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MSI Analysis System, Version 1.2 Technical Manual, TM255

1. ls Elles alg 4637CA T Figure 4 The Plate View tab 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 capillaries will take approximately 45 minutes Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Printed in USA Page 14 Revised 9 07 VII 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 Promega Materials to Be Supplied by the User e 95 C dry heating block water bath or thermal cycler e crushed ice e aerosol resistant pipette tips e 3100 or 3130 capillary array 36cm e performance optimized polymer 4 POP 4 Applied Biosystems 10X genetic analyzer buffer with EDTA Applied Biosystems e MicroAmp optical 96 well plate and septa Applied Biosystems e Hi Di formamide Applied Biosystems Cat 4311320 e PowerPlex Matrix Standards 3100 3130 Cat DG4650 The quality of the formamide is critical Use Hi Di formamide with a conductivity less than 100pS cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storag
2. Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 15 C VILB Sample Preparation 2 The Internal Lane Standard 600 is included in each experiment to standardize Promega 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 5pl ILS 600 x injections 9 5p1 Hi Di formamide x injections Note The volume of internal lane standard used in the loading cocktail can also 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 01 of ILS 600 and 9 0p1 of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 2511 of ILS 600 and 9 7511 of formamide 2 Mix for 10 15 seconds using a vortex mixer 3 Pipet 10pl of formamide internal lane standard mix into each well 4 Add 1p of amplified sample Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time 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
3. BAT 26 U41210 A og 103 115 113 FL BAT 25 L04143 A os 114 124 122 JOE NR 248 X60152 A o4 130 133 130 TMR MONO 27 AC007684 A or 142 154 150 JOE Penta C AL138752 AAAAG 3 143 194 164 174 TMR Penta D AC000014 AAAAG 7 135 201 168 187 FL 1Amplicon size range is based on genotyping 300 500 individuals from three different racial groups Caucasian American Asian American and African American Rare alleles outside these size ranges may exist Allele sizes determined using the ABI PRISM 3100 genetic analyzer 3See reference 1 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 2 Printed in USA Revised 9 07 The mononucleotide repeat markers included in the MSI Analysis System were CG selected for high sensitivity and specificity to alterations in tumor samples with o mismatch repair defects 1 3 These mononucleotide repeat markers are quasi monomorphic that is almost all individuals are homozygous for the same Promega common allele for a given marker see Section XI B for allele frequencies Use of monomorphic markers simplifies data interpretation The pentanucleotide repeat markers have been selected for their high level of polymorphism and low degree of MSI to uniquely identify samples helping confirm that tumor and matching normal samples are from the same individual 4586CC
4. If the peak heights are higher than desired the samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This may result in uneven allele peak heights across loci For best results use less DNA template in the amplification reactions or reduce the number of cycles in the amplification program by 2 4 cycles to achieve the desired signal intensity 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 for 3 minutes Denature the samples just prior to loading the instrument VILC 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 3130xl genetic analyzer with the following exceptions Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Printed in USA Page 16 Revised 9 07 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template va drop down list Confirm that the injection time
5. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 11 C VI B Sample Preparation 2 The Internal Lane Standard 600 is included in each experiment to standardize Promega 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 deionized formamide as follows 0 5ul ILS 600 x injections 9 5p1 deionized formamide x injections Note The volume of internal lane standard used in the loading cocktail can also 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 01 of ILS 600 and 9 0pl of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 2511 of ILS 600 and 9 751 of formamide Vortex for 10 15 seconds Pipet 101 of formamide internal lane standard mix into each well Add 111 of amplified sample Cover wells with appropriate septa Note Instrument detection limits vary therefore the injection time or the amount of product mixed with loading cocktail may need to be increased or decreased If peak heights are too high gt 4 000 6
6. Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 20 Printed in USA Revised 9 07 VIII B Importing Panel and Bin Files for GeneMapper Software Version 4 0 v To facilitate analysis of data generated with the MSI Analysis System Version 1 2 we have created panel and bin files to allow automatic assignment of genotypes Promega using GeneMapper software version 4 0 For GeneMapper software version 3 5 or 3 7 we recommend upgrading to version 4 0 Version 4 0 has been improved to allow analysis of the mononucleotide and pentanucleotide repeats amplified by the MSI Analysis System Panel and bin files for the MSI Analysis System Version 1 2 can be downloaded from the Promega web site at www promega com techserv tools msi Open the GeneMapper software version 4 0 Select Tools then Panel Manager Highlight the Panel Manager icon in the upper left tile navigation pane From the menu select File then Import Panels g e P Y Be Navigate to the file location where the panel and bin files have been saved on your computer Select the file Promega_Panels_MSI_GM4 0 0 txt then Import 6 Inthe navigation pane highlight the Promega MSI folder that you just imported 7 Select File then Import Bin Set 8 Navigate to the file location where the panel and bin files have been saved on your computer Select the file Promega_Bins_MSI
7. but the fragments have been assigned correctly the Sizing Quality can be overridden by selecting Override SQ The samples can then be reanalyzed For more information refer to Section IX or your GeneMapper software version 4 0 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 Promega Internal Lane Standard ILS 600 as shown in Figure 2 VIILG Reviewing Analyzed Sample Data The GeneMapper software version 4 0 provides a variety of options for sample data display The Promega MSI Analysis System panel and bin download package includes an informational document and an example plot settings file which illustrates one option for review of sample data For additional information on viewing sample plots please refer to your GeneMapper software version 4 0 documentation Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 24 Printed in USA Revised 9 07 IX Troubleshooting O Ua For questions not addressed here please contact your
8. 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 the 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 8 Select OK This new plate record will appear in the pending plate records table on the plate setup page of the collection software 9 Place the samples in instrument and close the instrument doors 10 Inthe pending plate records table click once on the name of the plate record you just created 11 Once the plate record is highlighted click the plate graphic corresponding to the plate on the autosampler that contains your amplified samples Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 13 VI C Instrument Preparation continued w 12 When the plate record is linked to the plate the plate graphic will change Promega from yellow to green the plate record moves from the pending plate records table to the linked plate records table and the Run Instrument button becomes enabled Figure 4
9. for colonic carcinogenesis Nature 363 558 61 8 Peltomaki P et al 1993 Microsatellite instability is associated with tumors that characterize the hereditary non polyposis colorectal carcinoma syndrome Cancer Res 53 5853 5 9 Thibodeau S N Bren G and Schaid D 1993 Microsatellite instability in cancer of the proximal colon Science 260 816 9 10 Boland C R et al 1998 A national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition Development of international criteria for the determination of microsatellite instability in colorectal cancer Cancer Res 58 5248 57 11 Umar A et al 2004 Revised Bethesda guidelines for hereditary nonpolyposis colorectal cancer Lynch syndrome and microsatellite instability J Natl Cancer Inst 96 261 8 12 Levinson G and Gutman G 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 13 Bacher J et al 1999 Pentanucleotide repeats Highly polymorphic genetic markers displaying minimal stutter artifact Proceedings from the Ninth International Symposium on Human Identification This can be viewed online at www promega com geneticidproc ussymp9proc default html 14 Bacher J and Schumm J 1998 Development of highly polymorphic pentanucleotide tandem repeat loci with low stutter Profiles in DNA 2 2 3 6 XI Appendix XLA Related Products Pro
10. is 5 seconds and the Pro ega 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 In the 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 3 In the 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 In the GeneMapper plate record enter sample names in the appropriate cells Scroll 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 ne
11. local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Promega Symptoms Causes and Comments Weak fluorescent signal Insufficient template DNA Make sure DNA is accurately for allele peaks 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 or use the MagneSil Genomic Fixed Tissue System Cat MD1490 Poor capillary electrophoresis injection ILS 600 peaks also affected Re inject sample Check instrument manual for instructions Poor quality formamide Use high quality formamide with conductivity of lt 100pS cm High salt concentration or altered pH DNA volume should not exceed 20 of the total reaction volume Carryover from DNA sample of K Na Mg or EDTA can have a deleterious effect on PCR Changes in pH may also 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 not properly denatured before loading Heat denature samples for recommended time and cool on crushed ice immediately prior to loading the capillary Amplification r
12. 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 29
13. to prevent cross contamination We recommend keeping all preamplification and postamplification 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 and MSI Analysis System 10X Primer Pair Mix 2 Mix these reagents by vortexing for 5 10 seconds before each use A precipitate may form in the Gold ST R Buffer If this occurs warm the buffer briefly at 37 C then vortex until it 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 2ml microcentrifuge tube for each reaction into a rack and label appropriately Alternatively use MicroAmp optical 96 well reaction plates Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 6 Printed in USA Revised 9 07 5 The following table shows the component volumes per sample when using a DNA template volume of 2p in a 10pl reaction volume If a va different template volume is require
14. using the MSI Analysis System Protocols for preparation and use of this internal lane standard are provided in Sections V B VI B and VII B 1 200 100 200 300 400 500 600 1 000 800 7 120 140 160 180 600 0 80 225 250 275 325 350 375 425 450 475 550 j 400 200 5751TA o Figure 2 Internal Lane Standard 600 ILS 600 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Printed in USA Page 4 Revised 9 07 II Product Components and Storage Conditions v Product Size Cat MSI Analysis System Version 1 2 _ 100 reactions 50 reaction pairs MD1641 For In Vitro Research Use Only Not For Use In Diagnostic Procedures Promega Each system contains sufficient reagents to perform 100 reactions 50 reaction pairs Includes Preamplification Components Box Yellow Label e 1 x100pl MSI 10X Primer Pair Mix e 1x 300p1 Gold ST R 10X Buffer e 1x125ml Nuclease Free Water t 1x 3pg K562 High Molecular Weight DNA 10ng l Postamplification Components Box Magenta Label e 1 x150pul Internal Lane Standard ILS 600 e 1 Protocol Storage Conditions Store all components at 20 C The MSI 10X Primer Pair Mix and the Internal Lane Standard 600 are light sensitive and must be stored in the dark Available Separately Product Size Cat PowerPlex Matri
15. 000RFU the samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail Centrifuge plate briefly to remove air bubbles from the wells if necessary Denature samples at 95 C for 3 minutes then immediately chill on crushed ice for 3 minutes Denature the samples just prior to loading the instrument VI C Instrument Preparation Refer to the ABI PRISM 3100 genetic analyzer user s manual for instructions on cleaning the pump blocks installing the capillary array performing a spatial calibration and adding polymer to the reserve syringe 1 2 Open the ABI PRISM 3100 data collection software Open a new plate record Name the plate and select GeneScan Select the plate size 96 well or 384 well Select Finish Complete the plate record spreadsheet for the well you have loaded Enter appropriate information into the Sample Name column Figure 3 In the Project Name column select 3100_Project1 from the pull down menu In the Dye Set column select F from the pull down menu Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 12 Printed in USA Revised 9 07 4769CA Figure 3 The Plate Editor 6 Inthe Run Module 1 column select GeneScan36_POP4DefaultModule from the pull down menu 7 To collect the
16. 9 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 10 Printed in USA Revised 9 07 VI 5 Select the appropriate matrix file Section V A v 6 To analyze the data automatically select the auto analyze check box and the appropriate analysis parameters and size standard Refer to the Promega ABI PRISM 310 genetic analyzer user s manual for specific information on these options 7 After loading the sample tray and closing the doors select Run to start the capillary electrophoresis system 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 Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 01 or 1 1 Materials to Be Supplied by the User dry heating block water bath or thermal cycler crushed ice aerosol resistant pipette tips 3100 capillary array 36cm performance optimized polymer 4 POP 4 Applied Biosystems 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 Note The quality of the formamide is critical Use Hi Di formamide with a conductivity less than 100pS cm Free
17. A DOE aires 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 Panel A An electropherogram shows 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 TMR labeled loci NR 24 and Penta C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 3 L B Microsatellite Instability Testing Overview Microsatellites are short tandemly repeated DNA sequences from 1 6 base pairs Promega in length 4 5 These repeats are distributed throughout the human 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 6 9 MSI is a change in length of a microsatellite allele due to insertion or deletion of repeating units during DNA replication and failure of the DNA misma
18. As MDT Arnal Sis Ov crew cscs innvansshssseandvassansviepnntsves dutesadsassaasboneanieandecaanassioani 19 B Importing Panel and Bin Files for GeneMapper Software Version 4 0 004 21 C Creating an Analysis Method Using GeneMapper Software Version 4 0 21 D Creating a Size Standards siniori Suicvas ti ssesbonincovanndenscastsaaaanang 23 E Processing Data siessnanimssusssiunnanoiaiaeanin anan 24 Eo Reviewing the Size Standard esius aE A ERa 24 G Reviewing Analyzed Sample Dab asstassccsescscissisesestanrvsiecesvasivnsmhniecnaiacnitiecs 24 IX Troubleshooting sscsissitasssnsscnsctnuennenesiorwtnriyesiar shvetivasesnnvinnteas iti dhnasiuanaien veined disieheaneniasebenies 25 Xe MRR N SOR rii ranted arate eee antes aceon 27 D Appendix AE E E E E eed 27 As elated Prod ttsurorossuennenieninsunenansnneeaa 27 B Allele Frequencies for Mononucleotide Repeat LOCi secsessseessssesssseeessses 28 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 1 O Promega I Description I A The MSI Analysis System The MSI Analysis System Version 1 2 9 is a fluorescent PCR based assay to detect microsatellite instability Typically MSI analysis involves comparison of allelic profiles of microsatellite markers generated by amplification of DNA from match
19. For best results use less DNA template in the amplification reactions The volume of ILS 600 can also be adjusted Denature the samples and ladder by heating at 95 C for 3 minutes and immediately chill on crushed ice for 3 minutes Denature the samples just prior to loading Assemble the tubes in the appropriate autosampler tray 48 or 96 tube Place the autosampler tray in the instrument and close the instrument doors V C Instrument Preparation Refer to the ABI PRISM genetic analyzer user s manual for instructions on cleaning the pump block installing the capillary calibrating the autosampler and adding polymer to the syringe i 2 Open the ABI PRISM 310 collection software 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 Create a new GeneMapper injection list Select the appropriate sample sheet by using the pull down menu Select the GS STR POP4 1ml A Module using the pull 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 with 1 2ng of template DNA Promega Corporation 2800 Woods Hollow Road Madison WI 53711 539
20. Technical Manual MSI Analysis System Version 1 2 INSTRUCTIONS FOR USE OF PRODUCT MD1641 This Technical Manual has been revised Please read it carefully e The MSI Analysis System Version 1 2 incorporates improved manufacturing processes e A new matrix must be generated using one of the new matrices PowerPlex Matrix Standards 310 Cat DG4640 or PowerPlex Matrix Standards 3100 3130 Cat DG4650 PRINTED IN USA Revised 9 07 Part TM255 MSI Analysis System Version 1 2 All technical literature is available on the Internet at www promega com tbs Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail techserv promega com IL D sipthion ssisssnai iaiia iiaa iaeiaiai ii N A a 2 As The MSI Analysis Syste sass ctavsassnsceeccanastactgaaceensaenensnvenuasten agian ga neramsandeaanesttie 2 B Microsatellite Instability Testing Overview ss sssssesrsssssssrsssssrsrssnsrsrrsseerrnsnseerrnnnne 4 C Thelnternal Lane Standard GW ccsecsnssciecnnnamacmiiernt ginamanumnens 4 II Product Components and Storage Conditions ccceessssessssseeeessseeeessseesssseeesseeeesey 5 I TINA Extraction MethodSsisininiriien iian AEE EE 5 IV DNA Amplification Using the MSI Analysis System 6 A Ampitication Setups sie aaora aa aE E A E 6 By Amplification Thermal Cyclin aa c
21. _GM4 0 0 txt then Import 9 At the bottom of the Panel Manager window select Apply then OK The panel manager window will close automatically VIILC Creating an Analysis Method Using GeneMapper Software Version 4 0 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 SS oO FP SP NY e Select the bin set corresponding to the Promega MSI System Promega_MSI 8 Ensure that the Use marker specific stutter ratio if available box is checked Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 21 O Promega VIII C Creating an Analysis Method Using GeneMapper Software Version 4 0 continued 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 RFU 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 o
22. 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Printed in USA Page 26 Revised 9 07 X References CG oS I Suraweera N et al 2002 Evaluation of tumor microsatellite instability using five quasimonomorphic mononucleotide repeats and pentaplex PCR Gastroenterology 123 1804 11 Promega 2 Bacher J W et al 2004 Development of a fluorescent multiplex assay for detection of MSI High tumors Dis Markers 20 237 50 3 Murphy K M et al 2006 Comparison of the microsatellite instability analysis system and the Bethesda panel for the determination of microsatellite instability in colorectal cancers J Mol Diagn 8 305 11 4 Tautz D 1989 Hypervariability of simple sequences as a general source for polymorphic DNA markers Nucleic Acids Res 17 6463 71 5 Weber J and May P 1989 Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction Am J Hum Genet 44 388 96 6 Aaltonen L et al 1993 Clues to the pathogenesis of familial colorectal cancer Science 260 812 6 7 Ionov Y et al 1993 Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism
23. assiesstessasastactaassedstasvstrainssaaastasiaensecesetneraiaseaiestadannsears 8 V Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer ssscsciascimnnicacamancionaiamammanaienamaniie 9 Pow Matrix Standardization ccoxencicioncasesiidsicansiianeacsaninvaneseshs niona 9 B SamplePrep ratiofisisusninininnuiinomusisimusninniieiiiiioniinan a 10 Co anstrument Prepara HON aae i A AAAA OR 10 VI Detection of Amplified Fragments Using the ABI PRISM 3100 Genetic Analyzer with Data Collection Software Version 1 0 1 or 1 1 11 As Spectral Caran ivcesa cis i taseanvucsnnasduneidvsaedstadansstuntasinsvtanidadbiesinestintastnistnaantnanaets 11 B Sample P repahi On ncchounmeanmansnnnanudnnnsntniainumamamaniunans 12 G Instrument Preparation ois cctsisessscuisnss seecstss annann aaa 12 VII 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 Analyzet ssss eissernir 15 A Spectral Calibration sovescepaiowniiniaiiniemaniciiiaenailiamenndaonandeoniuate 15 B Srauinple Pine para tnm asvcescussstivaseupesuiaesensenssscechavebenseansayshun epuceothsvsbiscaesbavstiesanccianitiviesces 16 Co nstrument Pre ParAtlON sakes decnstnscudusenasaswistascdyycpatctesducenedvssdetat duecealarsscthsentactitvadsteatnesres 16 VIL Data Analysts socciccaciionsacis iomsacsimenanaisannonioarmannunaaoananaden 18
24. concentration due to chelation by EDTA or other PCR inhibitors which may be present at low concentrations depending on the source of the template DNA and the extraction procedure used 9 For the positive amplification control dilute the K562 Genomic DNA sample 1 10 to Ing l in Nuclease Free Water Pipet 2ng of the diluted DNA into the bottom of the tube or well containing the 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 the 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 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 7 O Promega IV B Amplification Thermal Cycling ue Assemble the tubes or MicroAmp optical 96 well reaction plates in a thermal cycler Select and run a recommended protocol for the GeneAmp PCR system 9600 or the GeneAmp PCR system 9700 provided below Protocol for the Perkin Elmer 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 ho
25. d the water volume should be Pro adjusted accordingly me ga Amplification Mix for the MSI Analysis System Component Volume Per Sample Nuclease Free Water 5 85pl Gold ST R 10X Buffer 1 00p1 MSI Analysis System 10X Primer Pair Mix 1 00p1 AmpliTaq Gold DNA polymerase 5u 1 0 15 total reaction volume 8 00p1 Note Pipetting volumes smaller than 1p1 is inaccurate We recommend preparing enough Amplification Mix to avoid pipetting such small volumes 6 Combine the final volumes of Nuclease Free Water Gold ST R 10X Buffer MSI Analysis System 10X Primer Pair Mix and AmpliTag Gold DNA polymerase in a sterile 1 5ml amber tube Mix gently 7 Transfer 8pl of the Amplification Mix to the bottom of each reaction tube or well 8 Pipet 2pl of the template DNA 1 2ng for each sample into the bottom of the respective tube or well containing Amplification Mix Mix by pipetting several times Be sure that the template DNA is well mixed before transferring it into the bottom of the tube or well containing the Amplification 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
26. duct Size Cat MagneSil Genomic Fixed Tissue System 100 samples MD1490 ART 20P Pipet Tip 20 960 pk DY1071 ART 100 Pipet Tip 1001 960 pk DY1101 ART 100E Pipet Tip 100pl 960 pk DY1111 ART 200 Pipet Tip 2001 960 pk DY1121 ART 1000E Pipet Tip 1 000 800 pk DY1131 For Laboratory Use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 27 XI B Allele Frequencies for Mononucleotide Repeat Loci b m ON OGOOGO O ae O g CS WwW oOo SOA 8 0 0 0 NR 24 Size bp Caucasian African Asian 60 197 49 113 34 77 0 0 2 2 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Printed in USA Page 28 Revised 9 07 AUS Pat Nos 6 844 152 and 7 202 031 and Australian Pat No 2001290868 have been issued to Promega Corporation for detection of microsatellite instability and its use in diagnosing tumors Other patents are pending U S Pat Nos 6 238 863 and 6 767 703 and Korean Pat No 691195 have been issued to Promega Corporation for materials and methods for identifying and analyzing intermediate tandem repeat DNA markers Other patents are pending Licensed under U S Pat Nos 6 566 053 and 7 090 978 This product i
27. e at 4 C may cause a breakdown of the formamide Formamide with a conductivity greater than 100pS cm may contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution 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 VII A Spectral Calibration The PowerPlex Matrix Standards 3100 3130 Cat DG4650 are 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 For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 supplied with Cat DG4650 available upon request from Promega or online at www promega com tbs 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 matrix creation send an e mail to techserv promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330
28. eaction components not added to the bottom of the tube or well Gently tap the tube or plate to move the drop to bottom or centrifuge briefly Excessive fluorescent Too much template DNA signal for allele peaks e Make sure DNA is 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 25 IX Troubleshooting continued b Pro Symptoms Causes and Comments mega Extra peaks visible in Microsatellite artifacts i e stutter PCR amplification of one or all dye colors microsatellite loci generates artifacts that appear as smaller peaks 1bp above or below the prominent mononucleotide repeat allele or 5bp below a pentanucleotide repeat allele Stutter band peak heights will be higher if too much template DNA is used Excess amount of DNA One to two nanograms of DNA are recommended Amplification of gt 2ng DNA template may result in a higher number of stutter bands 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 peak heights are excessive or if a poor or incorrect matrix has been applied to the sa
29. ected people you would have to survey before you would find two with the same allelic pattern Using both pentanucleotide markers should allow detection of sample mix ups over 99 of the time For data analysis the GeneMapper analysis software from Applied Biosystems 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 quantitation of peak heights in RFU GeneMapper results should be reviewed to eliminate artifact peaks caused by bleedthrough stutter or CE spikes Figures 6 and 7 Refer to the GeneMapper analysis user s manual for detailed instructions on fragment analysis and genotyping To simplify data analysis we have created panel and bin files to allow automatic assignment of genotypes using GeneMapper software The proper panel and bin files for use with GeneMapper software can be obtained from the Promega web site at www promega com techserv tools msi BAT 26 BAT 25 MONO 27 Penta D 4634TA Figure 7 Bleedthrough or pull up peaks Smaller peaks arrows directly under 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 has been applied to the samples Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526
30. f these settings refer to your GeneMapper software version 4 0 documentation pa i pa pa pa 6975TA Figure 8 The Analysis Method Editor The PlusA ratio setting for Mono repeats is 0 9999 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 of between 50 and 200RFU are commonly used 11 Select OK to save your settings Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 22 Printed in USA Revised 9 07 6976TA Figure 9 The recommended settings for the Peak Detector tab VIIL 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 OK 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
31. fragments into the table 7 Select OK Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 23 O Promega VIILE 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 In the 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 VIII D 5 In the Panel column select Promega MSI from the Promega MSI folder This is the panel set that was imported in Section VIII C 6 Inthe Size Standard column select the size standard that was created in Section VILE 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 the data analysis VIILF 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
32. ing normal and tumor samples Alleles that are present in the tumor sample but not found in the 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 mix ups and or contamination Internal lane size standards Figure 2 are 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 3130xl genetic analyzer and the output data may be analyzed with the GeneMapper software Applied Biosystems to determine MSI status of tumor samples To simplify data analysis we have created panel and bin files to allow automatic assignment of genotypes using GeneMapper software The proper panel and bin files for use with GeneMapper software can be obtained from the Promega web site at www promega com techserv tools msi Table 1 The MSI Analysis System Locus Information Marker GenBank Major Repeat Size K562 Primer Name Number Sequence Range bp Alleles bp Dye NR 213 XM_033393 A o1 94 101 101 JOE
33. ion 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Printed in USA Page 18 Revised 9 07 The Bethesda guidelines 10 11 recommend that tumor samples in which 240 CG of microsatellite markers are altered 22 altered markers out of 5 be classified oe as MSI H Tumor samples with less than 40 mononucleotide repeat markers altered may be classified as nonMSI H However the occurrence of even one Promega altered mononucleotide marker in mismatch repair proficient tumors is uncommon and repeating the MSI assay or performing additional methods of analysis may be necessary to accurately classify these samples 3 Pentanucleotide markers are not intended for use in MSI classification Amplification of microsatellite markers will yield one or two major allele peaks depending upon whether the individual is homozygous one or heterozygous two for that marker In addition amplification of microsatellite markers often generates artifact peaks called stutter 12 Electropherograms of mononucleotide markers show a number of less intense stutter peaks at lbp intervals from the most prominent or true allele peak Figure 6 A shift in allele size of 3bp or more in the tumor samples compared to matching normal samples is usually scored as MSI positive for that marker The mononucleotide markers included in the MSI Analysis System are nearly mono
34. l Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 5 IV DNA Amplification Using the MSI Analysis System Materials to Be Supplied by the User thermal cycler GeneAmp system 9600 or 9700 Applied Biosystems e microcentrifuge e 0 2ml thin walled microcentrifuge tubes MicroAmp reaction tube strips or MicroAmp optical 96 well reaction plates Applied Biosystems e 15ml amber colored microcentrifuge tubes e aerosol resistant pipette tips e AmpliTag Gold DNA polymerase Applied Biosystems The MSI Analysis System is optimized to amplify 1 2ng of genomic DNA in a 101 reaction volume using the protocols detailed below However optimization of input DNA amounts should be performed in individual laboratories to adjust for variations in yield and quality of DNA resulting from differences in samples and or DNA isolation methods 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 The MSI Analysis System is optimized for use with the GeneAmp PCR system 9600 and 9700 thermal cyclers IV A Amplification Setup Note We strongly recommend using gloves and aerosol resistant pipette tips
35. ld for 30 seconds ramp 50 seconds to 70 C hold for 1 minute for 20 cycles then 60 C for 30 minutes 4 C soak Protocol for the Perkin Elmer GeneAmp PCR System 9700 Thermal Cycler Note Perkin Elmer 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM255 Page 8 Printed in USA Revised 9 07 3 After completion of the thermal cycling protocol store the samples at CG 20 C protected from light J Note DNA quantity and quality affect PCR yield Use the recommended Promega 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 V Detection of Amplified Fragments Using the ABI PRISM 310 Genetic Analyzer Materials to Be Supplied by the User e dry heating block water bath or thermal cycler e 310 capillaries 47cm x 50m Ap
36. llaries viewer window in the data collection software Each injection will take approximately 45 minutes VIII Data Analysis VIII A MSI Analysis Overview Detection of MSI is based on comparison of allelic profiles generated from amplification of matching normal and tumor DNA The appearance of novel alleles in the tumor DNA indicates microsatellite instability Figure 5 The MSI Analysis System allows the co amplification and detection of a panel of microsatellite markers that have been shown to be sensitive and specific for detection of MSI H tumors with mismatch repair deficiencies 2 3 Specific PCR product sizes and the respective fluorescent dyes used for labeling of each microsatellite marker are described in Table 1 NR 21 BAT 26 BAT 25 NR 24 Penta D Penta C f il A ull 4639TA Figure 5 Microsatellite instability assays using the MSI Analysis System Matching normal samples top panel and MSI positive colon tumor samples bottom panel were analyzed using the MSI Analysis System Two nanograms of genomic DNA were amplified and analyzed using an ABI PRISM 3100 genetic analyzer and the allelic patterns of the normal and tumor samples are shown The presence of new alleles in the tumor sample see arrows that were not present in the normal sample indicates MSI PCR product sizes and respective fluorescent dyes for loci contained in the MSI Analysis System are provided in Table 1 Promega Corporat
37. morphic so that most individuals are homozygous for the same common allele for a given marker see Section XI B for allele frequencies 4635TA Figure 6 Stutter artifact peaks Stutter peaks 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 can also be seen at the Penta C marker 5bp from true allele peaks Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 19 O Promega VIII A MSI Analysis Overview continued Two polymorphic pentanucleotide repeat markers Penta C and Penta D are included in the MSI Analysis System to help confirm that tumor and matching normal samples are from the same individual 13 14 Alleles found in the normal sample should also be present in the tumor if the tumor is from the same individual MSI has been observed in 14 of MSI H tumors in Penta C and 35 in Penta D so additional pentanucleotide alleles may also be present in some MSI H tumor samples 2 A matching probability between 1 10 and 1 16 was calculated for Penta C and between 1 18 to 1 33 for Penta D based upon allele frequency data 13 14 Matching probability is the average number of randomly sel
38. mples Increase the peak amplitude threshold i e 150 200RFU in the analysis method and reanalyze If problems persist generate a new matrix or perform a new spectral calibration and apply to samples Samples not properly denatured prior to loading Heat denature the samples for the recommended time and cool on crushed ice 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 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 Preferential amplification DNA is cross linked in sample DNA prepared from paraffin of smaller loci embedded samples often contain DNA that is cross linked with other DNA or protein molecules preventing amplification of longer DNA fragments Use the MagneSil Genomic Fixed Tissue System to purify DNA Degraded DNA DNA template is 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
39. plied Biosystems e performance optimized polymer 4 POP 4 Applied Biosystems e 10X genetic analyzer buffer with EDTA Applied Biosystems e sample tubes and septa Applied Biosystems e aerosol resistant pipette tips e Hi Di formamide Applied Biosystems Cat 4311320 PowerPlex Matrix Standards 310 Cat DG4640 e crushed ice Note The quality of the formamide is critical Use Hi Di formamide with a conductivity less than 100pS cm Freeze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with conductivity greater than 100p5 cm may contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution 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 V A Matrix Standardization The PowerPlex Matrix Standards 310 Cat DG4640 are required for matrix standardization 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 on matrix standardization see the PowerPlex Matrix S
40. s designed and sold for use in the multiplex PCR process covered by U S Pat No 5 582 989 Canadian Pat No 1 339 731 and Australian Pat No 634175 A limited license has been granted under the patent to use only this amount of the product to practice the multiplex PCR process and is conveyed to the purchaser by the purchase of this product 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 F rderung der Wissenschaften e V Germany The development and use of STR loci are covered by U S Pat No 5 364 759 Australian Pat No 670231 and other pending patents assigned to Baylor College of Medicine Houston Texas Patents for the foundational PCR process European Pat Nos 201 184 and 200 362 expired on March 28 2006 In the U S the patents covering the foundational PCR process expired on March 29 2005 2004 2007 Promega Corporation All Rights Reserved MagneSil MagneSphere and PowerPlex are registered trademarks of Promega Corporation ABI PRISM GeneMapper and MicroAmp are registered trademarks of Applera Corporation AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems Inc ART is a registered trademark of Molecular Bio Products Inc GenBank is a registered trademark of U S Department of Health and Human Services POP 4 and Hi Di are trademarks of Applera Corporation Products may be covered by pending
41. tandards 310 Technical Bulletin TBD021 supplied with Cat DG4640 available upon request from Promega or online at www promega com tbs Proper generation of a matrix file is critical to evaluate multicolor systems with the ABI PRISM 310 genetic analyzer A matrix must be generated for each ABI PRISM 310 genetic analyzer For answers to questions about matrix creation send an e mail message to techserv promega com Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 9 O Promega V B Sample Preparation The Internal Lane Standard 600 is included in each experiment to standardize the 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 deionized formamide as follows 1 0p1 ILS 600 24pl deionized formamide x injections Combine 25 l of the prepared loading cocktail and 1p of amplified sample Note Instrument detection limits vary therefore injection time or the amount of product mixed with loading cocktail may need to be increased or decreased If the peak heights are too high i e greater than 3 000RFU the samples can be diluted in Gold ST R 1X Buffer before mixing with loading cocktail This may result in uneven allele peak heights across loci
42. tch repair system to correct these errors MSI analysis can be used as a screening method to identify samples for further characterization In 1997 a National Cancer Institute NCI workshop recommended a panel of five microsatellite markers to detect MSI consisting of two mononucleotide markers and three dinucleotide repeats 10 Samples with instability in two or more of these markers are defined as MSI High MSI H whereas those with one unstable marker are designated as MSI Low MSI L Samples with no detectable alterations are MSI stable MSS Limitations in the original panel resulting from inclusion of dinucleotide repeats were addressed at a 2002 NCI workshop and revised recommendations for MSI detection were proposed 11 It was recommended to substitute mononucleotide for dinucleotide repeat markers to improve sensitivity and specificity for detection of MSI LC 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 3130x genetic analyzer The ILS 600 is designed for use in each CE injection to increase precision in analysis when
43. w 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 It is helpful to use names with similar initial characters when naming matched tumor and normal samples so that these samples remain together when analysis results are sorted 5 Place samples in the instrument and close the instrument doors 6 In the 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 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM255 Revised 9 07 Page 17 VILC Instrument Preparation continued v 10 Click on the green Run Instrument arrow on the toolbar to start the Promega sample run 11 Monitor electrophoresis by observing the run view array or capi
44. x Standards 310 1 each DG4640 PowerPlex Matrix Standards 3100 3130 1 each DG4650 Not for Medical Diagnostic Use II DNA Extraction Methods Obtaining sufficient high quality DNA from formalin fixed paraffin embedded tissues can be problematic DNA is often degraded due to prolonged or unsuitable fixation of the tissue sample before embedding in paraffin Therefore we recommend using the MagneSil Genomic Fixed Tissue System Cat MD1490 to extract DNA from paraffin embedded tissues when using the MSI Analysis System The MagneSil Genomic Fixed Tissue System provides a simple technique to prepare genomic DNA from formalin fixed paraffin embedded tissue After an overnight Proteinase K digestion genomic DNA can be manually purified from formalin fixed paraffin embedded thin tissue sections in less than an hour Amplifiable genomic DNA can be isolated from 10pm thin sections without the use of organic solvents or centrifugation of the lysate prior to purification Samples can be processed in the manual format using the MagneSphere Technology Magnetic Separation Stand twelve position Cat Z5342 DNA extraction using the MagneSil Genomic Fixed Tissue System can also be automated Please contact your local Promega Branch Office or Distributor contact information available at www promega com or email techserv promega com for more information Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Tol
45. ze formamide in aliquots at 20 C Multiple freeze thaw cycles or long term storage at 4 C may cause a breakdown of the formamide Formamide with conductivity greater than 100uS cm may contain ions that compete with DNA during injection This results in lower peak heights and reduced sensitivity A longer injection time may not increase the signal Caution 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 VI A Spectral Calibration The PowerPlex Matrix Standards 3100 3130 Cat DG4650 are 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 For protocols and additional information on spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 supplied with Cat DG4650 available upon request from Promega or online at www promega com tbs 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 matrix creation send an e mail to techserv promega com Promega Corporation 2800 Woods Hollow Road

MSI Analysis System, Version 1.2 Technical Manual, TM255

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