Biosystems 2 10X genetic analyzer buffer with EDTA Applied Biosystems MicroAmp optical 96 well plate and septa Applied Biosystems Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 7 A Spectral Calibration The PowerPlex Matrix Standards 3100 3130 Cat DG4650 is required for spectral calibration on the ABI PRISM 3100 Genetic Analyzer The PowerPlex Matrix Standards 310 Cat DG4640 cannot be used to perfo
31. ing Peak Amplitude Thresholds Fun Range fan Sizes B ho ho 100 R 100 Start Pt fo Start Size0 eT ficooo sd ae frooo G h 00 0 5o op Pt 10000 Stop Size 11000 Y h 00 p Smoothing and Baselining Min Peak Half Yicth 2 pts Polynomial Degree 3 Peak Window Size h 5 pts Slope Threshold Peak Start fo 0 Peak End f D Factory Defaults Smoothing None Light Heavy Baseline Vvindow f1 pts Size Calling Method 2nd Order Least Squares 3rd Order Least Squares Cubic Spline Interpolation Local Southern Method Global Southern Method x Cancel Figure 9 The recommended settings for the Peak Detector tab 6976TA 8 D Creating a Size Standard 1 Select Tools then GeneMapper Manager 2 Select the Size Standard tab 3 Select New 4 Select Basic or Advanced Figure 9 The type of analysis method selected must match the peak detection algorithm selected on the Peak Detector tab for the analysis method Select OR 5 Enter a detailed name such as ILS 600 in the Size Standard Editor Make sure that red is selected as the color for the size standard dye 6 Enter the sizes of the internal lane standard fragments into the table 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 and 600 bases Ta Select OR 22 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 53
32. iosystems aerosol resistant pipette tips Hi Di formamide Applied Biosystems Cat 4311320 o PowerPlex Matrix Standards 310 Cat DG4640 crushed ice or an ice water bath The quality of formamide is critical Use Hi Di formamide Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause breakdown of formamide Poor quality formamide may contain ions that compete with DNA during injection which results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Formamide is an irritant and a teratogen avoid inhalation and contact with skin Read the warning label and take appropriate precautions when handling this substance Always wear gloves and safety glasses when working with formamide 5 A Matrix Generation The PowerPlex Matrix Standards 310 Cat DG4640 is required for matrix generation on the ABI PRISM 310 Genetic Analyzer The PowerPlex Matrix Standards 3100 3130 Cat DG4650 cannot be used to generate a matrix on the ABI PRISM 310 Genetic Analyzer For protocols and additional information about generating a matrix see the PowerPlex Matrix Standards 310 Technical Bulletin TBD021 which is available online at www promega com protocols Proper matrix generation is critical to evaluate multicolor systems with the ABI PRISM 310 Genetic Analyzer A matrix must be generated for each ABI PRISM 310
33. k Protocol for the GeneAmp PCR System 9700 Thermal Cycler Note GeneAmp PCR System 9700 thermal cycling conditions are in 9600 emulation mode and use the silver block Cycling profile 95 C for 11 minutes then 96 C for 1 minute then ramp 100 to 94 C for 30 seconds ramp 29 to 58 C for 30 seconds ramp 23 to 70 C for 1 minute for 10 cycles then ramp 100 to 90 C for 30 seconds ramp 29 to 58 C for 30 seconds ramp 23 to 70 C for 1 minute for 20 cycles then 60 C for 30 minutes 4 C soak 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com O O Promega 3 After completion of the thermal cycling protocol store samples at 20 C protected from light Note DNA quantity and quality affect PCR yield Use the recommended DNA purification methods and accurate quantitation to minimize problems Cycle number may be modified to adjust for variations in DNA amount but should not exceed 32 cycles 5 Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User dry heating block water bath or thermal cycler 310 capillaries 47cm x 50um Applied Biosystems performance optimized polymer 4 POP 4 Applied Biosystems 10X genetic analyzer buffer with EDTA Applied Biosystems sample tubes and septa Applied B
34. mend using gloves and aerosol resistant pipette tips to prevent cross contamination We recommend keeping all pre amplification and post amplification reagents in separate rooms Prepare amplification reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for amplification setup 1 Thaw the Gold ST R 10X Buffer MSI 10X Primer Pair Mix and Nuclease Free Water 2 Mix these reagents by vortexing for 5 10 seconds before each use A precipitate may form in the Gold ST R 10X Buffer If this occurs warm the buffer briefly at 37 C then vortex until the precipitate is in solution 3 To prepare the Amplification Mix determine the number of reactions to be set up This should include positive and negative control reactions Add 1 or 2 reactions to this number to compensate for losses during pipetting This approach ensures that you will have enough PCR master mix for all samples It also ensures that each reaction contains the same master mix 4 Place one 0 2m microcentrifuge tube for each reaction into a rack and label appropriately Alternatively use MicroAmp optical 96 well reaction plates 6 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega 5 The following table shows the component volumes per sample when using a DNA template volume of 2ul in a 10ul reaction volume I
35. n select F from the drop down menu In the Run Module 1 column select GeneScan36_POP4DefaultModule from the drop down menu To collect the data without autoanalyzing select No Selection in the Analysis Module 1 column Analysis parameters can be applied after data collection and during data analysis using the GeneMapper analysis software To analyze data during data collection an appropriate analysis module must be selected in the Analysis Module 1 column Refer to the ABI PRISM 3100 Genetic Analyzer user s manual for specific instructions on creating analysis modules Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software Place samples in the instrument and close the instrument doors In the pending plate records table click once on the name of the plate record you just created Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 13 www promega com TM255 Revised 8 14 O Promega 6 C Instrument Preparation continued 11 Once the plate record is highlighted click the plate graphic corresponding to the plate on the autosampler that contains your amplified samples 12 When the plate record is linked to the plate the plate graphic will change from yellow to green the plate record moves from the pending plate records table to the linke
36. ndow in the data collection software Each injection will take approximately 45 minutes 8 Data Analysis 8 A MSI Analysis Overview Detection of MSI is based on comparing allelic profiles generated by amplifying matching normal and test sample DNA The appearance of novel alleles in the test sample indicates microsatellite instability Figure 5 The MSI Analysis System allows co amplification and detection of a panel of microsatellite markers that have been shown to be sensitive and specific for detection of MSI H samples with mismatch repair deficiencies Specific PCR product sizes and the respective fluorescent dyes used for labeling of each microsatellite marker are described in Table 1 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 17 www promega com TM255 Revised 8 14 3G 4639TA x 144 Y 2444 P Figure 5 Microsatellite instability assays using the MSI Analysis System Matching normal sample top panel and MSI positive test sample bottom panel were analyzed using the MSI Analysis System Two nanograms of genomic DNA was amplified and analyzed using an ABI PRISM 3100 Genetic Analyzer with POP 4 polymer and a 36cm capillary and the allelic patterns of the normal and test samples are shown The presence of new alleles in the test sample indicated by arrows that were not present in the normal sample indicates MSI PCR product siz
37. rated with the MSI Analysis System Version 1 2 we have created panels and bins text files to allow automatic assignment of genotypes using GeneMapper software versions 4 0 and 4 1 For GeneMapper software version 3 5 or 3 7 we recommend upgrading to version 4 0 or 4 1 Versions 4 0 and 4 1 allow improved analysis of the mononucleotide and pentanucleotide repeats amplified by the MSI Analysis System Note For more information on GeneMapper ID or ID X contact Promega Technical Service at genetic promega com For more information refer to your GeneMapper ID or ID X software documentation Panels and bins text files for the MSI Analysis System Version 1 2 can be downloaded from the Promega web site at www promega com resources tools msi panels and bins for genemapper software Open the GeneMapper software version 4 0 or 4 1 Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left pane From the menu select File then Import Panels oe a SS Navigate to the file location where the panels and bins text files were saved on your computer Select the file Promega_Panels_MSI_GM4 X txt then Import In the navigation pane highlight the Promega MSI folder that you just imported Select File then Import Bin Set Navigate to the file location where the panels and bins text files have been saved on your computer Select the file Promega_Bins_MSI_GM4
38. rm a spectral calibration on the ABI PRISM 3100 Genetic Analyzer For protocols and additional information about spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 which is available online at www promega com protocols Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 Genetic Analyzer Spectral calibration must be performed for each ABI PRISM 3100 Genetic Analyzer For answers to questions about spectral calibration contact Promega Technical Services at genetic promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 15 www promega com TM255 Revised 8 14 7 B Sample Preparation The Internal Lane Standard 600 is included in each CE injection to standardize analysis of amplified samples and uses a fourth color 1 Prepare a loading cocktail by combining and mixing the internal lane standard and Hi Di formamide as follows 0 5ul ILS 600 9 5ul Hi Di formamide x injections Note The volume of internal lane standard used in the loading cocktail can be increased or decreased to adjust the intensity of the size standard peaks The optimal peak height for the 100 base fragment of the internal lane standard is 500 1 000RFU If the peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0ul of
39. s erosnesauccanssenssenetaisscpastusnencs sun stak casiossautannaenenaeaessan NAAA Nainiai 17 8 B Importing Panels and Bins Text Files for GeneMapper Software Versions 4 0 and 4 1 06 20 8 C Creating an Analysis Method Using GeneMapper Software Versions 4 0 and 4 1 ssceseceees 21 Be Creatine a Size Standard cesassansessutaacasansacisacdwsdiuncasscosteossuoudonagsacouecauttesatetuuneouhinsavecdeasaserenseeuacers 22 Sele Progrssiho Daili eeren aE EE OO E N E OETA OOE 23 8 F Reviewing the Size Standard sessessessessesscseosessesscsecsecseeseoscsecsecsecsecseoscsecsecsecseoseoseoscseoseoseose 23 8 G Reviewing Analyzed Sample Data sessessesseseeseosessesscsscseeseosesecsecsecsecseoseseesecseoseoseoseososeoseoseose 23 S O MOONS EEE T A PE PEO EAT ONEA I E vee IAE AE E EE AO E A T 24 O FOO a E E E E E E AE 26 UE N o E E A E E A NEA A E A N O NEA A 26 TOE Ree TTO C S socscanestnsasnaceaseonsauutaesiaedicessactiyseaeuddarnipacsnpsayseuatisyeraeatesdineiavenspermenuebamenieiacsinet 26 10 C Allele Frequencies for Mononucleotide Repeat Loci ccssccssccssccssccsscnsccnsccsscesccesccsscesscesees 27 10 0 Summary of Chant eS ve cienveneresnuetoucedeatuasauseaueecvasessdasecqancatencsnanevssaverisesgueetutaroieiaseseseinseerneuatsume 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 1 www promega com TM255
40. sessessessessesecsecsseoseosessesseseoscoscssesecsecseoscsecsecsecseoseoseoeoseoseoseose 8 5 Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer sccsssccsssccnseonees 9 SA Maltis GOTO PALO Woes sciaseeinydvcreceniivacsensasnsnvn cus yauves yovied dur E E Vote coun vet eae sneegeaveouns uve nuaaeeuineiaes 9 5 B Sample Preparation lt cscsesisacivesecacavwaceucoussiueseuessrwosnsivdvesacavivanectavessceseiwssaueciustetndsnsistensasaeceeeieeacwess 10 SC Insitument Preparati epecaecececntecnsu cal acstecuiessens ne saandesa E a a i ai netts 10 6 Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 ccccscsscsccscsccsccccsccsceccscsccscsccsccscsccsceccscscessecs 11 Ce Spectral Calibrator ciccsivincecous ced enian ein un puns ENNE EEEE EENEN E EEN 11 0 B Sample Preparati cerieirar NN EER 12 0C Insiumen Pe AAU OM gece crac eee a EEE EEE O 12 7 Detection of Amplified Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130x Genetic Analyzer 15 Ths ODEO Al C ANDTAU OM rr E A AEE ET E E OA OOO AEA 15 Dae Sample Preparati Oissar T Ei 16 TC TAS UUMMETIL Prepa a OD eccsrernirrscissir terrines i NENE Ea ETEO TEENIE EENEN E T anen 16 S Data Analysis senri EANN ara EE E EErEE ANENE AASER 17 8 A MSL Aialysis Overview osacecacen je
41. the extraction procedure used O Be sure that the template DNA is mixed well before transferring it to the tube or well containing Amplification 9 For the positive amplification control dilute the K562 High Molecular Weight DNA 1 10 to 1ng ul in Nuclease Free Water Pipet 2ng of the diluted DNA into the bottom of the tube or well containing Amplification Mix Mix by pipetting several times 10 For the negative amplification control pipet Nuclease Free Water instead of template DNA into a microcentrifuge reaction tube or well containing Amplification Mix Mix by pipetting several times Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 7 www promega com TM255 Revised 8 14 O 4 B Amplification Thermal Cycling 1 Place the tubes or MicroAmp optical 96 well reaction plates in a thermal cycler 2 Select and run the recommended protocol for the GeneAmp PCR System 9600 or GeneAmp PCR System 9700 provided below Protocol for the GeneAmp PCR System 9600 Thermal Cycler Cycling profile 95 C for 11 minutes then 96 C for 1 minute then 94 C for 30 seconds ramp 68 seconds to 58 C hold for 30 seconds ramp 50 seconds to 70 C hold for 1 minute for 10 cycles then 90 C for 30 seconds ramp 60 seconds to 58 C hold for 30 seconds ramp 50 seconds to 70 C hold for 1 minute for 20 cycles then 60 C for 30 minutes 4 C soa
42. ur GeneMapper software version 4 0 or 4 1 documentation 1 Select all samples in the Samples tab by selecting Edit then Select All 2 Open the Size Match Editor by selecting Analysis then Size Match Editor 3 View the Size Matches tab to review the Size Quality score size standard peaks and size standard peak labels for each sample 4 Determine if all peaks in the size standard are present and labeled correctly for the ILS 600 as shown in Figure 2 8 G Reviewing Analyzed Sample Data The GeneMapper software provides a variety of options for sample data display The Promega MSI Analysis System panels and bins text files allow automatic assignment of genotypes using GeneMapper software The proper panels and bins text files for use with GeneMapper software can be obtained from the Promega web site at www promega com resources tools msi panels and bins for genemapper software For additional information on reviewing data refer to the GeneMapper Software Version 4 0 or 4 1 Installation and Administration Guide Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 23 www promega com TM255 Revised 8 14 9 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail genetic promega com For troubl
43. ven marker see Section 10 C for allele frequencies 18 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM255 Revised 8 14 www promega com Promega Penta C 4635TA Figure 6 Stutter peaks Stutter peaks indicated by arrows generated by polymerase slippage during PCR amplification of short tandem repeats are clearly visible at the mononucleotide marker NR 24 left at 1bp intervals from the true or tallest allele peak Much smaller stutter peaks also can be seen at the Penta C marker 5bp from true allele peaks Two polymorphic pentanucleotide repeat markers Penta C and Penta D are included in the MSI Analysis System to help confirm that the test sample and matching normal samples are from the same individual Alleles found in the normal sample also should be present in the test sample if both samples are from the same individual MSI has been observed in 14 of MSI H samples in Penta C and 35 in Penta D so additional pentanucleotide alleles also may be present in some MSI H samples A matching probability between 1 10 and 1 16 was calculated for Penta C and between 1 18 and 1 33 for Penta D based upon allele frequency data Matching probability is the average number of randomly selected people you would have to survey before you find two with the same allelic pattern Using both pentanucleotide markers should allow detection of sample mixups
44. y DNA from formalin fixed paraffin embedded FFPE tissues can be problematic DNA is often degraded due to prolonged or unsuitable fixation of the tissue sample before embedding in paraffin Promega offers automated and manual genomic DNA extraction systems for purifying high quality DNA from FFPE tissue sections The Maxwell 16 FFPE Tissue LEV DNA Purification Kit Cat AS1130 provides an easy automated method for purifying genomic DNA from one to ten sections 5um of FFPE tissue samples without the use of hazardous organic solvents such as xylene The ReliaPrep FFPE gDNA Miniprep System Cat A2352 provides a flexible all inclusive manual method for purifying genomic DNA from FFPE tissue without hazardous solvents or overnight digestion The MagneSil Genomic Fixed Tissue System Cat 4 MD1490 is a MagneSil Paramagnetic Particles based technique for extracting genomic DNA from FFPE tissue Please contact your local Promega Branch Office or Distributor contact information available at www promega com or email genetic promega com for more information Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 5 www promega com TM255 Revised 8 14 4 DNA Amplification Using the MSI Analysis System Materials to Be Supplied by the User thermal cycler GeneAmp System 9600 or 9700 Applied Biosystems o microcentrifuge 0 2ml thin walled
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