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GenePrint® 10 System

1. 10 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer 10 5 B Detection of Ampli ed Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 22 6 Data Analysis 24 6 A Importing GenePrint 10 Panels and Bins Text Files with GeneMapper Software Version 4 0 25 6 B Importing the ILS 600 Size Standard into GeneMapper Software Version 4 0 25 6 C Importing the GenePrint 10 GeneMapper Analysis Method into GeneMapper Software Version 4 0 26 6 D Processing Data with GeneMapper Software Version 4 0 26 6 E Importing GenePrint 10 Panels and Bins Text Files with GeneMapper ID Softwa
2. Cell Viability and Cytotoxicity Assays Product Size Cat CytoTox Glo Cytotoxicity Assay 10ml G9290 MultiTox Fluor Multiplex Cytotoxicity Assay 10ml G9200 CytoTox Fluor Cytotoxicity Assay 10ml G9260 CellTiter Glo Luminescent Cell Viability Assay 10ml G7570 Additional Sizes Available Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 57 www promega com TM392 Revised 7 14 Apoptosis Assays Product Size Cat Apo ONE Homogeneous Caspase 3 7 Assay uorescent 10ml G7790 Caspase Glo 3 7 Assay 10ml G8091 Caspase Glo 8 Assay 10ml G8201 Caspase Glo 9 Assay 10ml G8211 Additional Sizes Available 10 Summary of Changes The following changes were made to the 7 14 revision of this document 1 Expired patent or license statements were removed 2 The document design was updated 58 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com a U S Pat Nos 5 843 660 and 6 221 598 Australian Pat No 724531 Canadian Pat No 2 118 048 Korean Pat No 290332 Singapore Pat No 57050 Japanese Pat No 3602142 and other patents pending b Allele sequences for one or more of the loci vWA FGA D8S1179 D21S11 and D18S51 in allelic ladder mixtures is licens
3. 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 window in the data collection software Each injection will take approximately 45 minutes Note If peaks are low or absent the sample can be reinjected with an increased injection time If the ILS 600 is also a ected check the laser power 6 Data Analysis Due to the structure of the hid les generated on the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 polymer and a 36cm array HID application data generated on this electrophoresis platform with this con guration can only be analyzed using GeneMapper ID X software version 1 2 or later Data generated on the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with POP 4 polymer or Applied Biosystems 3130 or 3130xl Genetic Analyzer with POP 4 polymer can be analyzed using GeneMapper 4 0 software or GeneMapper ID software version 3 2 or 3 2 1 See Table 2 Table 2 Capillary Electrophoresis Instrument and Data Analysis Compatibility Capillary Electrophoresis Instru ment Poly mer Array Length Data Analysis Software ABI PRISM 3100 and 3100 Avant Genetic Analyzer 4 or 16 capillaries POP 4 36cm GeneMapper soft
4. n 4 peaks stutter or shadow bands is due to the loss of a repeat unit during DNA ampli cation somatic variation within the DNA sample material or both The amount of this artifact observed depends primarily on the locus and the DNA sequence being ampli ed Terminal nucleotide addition 12 13 occurs when a nonproofreading thermostable DNA polymerase adds a nucleotide generally adenine to the 3 ends of ampli ed DNA fragments in a template independent manner The e ciency with which this occurs varies with di erent primer sequences Thus an artifact peak one base shorter than expected i e missing the terminal addition is sometimes seen We have modi ed primer sequences and added a nal extension step of 60 C for 10 minutes 14 to the ampli cation protocols to provide conditions for essentially complete terminal nucleotide addition when recommended amounts of template DNA are used The presence of microvariant alleles alleles di ering from one another by lengths other than the repeat length complicates interpretation and assignment of alleles There appears to be a correlation between a high degree of polymorphism a tendency for microvariants and increased mutation rate 15 16 D21S11 displays numerous relatively common microvariants CE related artifacts are occasionally seen in one or all color channels Minor voltage changes or urea crystals passing by the laser can cause spikes or un
5. Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality rede ne the size standard for the sample to skip these peaks No alleles called but no error message appears Panels text le was not selected for sample In the Panel column select the appropriate panels text le for the GenePrint 10 System No size standard was selected In the Size Standard column be sure to select the appropriate size standard Size standard was not correctly de ned or size peaks were missing Rede ne size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be agged as red and no allele sizes will be called Signi cantly raised baseline Poor spectral calibration Perform a new spectral calibra tion and re run the samples Re run and optimize the matrix Make sure that the matrix applied was generated on the same instrument Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 43 www promega com TM392 Revised 7 14 Symptoms
6. OK The Panel Manager window will close automatically 6 F Importing the ILS 600 Size Standard into GeneMapper ID Software Versions 3 2 and 3 2 1 1 Save the ILS_600 xml le to a known location on your computer 2 Select Tools then GeneMapper Manager 3 Select the Size Standard tab 4 Select Import 5 Browse to the location of the ILS_600 xml le 6 Highlight the le then select Import 7 Select Done to save changes and exit the GeneMapper Manager 6 G Importing the GenePrint 10 GeneMapper ID Analysis Method into GeneMapper ID Software Versions 3 2 and 3 2 1 1 Save the GenePrint 10 GMID 3 2 Analysis Method xml le to a known location on your computer Note This analysis method is compatible with GeneMapper ID software versions 3 2 and 3 2 1 2 Select Tools then GeneMapper Manager 3 Select the Analysis Methods tab 4 Select Import 5 Browse to the location of the GenePrint 10 GMID 3 2 Analysis Method xml le 6 Highlight the le then select Import 7 Select Done to save changes and exit the GeneMapper Manager 6 H Processing Data with GeneMapper ID Software Versions 3 2 and 3 2 1 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to location of the run les Highlight desired les then select Add to List
7. accuracy Figure 1 outlines the GenePrint 10 System protocols in this manual for amplifying STR loci and detecting ampli ed products These protocols were tested at Promega The alleles are resolved using capillary electro phoresis CE and the resulting CE data are analyzed using genotyping software and the parameters given on the Promega web site at www promega com resources tools geneprint 10 system software panels and bin les This site provides instructions and applications to set the report parameters in the GeneMapper software to make genotyping easier and more accurate 4 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 1 Description continued Ampli cation Setup Sections 4 and 9 B Thermal Cycling Sections 4 and 9 B GeneAmp PCR System 9700 Instrument Setup and Sample Preparation Section 5 Applied Biosystems 3500 or 3500xL Genetic Analyzer Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 Data Analysis Section 6 GeneMapper Software Version 4 0 GeneMapper ID X Software Versions 1 2 and Later GeneMapper ID Software Versions 3 2 and 3 2 1 Figure 1 An overview of the GenePrint 10 System protocol P
8. 10 System continued 4 Add the nal volume of each reagent listed in Table 1 to a sterile tube Add Water Ampli cation Grade to the tube rst then add GenePrint 10 5X Master Mix and GenePrint 10 5X Primer Pair Mix The template DNA will be added at Step 6 Table 1 PCR Ampli cation Mix for Ampli cation of Extracted DNA 5 Vortex the PCR ampli cation mix for 5 10 seconds then pipet PCR ampli cation mix into each reaction well Failure to vortex the PCR ampli cation mix su ciently can result in poor ampli cation or locus to locus imbalance 6 Add template DNA 10ng for each sample to the respective well containing PCR ampli cation mix 7 For the positive ampli cation control vortex the tube of 2800M Control DNA then add 1 0 l of 2800M Control DNA to a reaction well containing PCR ampli cation mix 8 For the negative ampli cation control pipet Water Ampli cation Grade or TE 4 bu er instead of template DNA into a reaction well containing PCR ampli cation mix 9 Seal the plate Optional Brie y centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles PCR Ampli cation Mix Component Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade to a nal volume of 25 0 l GenePrint 10 5X Master Mix 5 0 l GenePrint 10 5X Primer Pair Mix 5 0 l templa
9. 106 112 X Y vWA TMR 123 171 10 22 TPOX TMR 262 290 6 13 1The length of each allele in the allelic ladder has been con rmed by sequence analysis 2When using an internal lane standard such as the Internal Lane Standard 600 the calculated sizes of allelic ladder components may di er from those listed This occurs because di erent sequences in allelic ladder and ILS components may cause di erences in migration The dye label also a ects migration of alleles 3HeLa cells have a microvariant allele 13 3 at the D13S317 locus This will appear as an o ladder allele see www cstl nist gov strbase var_D13S317 htm Tri Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 51 www promega com TM392 Revised 7 14 Note Samples on nonFTA cards must be pretreated with the PunchSolution Kit Cat DC9271 to lyse samples before adding PCR ampli cation mix For more information see the PunchSolution Kit Technical Manual TMD038 Failure to pretreat these samples may result in incomplete pro les 1 Prepare cell suspensions in phosphate bu ered saline 1X at a density of 106 cells ml based on cell counting 2 While wearing gloves spot a 20 l aliquot of the cell suspension on an FTA or nonFTA e g Fitzco or S amp S 903 storage card The use of indicating paper ensures that you obtain a punch from the
10. Causes and Comments Error message after attempting to import panels There was a con ict between di erent sets of panels and and bins text les Unable to save panel data bins text les Check to be sure that the bins are installed java SQLEException ORA 00001 unique properly If not delete all panels and bins text les and constraint IFA CKP_NNN violated re import les in a di erent order Samples in the project not analyzed The GenePrint 10 GeneMapper 4 0 analysis method can be imported into GeneMapper software version 4 1 However while the method can be imported it is not possible to use this analysis method in GeneMapper software version 4 1 as it is not an available method in the Analysis Method column of the Project window 7 E GeneMapper ID Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID software at least one allelic ladder must be de ned per folder of sample les being analyzed in the project An insu cient number of ILS 600 fragments was de ned Be sure to de ne at least two ILS 600 fragments smaller than the smallest sample peak or allelic ladder peak and at least two ILS 600 fragments larger than the largest sample peak or allelic ladder peak Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks de ned in the size standard were detected during the run Create a new size st
11. Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 15 www promega com TM392 Revised 7 14 Figure 4 The Edit Size Standard window 11271TA 16 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer continued 4 To create a new QC Protocol navigate to the Library Select QC Protocols then select Create Alterna tively a previously created QC Protocol may be used Assign a descriptive protocol name Select the size standard created in Step 3 The settings for the QC protocol should be based on the internally validated conditions for the GenePrint 10 System on the Applied Biosystems 3500 or 3500xL Genetic Analyzer Figure 5 shows one option for these settings 11272TA Figure 5 The Create New QC Protocol window Promega Corpora on 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 TM392 Revised 7 14 5 To create a new Assay navigate to the Library Select Assays then select Create Alternatively a previ ously created Assay may be used In the Create New Assay window Figure 6 select the Instrument Protoco
12. Revised 7 14 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer continued When creating an Instrument Protocol be sure to select the same dye set that was used to perform the Promega 4 dye spectral calibration We recommend using a run time of 1 210 1 500 seconds and the default injection conditions Run time and other instrument settings should be optimized and validated in your laboratory When optimizing injection conditions in your laboratory you may choose to create speci c Instrument Protocols for each condition tested If a single Instrument Protocol is used follow the instructions in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide to edit a library entry Assign a descriptive protocol name Note For more detailed information refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide 3 To create a new Size Standard for the QC protocol navigate to the Library Select Size Standards then select Create Alternatively a previously created Size Standard may be used Assign the size standard the name Promega_ILS600 or another appropriate name Choose Red as the dye color The fragments in the size standard are 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 and 500 bases See Figure 4 Promega Corpora on 2800 Woods Hollow Road
13. direct ampli cation samples and negative control reactions pipet 25 l of the PCR ampli cation mix prepared in Step 4 into the appropriate reaction wells For the positive control pipet 20 l of the PCR ampli cation mix prepared in Step 5 into the appropriate reaction well Failure to vortex the PCR ampli cation mixes su ciently can result in poor ampli cation or locus to locus imbalance 7 For FTA storage cards add one 1 2mm punch from a card containing human tissue culture cells directly to the appropriate wells of the reaction plate For nonFTA card punches add the PCR ampli cation mix to the PunchSolution Reagent pretreated punch Note It also is acceptable to add the FTA card punch rst then add the PCR ampli cation mix 8 For the positive ampli cation control vortex the tube of 2800M Control DNA then add 5 l of 2800M Control DNA to a reaction well containing 20 l of PCR ampli cation mix Notes 1 Do not include blank storage card punches in the positive control reactions 2 Optimization of the amount of control DNA may be required depending on cycling conditions and laboratory preferences See Step 5 9 Reserve a well containing PCR ampli cation mix as a negative ampli cation control Note An additional negative control with a blank punch may be performed to detect contamination from the storage card or punch device 10 Seal the plate and brie y centrifuge t
14. followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated Ladder in the Sample Type column for proper genotyping 28 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 6 H Processing Data with GeneMapper ID Software Versions 3 2 and 3 2 1 continued 5 In the Analysis Method column select the GenePrint 10 GMID 3 2 Analysis Method xml le imported in Section 6 G 6 In the Panel column select the panels text le that was imported in Section 6 E 7 In the Size Standard column select the size standard that was imported in Section 6 F 8 Select Analyze green arrow button to start data analysis 6 I Importing GenePrint 10 Panels Bins and Stutter Text Files with GeneMapper ID X Software Versions 1 2 and Later 1 Save the GenePrint_10_Panels_IDX_v1 0 txt GenePrint_10_Bins_IDX_v1 0 txt and GenePrint_10_Stutter_IDX_v1 0 txt les to a known location on your computer 2 Open the GeneMapper ID X software 3 Select Tools then Panel Manager 4 Highlight the Panel Manager icon in the upper left navigation pane 5 Sel
15. gloves and safety glasses when working with formamide Promega Corpora on 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 TM392 Revised 7 14 Sample Preparation 1 Thaw the Internal Lane Standard 600 Note Centrifuge tube brie y to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing the Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 samples 9 5 l Hi Di formamide samples 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 If the peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9 75 l of Hi Di formamide 3 Vortex for 10 15 seconds to mix 4 Pipet 10 l of formamide internal lane standard mix into each well 5 Add 1 l of ampli ed sample or 1 l of GenePrint 10 Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limi
16. on Promega products
17. online at www promega com protocols Promega Corpora on 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 TM392 Revised 7 14 4 Protocol for DNA Ampli cation Using the GenePrint 10 System The GenePrint 10 System is optimized for the GeneAmp PCR System 9700 thermal cycler The use of gloves and aerosol resistant pipette tips is highly recommended to prevent cross contamination Keep all pre ampli cation and post ampli cation reagents in separate rooms Prepare ampli cation reactions in a room dedicated for reaction setup Use equipment and supplies dedicated for ampli cation setup Meticulous care must be taken to ensure successful ampli cation A guide to ampli cation troubleshooting is provided in Section 7 The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm Quanti cation of this control DNA by other methods such as qPCR may result in a di erent value Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler with a silver or gold plated silver sample block Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips see Section 9 E The GenePrint 10 System was optimized to amplify 10ng of template DNA in a 25 l reaction using the protocol detailed below Pre
18. 11 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 Instrument Setup and Sample Preparation 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3500 3500xL capillary array 36cm 96 well retainer and base set standard Applied Biosystems Cat 4410228 POP 4 polymer in a pouch for the Applied Biosystems 3500 or 3500xL Genetic Analyzer anode bu er container cathode bu er container MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 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
19. 26 608 274 4330 Fax 608 277 2516 29 www promega com TM392 Revised 7 14 5 In the Choose Security Group window select the applicable security group from the drop down menu then select OK 6 Browse to the location of the ILS_600 xml le 7 Highlight the le then select Import 8 Select Done to save changes and exit the GeneMapper ID X Manager 6 K Importing the GenePrint 10 GeneMapper ID X Analysis Method into GeneMapper ID X Software Versions 1 2 and Later 1 Save the GenePrint 10 GMIDX Analysis Method xml le to a known location on your computer 2 Select Tools then GeneMapper ID X Manager 3 Select the Analysis Methods tab 4 Select Import 5 In the Choose Security Group window select the applicable security group from the drop down menu then select OK 6 Browse to the location of the GenePrint 10 GMIDX Analysis Method xml le 7 Highlight the le then select Import 8 Select Done to save changes and exit the GeneMapper ID X Manager 6 L Processing Single Source Data with GeneMapper ID X Software Versions 1 2 and Later 1 Select File then New Project 2 Select Edit then Add Samples to Project 3 Browse to location of the run les Highlight desired les then select Add to List followed by Add 4 In the Sample Type column use the drop do
20. 330 Fax 608 277 2516 TM392 Revised 7 14 www promega com Table 5 The GenePrint 10 System Allelic Ladder Information 9 B Direct Ampli cation of DNA from Storage Card Punches Materials to Be Supplied by the User GeneAmp PCR System 9700 thermal cycler with a silver or gold plated silver sample block Applied Biosystems microcentrifuge MicroAmp optical 96 well reaction plate Applied Biosystems aerosol resistant pipette tips see Section 9 E PunchSolution Kit Cat DC9271 for nonFTA card punches 1 2mm Harris Micro Punch or equivalent manual punch and cutting mat or automated punch system phosphate bu ered saline 1X storage cards e g FTA Fitzco S amp S 903 This section contains protocols for depositing human tissue culture cells onto a storage card and direct ampli cation of DNA from storage card punches using the GenePrint 10 System and GeneAmp PCR System 9700 thermal cycler STR Locus Label Size Range of Allelic Ladder Components1 2 bases Repeat Numbers of Allelic Ladder Components TH01 FL 156 195 4 9 9 3 10 11 13 3 D21S11 FL 203 259 24 24 2 25 25 2 26 28 28 2 29 29 2 30 30 2 31 31 2 32 32 2 33 33 2 34 34 2 35 35 2 36 38 D5S818 JOE 119 155 7 16 D13S317 JOE 176 208 7 15 D7S820 JOE 215 247 6 143 D16S539 JOE 264 304 5 8 15 CSF1PO JOE 321 357 6 15 Amelogenin TMR
21. 75 l of Hi Di formamide 3 Vortex for 10 15 seconds to mix 4 Pipet 10 l of formamide internal lane standard mix into each well Promega Corpora on 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 TM392 Revised 7 14 5 Add 1 l of ampli ed sample or 1 l of GenePrint 10 Allelic Ladder Mix Cover wells with appropriate septa Note Instrument detection limits vary therefore injection time or the amount of sample mixed with loading cocktail may need to be adjusted Use the Module Manager in the data collection software to modify the injection time in the run module see Instrument Preparation below Alternatively use less DNA template in the ampli cation reactions or reduce the number of cycles in the ampli cation program by 2 4 cycles to achieve the desired signal intensity 6 Centrifuge the plate brie y to remove air bubbles from the wells 7 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 Instrument Preparation Refer to the instrument users manual for instructions on cleaning installing the capillary array performing a spatial calibration and adding polymer Analyze samples as described in the user s manual for the ABI PRISM 3100 or 3100 Avant Genetic Analyzer wi
22. Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks de ned in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality rede ne the size standard for the sample to skip these peaks No alleles called but no error message appears Panels text le was not selected for sample In the Panel column select the appropriate panels text le for the GenePrint 10 System No size standard was selected In the Size Standard column be sure to select the appropriate size standard Size standard was not correctly de ned or size peaks were missing Rede ne size standard to include only peaks present in your sample Terminating analysis early or using short run times will cause larger ladder peaks to be missing This will cause your sizing quality to be agged as red and no allele sizes will be called Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 45 www promega com TM392 Revised 7 14 Symptoms Cau
23. Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 B Ampli cation of Extracted DNA continued Symptoms Causes and Comments Peak height imbalance Excessive amount of DNA Ampli cation of gt 10ng of template per 25 l reaction can result in an imbalance with smaller loci showing more product than larger loci Decrease the number of cycles Degraded DNA sample DNA template was degraded and larger loci showed diminished yield Repurify template DNA if possible Insu cient template DNA Use the recommended amount of template DNA if available Stochastic e ects can occur when amplifying low amounts of template Impure template DNA Inhibitors that may be present in samples can lead to allele dropout or imbalance 7 C Direct Ampli cation of DNA from Storage Card Punches The following information is speci c to direct ampli cation of DNA from storage card punches For additional information about general ampli cation and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks The reaction volume was too low This system is optimized for a nal reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume may result in suboptimal performance Poor sample deposition Collection of cells was variable Increase cycle number Poor sample transfer to storage
24. Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 41 www promega com TM392 Revised 7 14 7 D GeneMapper Software Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper software at least one allelic ladder must be de ned per folder of sample les being analyzed in the project An insu cient number of ILS 600 fragments was de ned Be sure to de ne at least two ILS 600 fragments smaller than the smallest sample peak or allelic ladder peak and at least two ILS 600 fragments larger than the largest sample peak or allelic ladder peak Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks de ned in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis O ladder alleles An allelic ladder from a di erent run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 D
25. Internal Lane Standard 600 The Internal Lane Standard 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 15 Each fragment is labeled with carboxy X rhodamine CXR and may be detected separately as a fourth color in the presence of GenePrint 10 System ampli ed material The ILS 600 is designed for use in each CE injection to increase precision in analyses when using the GenePrint 10 System Protocols to prepare and use this internal lane standard are provided in Section 5 5751TA 1 200 1 000 800 600 400 200 0 60 80 100 120 140 160 180 200 225 250 275 300 325 350 375 400 425 450 475 500 550 600 Figure 15 An electropherogram showing the fragments of the Internal Lane Standard 600 9 D Composition of Bu ers and Solutions Phosphate bu ered saline 1X 8g NaCl 0 2g KCl 1 44g Na2HPO4 2H2O 0 24g KH2HPO4 Dissolve chemicals in 800ml of deionized water Adjust to pH 7 4 with HCl Bring the nal volume to 1 liter with deionized water Sterilize by autoclaving TE 4 bu er 10mM Tris HCl 0 1mM EDTA pH 8 0 1 21g Tris base 0 037g EDTA Na2EDTA 2H2O Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Bring the nal volume to 1 liter with deionized water TE 4 bu er with 20 g ml glycogen 1 21g T
26. Panels text le selected for analysis was incorrect for the STR system used Assign correct panels text le for the GenePrint 10 System The allelic ladder was not identi ed as an allelic ladder in the Sample Type column The internal lane standard was not properly identi ed in the sample Manually rede ne the sizes of the size standard fragments in the sample 42 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 D GeneMapper Software continued Symptoms Causes and Comments O ladder alleles continued Incorrect analysis method used Only the GenePrint 10 System GeneMapper 4 0 analysis method is capable of calibrating the bins based on sizes of alleles in the allelic ladder analyzed using the speci c con guration of instrument polymer and capillary length Creating an analysis method within GeneMapper software version 4 0 does not enable this functionality and will result in o ladder allele calls Size standard not called correctly Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks de ned in the size standard were detected during the run
27. Print 10 5X Primer Pair Mix 2800M Control DNA and Water Ampli cation Grade are provided in a separate box and should be stored separately from those used following ampli cation GenePrint 10 Allelic Ladder Mix and Internal Lane Standard 600 Always include a negative control reaction i e no template to detect reagent contamination We highly recommend the use of gloves and aerosol resistant pipette tips e g ART tips Some of the reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly 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 3 B Spectral Calibration Proper spectral calibration is critical to evaluate multicolor systems with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers A spectral calibration must be performed for each individual instrument Very high peak heights may not be perfectly separated spectrally and an allele peak in one color channel can bleed into another color channel Use of a poor or incorrect matrix will allow this as well For protocols and additional information about spectral calibration see the PowerPlex Matrix Standards 3100 3130 Technical Bulletin TBD022 This manual is available
28. Revised 7 14 TM392 T E C H N I C A L M A N U A L GenePrint 10 System Instruc ons for Use of Product B9510 Promega Corpora on 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 TM392 Revised 7 14 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 genetic promega com GenePrint 10 System 1 Description 2 2 Product Components and Storage Conditions 5 3 Before You Begin 6 3 A Precautions 6 3 B Spectral Calibration 6 4 Protocol for DNA Ampli cation Using the GenePrint 10 System 7 5 Instrument Setup and Sample Preparation
29. a locus For example K562 DNA has a tri allelic pattern at the D21S11 locus The STR genotype of a cell line can evolve over multiple passages Users can genotype cell line DNA regularly with the GenePrint 10 System to monitor any change in the STR genotype 11349TA Figure 13 Representative data for the GenePrint 10 System Ten nanograms of 2800M Control DNA was ampli ed using the GenePrint 10 System Ampli cation products were mixed with Internal Lane Standard 600 and analyzed using an Applied Biosystems 3130 Genetic Analyzer and a 3 0kV 4 second injection The results were analyzed using GeneMapper ID X software version 1 2 and the appropriate panels bins and stutter les 32 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 11348TA Figure 14 The GenePrint 10 Allelic Ladder The GenePrint 10 Allelic Ladder was analyzed using an Applied Biosystems 3130 Genetic Analyzer and a 3kV 4 second injection The sample le was analyzed using GeneMapper ID X software version 1 2 and the appropriate panels bins and stutter les Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 33 www promega com TM392 Revised 7 14 Artifacts and Stutter Stutter products are a common ampli c
30. again then view the label edit table Marker header bar for some loci are gray When an edit is made to a locus the quality ags and marker header bar automatically change to gray To change the GQ and marker header bar for a locus to green override the GQ in the plot window 46 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 F GeneMapper ID X Software continued Symptoms Causes and Comments Alleles not called To analyze samples with GeneMapper ID X software at least one allelic ladder must be de ned per folder of sample les being analyzed in the project An insu cient number of ILS 600 fragments was de ned Be sure to de ne at least two ILS 600 fragments smaller than the smallest sample peak or allelic ladder peak and at least two ILS 600 fragments larger than the largest sample peak or allelic ladder peak In this instance the allelic ladder would have failed the allelic ladder quality check Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks de ned in the size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic la
31. andard using the internal lane standard fragments present in the sample Re run samples using a longer run time A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis O ladder alleles An allelic ladder from a di erent run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 H 44 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 E GeneMapper ID Software continued Symptoms Causes and Comments O ladder alleles continued Panels text le selected for analysis was incorrect for the STR system used Assign correct panels text le for the GenePrint 10 System The allelic ladder was not identi ed as an allelic ladder in the Sample Type column The internal lane standard was not properly identi ed in the sample Manually rede ne the sizes of the size standard fragments in the sample Size standard not called correctly Extra peaks in advanced mode size standard Open the Size Match Editor Highlight the extra peak select
32. 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 Note 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 5 Place samples in the instrument and close the instrument doors 24 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 B Detection of Ampli ed Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 continued 6 In the spectral viewer select dye set F and con rm that the active dye set is the le generated for the GenePrint 10 chemistry 7 In the 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 ampli ed samples
33. arate PCR ampli cation mix will be prepared in Step 5 for the positive control reaction Increase the number of reactions by 10 15 to compensate for pipetting error e g for 96 reactions add 10 to 15 additional reactions While this approach does consume a small amount of each reagent it ensures that you will have enough PCR ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cation mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 52 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 9 B Direct Ampli cation of DNA from Storage Card Punches continued 4 Prepare a PCR ampli cation mix for the direct ampli cation samples and negative controls by adding the nal volume of each reagent listed in Table 6 to a sterile tube Add Water Ampli cation Grade to the tube rst then add GenePrint 10 5X Master Mix and GenePrint 10 5X Primer Pair Mix For FTA card punches the template DNA will be added at Step 7 Table 6 PCR Ampli cation Mix for Direct Ampli cation of DNA from Storage Card Punches 5 For the positive control reaction prepare a PCR ampli cation mix for ampli cation of the 2800M Control DNA by adding the nal volume of each reagent listed in Table 7 to a sterile tube Add Wa
34. ation artifact associated with STR analysis Stutter products are often observed one repeat unit below the true allele peak and occasionally two repeat units smaller or one repeat unit larger than the true allele peak Frequently alleles with a greater number of repeat units will exhibit a higher percent stutter The pattern and intensity of stutter can di er slightly between primer sets for the same loci In addition to stutter peaks other artifact peaks can be observed at some GenePrint 10 System loci Low level products can be seen in the n 2 and n 2 positions two bases below and above the true allele peak respectively with some loci such as D21S11 Samples may show low level artifacts in the noncalling regions between the D7S820 and D16S539 allele ranges Occasionally an o ladder artifact can be observed in the 270 271bp position in the JOE dye channel One or more extra peaks that are not directly related to ampli cation may be observed at positions 8 26 bases smaller than TPOX alleles These extra peaks occur when the ampli ed peaks are particularly intense high signal level or template amount the formamide polymer or capillary was of poor quality or denaturation was ine ective We have carefully selected STR loci and primers to avoid or minimize artifacts including those associated with thermostable DNA polymerases such as repeat slippage and terminal nucleotide addition Repeat slippage 10 11 sometimes called
35. card or variable sampling from storage card Take punches from a di erent portion of the card Increasing cycle number can improve low peak heights DNA was not accessible on nonlytic material Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 39 www promega com TM392 Revised 7 14 Symptoms Causes and Comments Faint or absent allele peaks continued Too much sample in the reaction Use one 1 2mm storage card punch per 25 l reaction Follow the manufacturer s recommendations when depositing sample onto the storage card With storage cards reducing the reaction volumes below 25 l may result in ampli cation failure Active PunchSolution Reagent carried over into the ampli cation reaction when using nonFTA card punches Ensure that the heat block was set at 70 C and samples were incubated for 30 minutes Incubation for shorter time periods may result in incomplete inactivation of the PunchSolution Reagent We have not tested longer incubation times Inactive PunchSolution Reagent Thaw the PunchSolu tion Reagent at 2 10 C Do not store reagents in the refrigerator door where the temperature can uctuate Avoid multiple freeze thaw cycles as this may reduce activity Faint or absent peaks for If t
36. cation mix for 5 10 seconds before dispensing into the reaction plate 7 B Ampli cation of Extracted DNA The following information is speci c to ampli cation of extracted DNA For information about general ampli cation and detection see Section 7 A Symptoms Causes and Comments Faint or absent allele peaks Impure template DNA Because of the small amount of template used this is rarely a problem Depending on the DNA extraction procedure used and sample source inhibitors might be present in the DNA sample Insu cient template Use the recommended amount of template DNA if available High salt concentration or altered pH If the DNA template is stored in TE bu er 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 of K Na Mg2 or EDTA from the DNA sample can negatively a ect PCR A change in pH also may a ect PCR Store DNA in TE 4 bu er 10mM Tris HCl pH 8 0 0 1mM EDTA TE 4 bu er with 20 g ml glycogen or nuclease free water Extra peaks visible in one or all color channels Artifacts of STR ampli cation Ampli cation of excess amounts of puri ed DNA can result in a higher number of artifact peaks Use the recommended amount of template DNA See Section 6 N for additional information on stutter and artifacts 38 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll
37. ce1 5 3 TH01 FL 11p15 5 HUMTH01 human tyrosine hydroxylase gene AATG 17 D21S11 FL 21q11 21q21 HUMD21LOC TCTA Complex 17 D5S818 JOE 5q23 3 32 NA AGAT D13S317 JOE 13q22 q31 NA TATC D7S820 JOE 7q11 21 22 NA GATA D16S539 JOE 16q24 qter NA GATA CSF1PO JOE 5q33 3 34 HUMCSF1PO human c fms proto oncogene for CSF 1 receptor gene AGAT Amelogenin2 TMR Xp22 1 22 3 and Y HUMAMEL human Y chromosomal gene for Amelogenin like protein NA vWA TMR 12p13 31 HUMVWFA31 human von Willebrand factor gene TCTA Complex 17 TPOX TMR 2p23 2pter HUMTPOX human thyroid peroxidase gene AATG 1The August 1997 report 18 19 of the DNA Commission of the International Society for Forensic Haemogenetics ISFH states 1 for STR loci within coding genes the coding strand shall be used and the repeat sequence motif de ned using the rst possible 5 nucleotide of a repeat motif and 2 for STR loci not associated with a coding gene the rst database entry or original literature description shall be used 2Amelogenin is not an STR but displays a 106 base X speci c band and a 112 base Y speci c band TMR carboxy tetramethylrhodamine FL uorescein JOE 6 carboxy 4 5 dichloro 2 7 dimethoxy uorescein NA not applicable 50 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4
38. color Re inject samples to con rm 36 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 A Ampli cation and Fragment Detection continued Symptoms Causes and Comments Extra peaks visible in one or all color channels Incorrect spectral was active Re run samples and con rm continued that the spectral is set for the dye set being used See instructions on instrument preparation in Section 5 Pull up or bleedthrough Pull up can occur when peak heights are too high or if a poor or incorrect matrix is applied to the samples Perform a new spectral calibration and re run the samples Instrument sensitivities can vary Optimize the injection conditions See Section 5 CE related artifacts contaminants Contaminants in the water used with the instrument or to dilute the 10X genetic analyzer bu er may generate peaks in the uorescein and JOE channels Use autoclaved deionized water change vials and wash bu er reservoir Repeat sample preparation using fresh formamide Long term storage of ampli ed samples in formamide can result in degradation The CE polymer was beyond its expiration date or polymer was stored at room temperature for more than one week Maintain instrumentation on a daily or weekly basis as recommended by the manufacturer Allelic ladder not r
39. cts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 10 minute extension step at 60 C after thermal cycling Section 9 B Decrease cycle number Increase the nal extension time Peak height imbalance Excessive amount of DNA Ampli cation of gt 20ng of template can result in an imbalance with smaller loci showing more product than larger loci Use one 1 2mm punch from a storage card per 25 l reaction Follow the manufacturer s recommendations when depositing sample onto the card Decrease cycle number The reaction volume was too low This system is optimized for a nal reaction volume of 25 l to overcome inhibitors present in FTA cards and PunchSolution Reagent Decreasing the reaction volume can result in suboptimal performance DNA was not accessible on nonlytic material Small loci may amplify preferentially with large loci dropping out Pretreat nonFTA materials with PunchSolution Reagent to ensure that DNA is liberated from cellular proteins Extreme variability in sample to sample There can be signi cant individual to individual variability peak heights in the deposition of cells onto a storage card resulting in peak height variability The PunchSolution Kit increases the recovery of ampli able DNA from samples but does not normalize the amount of DNA present Promega Corpora on 2800 Woods
40. dders are used for analysis O ladder alleles An allelic ladder from a di erent run than the samples was used Re analyze samples with an allelic ladder from the same run The GeneMapper ID X software requires that the allelic ladder be imported from the same folder as the sample Be sure that the allelic ladder is in the same folder as the sample Create a new project and re analyze as described in Section 6 L Panels text le selected for analysis was incorrect for the STR system used Assign correct panels text le for the GenePrint 10 System The allelic ladder was not identi ed as an allelic ladder in the Sample Type column The internal lane standard was not properly identi ed in the sample Manually rede ne the sizes of the size standard fragments in the sample A low quality allelic ladder was used during analysis Ensure that only high quality allelic ladders are used for analysis Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 47 www promega com TM392 Revised 7 14 Symptoms Causes and Comments Size standard not called correctly Extra peaks in size standard Open the Size Match Editor Highlight the extra peak select Edit and select delete size label Select auto adjust sizes Run was too short and larger peaks in ILS were not captured Not all ILS 600 peaks de ned in th
41. dditional STR Locus Information 48 B Direct Ampli cation of DNA from Storage Card Punches 50 C The Internal Lane Standard 600 55 D Composition of Bu ers and Solutions 55 E Related Products 56 10 Summary of Changes 57 1 Description Cell line misidenti cation is an important concern for researchers In some cases laboratories have invested substantial time and e ort researching cell lines that were later revealed to be misidenti ed 1 This has prompted the National Institutes of Health to issue a notice to researchers strongly recommending authentication procedures when using cultured cells 2 Genetic pro ling can be used for human cell line authentication using short tandem repeat STR loci 3 6 Capes Davis et al published a list of misidenti ed or contaminated human cell lines 7 the updated list can be found at http standards atcc org kwspub home the_international_cell_line_au
42. e size standard were detected during the run Create a new size standard using the internal lane standard fragments present in the sample Re run samples using a longer run time Peaks in size standard missing If peaks are below threshold decrease the peak amplitude threshold in the analysis method for the red channel to include peaks If peaks are low quality rede ne the size standard for the sample to skip these peaks Signi cantly raised baseline Poor spectral calibration Perform a new spectral calibra tion and re run the samples Re run and optimize the matrix Make sure that the matrix applied was generated on the same instrument 8 References 1 Chatterjee R 2007 Cell biology Cases of mistaken identity Science 315 928 31 2 Ruiz Bravo N and Gottesman M 2007 Notice regarding authentication of cultured cell lines This can be viewed online at http grants nih gov grants guide notice les NOT OD 08 017 html 3 Yoshino K et al 2006 Essential role for gene pro ling analysis in the authentication of human cell lines Human Cell 19 43 8 4 Szibor R et al 2003 Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sci Int 138 37 43 5 Dirks W G et al 2005 Short tandem repeat DNA typing provides an international reference standard for authentication of human cell lines ALTEX 22 103 9 6 Masters J R et al 2001 S
43. ect File then Import Panels 6 In the Choose Security Group window select the applicable security group from the drop down menu then select OK 7 Navigate to the panels text le saved to your computer above Select the le then Import 8 In the navigation pane highlight the GenePrint 10 panels folder that you just imported in Step 5 9 Select File then Import Bin Set 10 Navigate to the bins text le saved to your computer above Select the le then Import 11 In the navigation pane highlight the GenePrint 10 panels folder that you just imported in Step 5 12 Select File then Import Marker Stutter A warning box will appear asking if you want to overwrite current values Select Yes 13 Navigate to the stutter text le saved to your computer above Select the le then Import 14 At the bottom of the Panel Manager window select OK This will save the panels bins and stutter text les then close the window automatically 6 J Importing the ILS 600 Size Standard into GeneMapper ID X Software Versions 1 2 and Later 1 Save the ILS_600 xml le to a known location on your computer 2 Select Tools then GeneMapper ID X Manager 3 Select the Size Standard tab 4 Select Import Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 95
44. ed under U S Pat Nos 7 087 380 7 645 580 Australia Pat No 2003200444 and corresponding patent claims outside the US c 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 Gesellscha zur F rderung der Wissenscha en e V Germany 2013 2014 Promega Corpora on All Rights Reserved Apo ONE Caspase Glo CellTiter Glo GenePrint MagneSil Maxwell PowerPlex and Wizard are registered trademarks of Promega Corpora on CytoTox Fluor CytoTox Glo DNA IQ PunchSolu on and Slicprep are trademarks of Promega Corpora on ABI PRISM and MicroAmp are registered trademarks of Applera Corpora on Applied Biosystems GeneMapper and POP 4 are registered trademarks of Applied Biosystems ART is a registered trademark of Molecular Bio Products Inc FTA is a registered trademark of Flinders Technologies Pty Ltd and is licensed to Whatman GeneAmp is a registered trademark of Roche Molecular Systems Inc GenBank is a registered trademark of U S Dept of Health and Human Services Hi Di is a trademark of Applera Corpora on Products may be covered by pending or issued patents or may have certain limita ons Please visit our Web site for more informa on All prices and speci ca ons are subject to change without prior no ce Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date informa on
45. expected peaks Spikes sometimes appear in one color but often are easily identi ed by their presence in more than one color Re inject the samples to con rm 34 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 Troubleshooting For questions not addressed here please contact your local Promega Branch O ce or Distributor Contact information available at www promega com E mail genetic promega com 7 A Ampli cation and Fragment Detection This section provides information about general ampli cation and detection For questions about ampli cation of extracted DNA see Section 7 B For questions about direct ampli cation see Section 7 C Symptoms Causes and Comments Faint or absent allele peaks The GenePrint 10 5X Master Mix was not vortexed well before use Vortex the 5X Master Mix for 15 seconds before dispensing into the PCR ampli cation mix An air bubble formed at the bottom of the reaction well Use a pipette to remove the air bubble or centrifuge the reactions brie y before thermal cycling The reaction volume was too low This system is optimized for a nal reaction volume of 25 l Decreasing the reaction volume may result in suboptimal performance Improper storage of the 2800M Control DNA Thermal cycler or plate problems Review the thermal cycling prot
46. ferential ampli cation of smaller loci can occur Expect to see high peak heights for smaller loci and relatively lower peak heights for larger loci if more than the recommended amount of template is used Reduce the amount of template DNA or number of cycles to correct this Ampli cation Setup 1 Thaw the GenePrint 10 5X Master Mix and GenePrint 10 5X Primer Pair Mix completely Note Centrifuge tubes brie y to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Determine the number of reactions to be set up This should include positive and negative control reactions Increase the number of reactions by 10 15 to compensate for pipetting error e g for 96 reactions add 10 to 15 additional reactions While this approach does consume a small amount of each reagent it ensures that you will have enough PCR ampli cation mix for all samples It also ensures that each reaction contains the same PCR ampli cations mix 3 Use a clean MicroAmp plate for reaction assembly and label appropriately 8 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 4 Protocol for DNA Ampli cation Using the GenePrint
47. he Consumables Information and Maintenance Noti cations are acceptable Set the oven temperature to 60 C then select Start Pre Heat at least 30 minutes prior to the rst injection to preheat the oven 9247TA Figure 2 The Dashboard 2 To create a new Instrument Protocol navigate to the Library select Instrument Protocol then select Create Alternatively a previously created Instrument Protocol may be used Figure 3 shows the settings used at Promega for the Applied Biosystems 3500xL Genetic Analyzer for the application type dye set capillary length polymer run module and appropriate protocol information The only setting that was changed from the default settings is dye set Promega Corpora on 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 TM392 Revised 7 14 11270TA Figure 3 The Edit Instrument Protocol window The recommended settings are Application Type HID Capillary Length 36cm Polymer POP 4 Dye Set Promega 4Dye Run Module HID36_POP4 xl Injection Time1 24 seconds Injection Voltage 1 2kV Run Time 1 210 1 500 seconds 1Injection time may be modi ed 2 24 seconds to increase or decrease peak heights 14 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392
48. he plate to bring storage card punches to the bottom of the wells Thermal Cycling This manual contains a protocol for use of the GenePrint 10 System with the GeneAmp PCR System 9700 thermal cycler We have not tested other thermal cyclers For information about other thermal cyclers please contact Promega Technical Services by e mail genetic promega com Ampli cation and detection instrumentation may vary You will need to optimize protocols including cycle number 25 27 cycles injection time and loading volume for your laboratory instrumentation Testing at Promega shows that 26 cycles works well for a variety of sample types 1 Place the MicroAmp plate in the thermal cycler 54 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 9 B Direct Ampli cation of DNA from Storage Card Punches continued 2 Select and run the recommended protocol Be sure that Max mode is selected as the ramp speed The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycle time is 1 5 hours 3 After completion of the thermal cycling protocol store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts PCR Optimization Cycle number should be opt
49. he positive control reaction failed to amplify check to the positive control reaction make sure that the correct amount of 2800M Control DNA was added to the reaction We recommend 5 l 50ng of 2800M Control DNA per 25 l ampli cation reaction Do not include a blank punch in the positive control reaction Presence of a blank punch may inhibit ampli cation of 2800M Control DNA Improper storage of the 2800M Control DNA The appropriate PCR ampli cation mix was not used for the positive control reaction with the 2800M Control DNA Prepare the appropriate PCR ampli cation mix as described in Table 7 of Section 9 B and add 5 l 50ng of 2800M Control DNA to a well containing PCR ampli ca tion mix 40 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 7 C Direct Ampli cation of DNA from Storage Card Punches continued Symptoms Causes and Comments Extra peaks visible in one or all color channels Punch may be contaminated Take punches from blank paper between samples Artifacts of STR ampli cation Direct ampli cation of gt 20ng of template can result in a higher number of artifact peaks Use the recommended punch size and number See Section 6 N for additional information on stutter and artifacts Artifacts of STR ampli cation Ampli cation of STRs can result in artifa
50. hort tandem repeat pro ling provides an international reference standard for human cell lines Proc Natl Acad Sci USA 98 8012 7 7 Capes Davis A et al 2010 Check your cultures A list of cross contaminated or misidenti ed cell lines Int J Cancer 127 1 8 8 ANSI ATCC ASN 0002 2011 Authentication of human cell lines Standardization of STR pro ling ANSI eStandards Store 2012 48 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 8 References continued 9 Kline M C et al 2005 Results from the NIST 2004 DNA quantitation study J Forensic Sci 50 570 8 10 Levinson G and Gutman G A 1987 Slipped strand mispairing A major mechanism for DNA sequence evolution Mol Biol Evol 4 203 21 11 Schl tterer C and Tautz D 1992 Slippage synthesis of simple sequence DNA Nucleic Acids Res 20 211 5 12 Smith J R et al 1995 Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase Genome Res 5 312 7 13 Magnuson V L et al 1996 Substrate nucleotide determined non templated addition of adenine by Taq DNA polymerase Implications for PCR based genotyping and cloning BioTechniques 21 700 9 14 Walsh P S Fildes N J and Reynolds R 1996 Sequence analysis and characterization of stutter produc
51. imized based on the results of an initial experiment to determine the sensitivity with your collection method sample types and instrumentation 1 Choose several samples that represent typical samples you encounter in the laboratory Prepare them as you would using your normal work ow 2 Be sure to pretreat nonFTA samples with the PunchSolution Kit Cat DC9271 3 Prepare three identical reaction plates with punches from the same samples 4 Amplify samples using the thermal cycling protocol provided above but subject each plate to a di erent cycle number 25 27 cycles 5 Following ampli cation use your laboratory s validated separation and detection protocols to determine the optimal cycle number Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 26 cycles then 60 C for 10 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and enter the reaction volume Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 55 www promega com TM392 Revised 7 14 9 C The
52. ine human loci including the ASN 0002 loci TH01 TPOX vWA Amelogenin CSF1PO D16S539 D7S820 D13S317 and D5S818 as well as D21S11 These loci collectively provide a genetic pro le with a random match probability of 1 in 2 92 109 The GenePrint 10 System is compatible with the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers You may need to optimize protocols including the amount of template DNA cycle number injection conditions and loading volume for your laboratory instrumentation The GenePrint 10 System contains all materials necessary to amplify STR regions of human genomic DNA including a hot start thermostable DNA polymerase which is a component of the GenePrint 10 5X Master Mix An internal lane standard ILS and allelic ladder are provided for sizing and genotyping of ampli ed fragments and the 2800M Control DNA is supplied as a positive control The ILS is added to every sample after ampli cation and used within each capillary electrophoresis run to determine the size of each ampli ed product The allelic ladder consists of the most common alleles at a particular locus and is used as a standard to positively identify each allele GenePrint 10 Allelic Ladder Mix information including the size range and repeat numbers for each allele can be found in Section 9 A The 2800M Control DNA has a known genotype and can be used to verify genotyping
53. l created in Step 2 and the QC Protocol created in Step 4 Assign a descriptive assay name Select the application type HID An Assay is required for all named samples on a plate 11327TA Figure 6 The Create New Assay window 18 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer continued 6 To create a new File Name Convention Figure 7 navigate to the Library Select File Name Conventions then select Create Alternatively a previously created File Name Convention may be used Select the File Name Attributes according to laboratory practices and save with a descriptive name 9252TA Figure 7 The Create New File Name Convention window Promega Corpora on 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 TM392 Revised 7 14 7 To create a new Results Group Figure 8 navigate to the Library Select Results Group then select Create Alternatively a previously created Results Group may be used Select the Results Group Attributes according to laboratory practices Save with a descriptive name 9253TA Figure 8 The Create New Results Group wind
54. mamide 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 Sample Preparation 1 Thaw the Internal Lane Standard 600 Note Centrifuge tube brie y to bring contents to the bottom then vortex for 15 seconds before each use Do not centrifuge after vortexing as this may cause the size standard to be concentrated at the bottom of the tube 2 Prepare a loading cocktail by combining and mixing the Internal Lane Standard 600 and Hi Di formamide as follows 0 5 l ILS 600 samples 9 5 l Hi Di formamide samples 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 If the peak heights are too low we recommend altering the formamide internal lane standard mix to contain 1 0 l of ILS 600 and 9 0 l of Hi Di formamide If the peak heights are too high we recommend altering the loading cocktail to contain 0 25 l of ILS 600 and 9
55. nd must be stored in the dark We strongly recommend that pre ampli cation and post ampli cation reagents be stored and used separately with di erent pipettes tube racks etc Available Separately Matrix standards are required for initial setup of the color separation matrix see Section 3 B The matrix standards are sold separately and are available for the ABI PRISM 3100 and 3100 Avant Genetic Analyzers and Applied Biosystems 3130 3130xl 3500 and 3500xL Genetic Analyzers PowerPlex Matrix Standards 3100 3130 Cat DG4650 6 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 3 Before You Begin 3 A Precautions The quality of puri ed DNA or direct ampli cation samples small changes in bu ers ionic strength primer concentrations choice of thermal cycler and thermal cycling conditions can a ect PCR success We suggest strict adherence to recommended procedures for ampli cation as well as electrophoresis and uorescence detection PCR based STR analysis is subject to contamination by very small amounts of human DNA Take extreme to avoid cross contamination when preparing template DNA handling primer pairs assembling ampli cation reactions and analyzing ampli cation products Reagents and materials used prior to ampli cation GenePrint 10 5X Master Mix Gene
56. ocol in Section 4 or 9 B We have not tested other reaction plates or thermal cyclers Calibrate the thermal cycler heating block if necessary Primer concentration was too low Use the recommended primer concentration Vortex the GenePrint 10 5X Primer Pair Mix for 15 seconds before use Samples were not denatured completely Heat denature samples for the recommended time then cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do not cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Poor capillary electrophoresis injection ILS 600 peaks also a ected Re inject the 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 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 35 www promega com TM392 Revised 7 14 Symptoms Causes and Comments Extra peaks visible in one or all color channels Contamination with another template DNA or previously ampli ed DNA Cross contamination can be a problem Use aerosol resistant pipette tips and change gloves regularly Samples were not denatured completely Heat denature samples for the recommended time and cool on crushed ice or in an ice water bath immediately prior to capillary electrophoresis Do n
57. ontact Promega Technical Services by e mail genetic promega com Ampli cation and detection instrumentation may vary You may need to optimize protocols including the amount of template DNA cycle number injection time and loading volume for your laboratory instrumentation Testing at Promega shows that 30 cycles work well for 10ng of puri ed DNA template 1 Place the MicroAmp plate in the thermal cycler 2 Select and run the recommended protocol Be sure that Max mode is selected as the ramp speed The preferred protocol for use with the GeneAmp PCR System 9700 thermal cycler is provided below The estimated total cycling time is 1 5 hours 3 After completion of the thermal cycling protocol store ampli ed samples at 20 C in a light protected box Note Long term storage of ampli ed samples at 4 C or higher may produce artifacts Thermal Cycling Protocol1 96 C for 1 minute then 94 C for 10 seconds 59 C for 1 minute 72 C for 30 seconds for 30 cycles then 60 C for 10 minutes 4 C soak 1When using the GeneAmp PCR System 9700 thermal cycler the program must be run with Max mode as the ramp speed This requires a silver or gold plated silver sample block The ramp speed is set after the thermal cycling run is started The Select Method Options screen appears Select Max for the ramp speed and enter the reaction volume 10 Promega Corpora on 2800 Woods Hollow Road Madison WI 537
58. ot cool samples in a thermal cycler set at 4 C as this may lead to artifacts due to DNA re annealing Artifacts of STR ampli cation Ampli cation of STRs can result in artifacts that appear as peaks one base smaller than the allele due to incomplete addition of the 3 A residue Be sure to perform the 10 minute extension step at 60 C after thermal cycling Section 4 or 9 B Decrease the number of cycles Plasticware can alter heat transfer during ampli ca tion and prevent full adenylation Increase the nal extension time Artifacts The signal strength of certain artifacts increases with storage of the ampli cation plate at 4 C sometimes in as short a time period as overnight but more commonly when left at 4 C for a few days We recommend storing ampli cation products at 20 C Double stranded DNA migrates faster than single stranded DNA during capillary electrophoresis Appearance of shadow peaks migrating in front of the main peaks especially if the shadow peaks are separated by the same distance as the main peaks in a heterozygote can indicate the presence of double stranded DNA due to incomplete denaturation or post injection re annealing CE related artifacts spikes Minor voltage changes or urea crystals passing by the laser can cause spikes or unexpected peaks Spikes sometimes appear in one color but often are easily identi ed by their presence in more than one
59. ow 20 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer continued 8 To create a New Plate navigate to the Library and from the Manage menu select Plates then Create 9 Assign a descriptive plate name Select the plate type HID from the drop down menu Figure 9 9254TA Figure 9 De ning plate properties 10 Select Assign Plate Contents Figure 10 9255TA Figure 10 Assigning plate contents Promega Corpora on 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 TM392 Revised 7 14 11 Assign sample names to wells 12 In the lower left portion of the screen under Assays use the Add from Library option to select the Assay created in Step 5 or one previously created Click on the Add to Plate button and close the window 13 Under File Name Convention use the Add from Library option to select the File Name Convention created in Step 6 or one previously created Click on the Add to Plate button and close the window 14 Under Results Groups use the Add from Library option to select the Results Group created in Step 7 o
60. portion of the card that contains deposited cells 3 Allow the sample to air dry at room temperature 4 Use a manual punch tool with a 1 2mm tip to manually create sample disks from the storage card Place tip near the center of the sample spot and with a twisting or pressing action cut a 1 2mm disk Use the plunger to eject one 1 2mm sample disk into the appropriate well of a reaction plate Automated punchers also can be used to create sample disks Refer to the user s guide for your instrument for assistance with generating 1 2mm disks technical advice and troubleshooting information Note Static may be problematic when adding a punch to a well For FTA card punches adding PCR ampli cation mix to the well before adding the punch may help alleviate static problems For nonFTA card punches adding PunchSolution Reagent to the well before adding the punch during pretreatment may help alleviate static problems Ampli cation Setup 1 Thaw the GenePrint 10 5X Master Mix and GenePrint 10 5X Primer Pair Mix completely Note Centrifuge tubes brie y to bring contents to the bottom then vortex reagents for 15 seconds before each use Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after vortexing as this may cause the reagents to be concentrated at the bottom of the tube 2 Determine the number of reactions to be set up This should include negative control reactions but not positive control reactions a sep
61. r one previously created Click on the Add to Plate button and close the window 15 Highlight the sample wells then select the boxes in the Assays File Name Conventions and Results Groups that pertain to those samples 16 Select Link Plate for Run 17 The Load Plate window will appear Select Yes 18 In the Run Information window Figure 11 assign a Run Name Select Start Run not shown 9256TA Figure 11 Assigning a run name 22 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 B Detection of Ampli ed Fragments Using the ABI PRISM 3100 or 3100 Avant Genetic Analyzer with Data Collection Software Version 2 0 or the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 Materials to Be Supplied by the User 95 C dry heating block water bath or thermal cycler crushed ice or ice water bath centrifuge compatible with 96 well plates aerosol resistant pipette tips 3100 or 3130 capillary array 36cm performance optimized polymer 4 POP 4 for the 3100 or 3130 10X genetic analyzer bu er with EDTA MicroAmp optical 96 well plate and septa or equivalent Hi Di formamide Applied Biosystems Cat 4311320 The quality of formamide is critical Use Hi Di formamide Freeze for
62. r proper genotyping 5 In the Analysis Method column select the GenePrint 10 GeneMapper 4 0 analysis method imported in Section 6 C 6 In the Panel column select the panels text le that was imported in Section 6 A 7 In the Size Standard column select the size standard that was imported in Section 6 B 8 Select Analyze green arrow button to start the data analysis 6 E Importing GenePrint 10 Panels and Bins Text Files with GeneMapper ID Software Versions 3 2 and 3 2 1 1 Save the GenePrint_10_Panels_v1 0 txt and GenePrint_10_Bins_v1 0 txt les to a known location on your computer 2 Open the GeneMapper ID software version 3 2 or 3 2 1 3 Select Tools then Panel Manager 4 Highlight the Panel Manager icon in the upper left navigation pane 5 Select File then Import Panels Promega Corpora on 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 TM392 Revised 7 14 6 Navigate to the panels text le imported above Select the le then Import 7 In the navigation pane highlight the GenePrint 10 panels folder that you just imported in Step 6 8 Select File then Import Bin Set 9 Navigate to the bins text le saved to your computer above Select the le then Import 10 At the bottom of the Panel Manager window select
63. r the project name Choose the applicable security group from the drop down menu then select OK 6 M Controls 1 Examine the results for the negative control Using the protocols de ned in this manual the negative control should be devoid of ampli cation products 2 Examine the results for the 2800M Control DNA Compare the 2800M Control DNA allelic repeat sizes with the locus speci c allelic ladder The expected 2800M DNA allele designations for each locus are listed in Table 3 Table 3 Expected Allele Designations for the 2800M Control DNA STR Locus Alleles TH01 6 9 3 D21S11 29 31 2 D5S818 12 12 D13S317 9 11 D7S820 8 11 D16S539 9 13 CSF1PO 12 12 Amelogenin X Y vWA 16 19 TPOX 11 11 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 31 www promega com TM392 Revised 7 14 6 N Results Representative results of the GenePrint 10 System are shown in Figure 13 The GenePrint 10 Allelic Ladder Mix is shown in Figure 14 Locus to locus peak height imbalance will likely occur with cell line DNA Normal genomic DNA has equal copies of each locus and ampli cation will result in relatively even locus to locus balance Cell line DNA can have mutations that a ect the locus to locus allele peak height balance Additionally cell lines occasionally have tri allelic patterns at
64. re Versions 3 2 and 3 2 1 26 6 F Importing the ILS 600 Size Standard into GeneMapper ID Software Versions 3 2 and 3 2 1 27 6 G Importing the GenePrint 10 GeneMapper ID Analysis Method into GeneMapper ID Software Versions 3 2 and 3 2 1 27 6 H Processing Data with GeneMapper ID Software Versions 3 2 and 3 2 1 27 6 I Importing GenePrint 10 Panels Bins and Stutter Text Files with GeneMapper ID X Software Versions 1 2 and Later 28 6 J Importing the ILS 600 Size Standard into GeneMapper ID X Software Versions 1 2 and Later 28 2 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 6 K Importing the GenePrint 10 GeneMapper ID X Analysis Method into GeneMapper ID X Software Versions 1 2 and Later 29 6 L Processing Single Source Data with GeneMapper ID X Software Versions 1 2 and Later 29 6 M Cont
65. rint 10 data Note The analysis methods contain a 20 main peak lter 6 A Importing GenePrint 10 Panels and Bins Text Files with GeneMapper Software Version 4 0 1 Save the GenePrint_10_Panels_v1 0 txt and GenePrint_10_Bins_v1 0 txt les to a known location on your computer 2 Open the GeneMapper software version 4 0 3 Select Tools then Panel Manager 4 Highlight the Panel Manager icon in the upper left navigation pane 5 Select File then Import Panels 6 Navigate to the panels text le imported above Select the le then Import 7 In the navigation pane highlight the GenePrint 10 panels folder that you just imported in Step 6 8 Select File then Import Bin Set 9 Navigate to the bins text le saved to your computer above Select the le then Import 10 At the bottom of the Panel Manager window select OK The Panel Manager window will close automatically 6 B Importing the ILS 600 Size Standard into GeneMapper Software Version 4 0 1 Save the ILS_600 xml le to a known location on your computer 2 Select Tools then GeneMapper Manager 3 Select the Size Standard tab 4 Select Import 5 Browse to the location of the ILS_600 xml le 6 Highlight the le then select Import 7 Select Done to save changes and exit the GeneMapper Manager 26 Promega Co
66. ris base 0 037g EDTA Na2EDTA 2H2O 20 g ml glycogen Dissolve Tris base and EDTA in 900ml of deionized water Adjust to pH 8 0 with HCl Add glycogen Bring the nal volume to 1 liter with deionized water 56 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 9 E Related Products Sample Preparation Systems Product Size Cat Wizard SV Genomic DNA Puri cation System 50 preps A2360 250 preps A2361 Wizard Genomic DNA Puri cation Kit 100 isolations 300 l A1120 500 isolations 300 l A1125 100 isolations 10ml A1620 MagneSil Genomic Fixed Tissue System 100 samples MD1490 DNA IQ System 100 reactions DC6701 400 reactions DC6700 PunchSolution Kit 100 preps DC9271 Slicprep 96 Device 10 pack V1391 Maxwell Automated Nucleic Acid Puri cation Product Size Cat Maxwell 16 Tissue DNA Puri cation Kit 48 preps AS1030 For more information about other Maxwell nucleic acid puri cation kits visit www promega com maxwell16 Accessory Components Product Size Cat PowerPlex Matrix Standards 3100 3130 25 l each dye DG4650 Internal Lane Standard 600 150 l DG1071 Water Ampli cation Grade 6 250 l 5 1 250 l DW0991 2800M Control DNA 10ng l 25 l DD7101 Not for Medical Diagnostic Use
67. rols 30 6 N Results 31 7 Troubleshooting 34 A Ampli cation and Fragment Detection 34 B Ampli cation of Extracted DNA 37 C Direct Ampli cation of DNA from Storage Card Punches 38 D GeneMapper Software 41 E GeneMapper ID Software 43 F GeneMapper ID X Software 45 8 References 47 9 Appendix 48 A A
68. romega Corpora on 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 TM392 Revised 7 14 2 Product Components and Storage Conditions P R O D U C T S I Z E C AT GenePrint 10 System 50 reac ons B9510 Cat B9510 contains su cient reagents for 50 reactions of 25 l each Includes Pre ampli cation Components Box 250 l GenePrint 10 5X Master Mix 250 l GenePrint 10 5X Primer Pair Mix 1 25ml Water Ampli cation Grade 25 l 2800M Control DNA 10ng l Post ampli cation Components Box 50 l GenePrint 10 Allelic Ladder Mix 150 l Internal Lane Standard 600 The GenePrint 10 Allelic Ladder Mix is provided in a separate sealed bag for shipping This component should be moved to the post ampli cation box after opening The Water Ampli cation Grade is provided in a separate sealed bag for shipping This component should be moved to the pre ampli cation box after opening Storage Conditions For long term storage store all components except the 2800M Control DNA at 30 C to 10 C in a nonfrost free freezer Store the 2800M Control DNA at 2 10 C For daily use the GenePrint 10 System components can be stored for up to 1 week at 2 10 C The GenePrint 10 5X Primer Pair Mix GenePrint 10 Allelic Ladder Mix and Internal Lane Standard 600 ILS 600 are light sensitive a
69. rpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 6 C Importing the GenePrint 10 GeneMapper Analysis Method into GeneMapper Software Version 4 0 Promega scientists have created an analysis method for GeneMapper software version 4 0 GenePrint 10 GM 4 0 Analysis Method xml 1 Save the analysis method xml le to a known location on your computer 2 Select Tools then GeneMapper Manager 3 Select the Analysis Methods tab 4 Select Import 5 Browse to the location of the GenePrint 10 GM 4 0 Analysis Method xml le 6 Highlight the le then select Import 7 Select Done to save changes and exit the GeneMapper Manager 6 D Processing Data with GeneMapper Software Version 4 0 1 Select File then New Project 2 Select Generic as the Project Type in the New Project window then select OK 3 Browse to the location of the run les Highlight the desired les then select Add to list followed by Add 4 In the Sample Type column use the drop down menu to select Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated Ladder in the Sample Type column fo
70. ses and Comments Signi cantly raised baseline Poor spectral calibration Perform a new spectral calibra tion and re run the samples Re run and optimize the matrix Make sure that the matrix applied was generated on the same instrument Error message after attempting to import panels There was a con ict between di erent sets of panels and and bins text les Unable to save panel data bins text les Check to be sure that the bins are installed java SQLEException ORA 00001 unique properly If not delete all panels and bins text les and constraint IFA CKP_NNN violated re import les in a di erent order 7 F GeneMapper ID X Software Symptoms Causes and Comments Stutter peaks not ltered Stutter le was not imported into the Panel Manager when the panels and bin text les were imported Be sure that the Use marker speci c stutter ratio and distance if available box is checked Samples in the project not analyzed The Analysis Requirement Summary window was not active and there was an analysis requirement that was not met Turn on Analysis Requirement Summary in the Options menu and correct the necessary analysis require ments to continue analysis Edits in label edit viewer cannot be viewed To view edits made to a project the project must rst be saved Close the plot view window go back to the main GeneMapper ID X page and save the project Display the plot window
71. te DNA 10ng 1 2 up to 15 l total reaction volume 25 l 1Store DNA templates in nuclease free water TE 4 bu er 10mM Tris HCl pH 8 0 0 1mM EDTA or TE 4 bu er with 20 g ml glycogen If the DNA template is stored in TE bu er that is not pH 8 0 or contains a higher EDTA concentration the volume of DNA added should not exceed 20 of the nal reaction volume PCR ampli cation e ciency 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 the template DNA and the extraction procedure used 2Apparent DNA concentrations can di er depending on the DNA quanti cation method used 9 The amount of DNA template recommended here is based on DNA concentrations determined by measuring absorbance at 260nm We strongly recommend that you perform experiments to determine the optimal DNA amount based on your DNA quanti cation method Promega Corpora on 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 TM392 Revised 7 14 Thermal Cycling This manual contains a protocol for use of the GenePrint 10 System with the GeneAmp PCR System 9700 thermal cycler We have not tested other thermal cyclers For information about other thermal cyclers please c
72. ter Ampli cation Grade to the tube rst then add GenePrint 10 5X Master Mix and GenePrint 10 5X Primer Pair Mix For FTA card punches the template DNA will be added at Step 7 Table 7 PCR Ampli cation Mix for Ampli cation of 2800M Control DNA PCR Ampli cation Mix Component Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 15 0 l GenePrint 10 5X Master Mix 5 0 l GenePrint 10 5X Primer Pair Mix 5 0 l total reaction volume 25 l PCR Ampli cation Mix Component Volume Per Reaction Number of Reactions Final Volume Water Ampli cation Grade 10 l GenePrint 10 5X Master Mix 5 0 l GenePrint 10 5X Primer Pair Mix 5 0 l 2800M Control DNA 10ng l1 5 0 l total reaction volume 25 l 1The volume of 2800M Control DNA may require optimization We recommend 2 5 5 l of 2800M Control DNA per positive control reaction depending on the number of ampli cation cycles If you are using a higher number of PCR cycles use less 2800M Control DNA and adjust the volume of Water Ampli cation Grade so that the nal reaction volume is 25 l Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 53 www promega com TM392 Revised 7 14 6 Vortex the PCR ampli cation mixes for 5 10 seconds For
73. th Data Collection Software Version 2 0 and the Applied Biosystems 3130 or 3130xl Genetic Analyzer with Data Collection Software Version 3 0 with the following exceptions 1 In the Module Manager select New Select Regular in the Type drop down list and select HIDFragmentAnalysis36_POP4 in the Template drop down list Con rm that the injection time is 5 seconds the injection voltage is 3kV and the run time is 1 800 seconds Give a descriptive name to your run module and select OK Note Instrument sensitivities can vary The injection time may be adjusted in the Module Manager A suggested range for the injection time is 3 22 seconds 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 Dye Set 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 appro priate 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
74. thentication_committee iclac_ Cross_Contaminations_v6_8 pdf STR loci consist of short repetitive sequence elements 3 7 base pairs in length These repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers which can be detected using the polymerase chain reaction Alleles of STR loci are di erentiated by the number of copies of the repeat sequence contained within the ampli ed region and are distinguished from one another using uorescence detection following electrophoretic separation Promega Corpora on 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 TM392 Revised 7 14 The American Tissue Culture Collection Standards Development Organization Workgroup recently issued standard ASN 0002 8 which recommends the use of at least eight STR loci TH01 TPOX vWA CSF1PO D16S539 D7S820 D13S317 and D5S818 plus Amelogenin for gender identi cation for human cell line authentication The International Cell Line Authentication Committee ICLAC has posted a worksheet that can be used to calculate the match criteria for human cell lines The worksheet can be found via the References link on their web site at http standards atcc org kwspub home the_international_cell_line_authentication_committee iclac_ The GenePrint 10 System a c allows co ampli cation and three color detection of n
75. ts at the tetranucleotide repeat locus vWA Nucleic Acids Res 24 2807 12 15 M ller A Meyer E and Brinkmann B 1994 Di erent types of structural variation in STRs HumFES FPS HumVWA and HumD21S11 Int J Leg Med 106 319 23 16 Brinkmann B M ller A and Wiegand P 1995 Structure of new mutations in 2 STR systems Int J Leg Med 107 201 3 17 Gri ths R A et al 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system Int J Legal Med 111 267 72 18 B r W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems Int J Legal Med 110 175 6 19 Gill P et al 1997 Considerations from the European DNA Pro ling Group EDNAP concerning STR nomenclature Forensic Sci Int 87 185 92 9 Appendix 9 A Additional STR Locus Information Additional information about the human STR loci ampli ed by the GenePrint 10 System can be found in Table 4 GenePrint 10 System allelic ladder information can be found in Table 5 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 49 www promega com TM392 Revised 7 14 Table 4 The GenePrint 10 System Locus Speci c Information STR Locus Label Chromosomal Location GenBank Locus and Locus De nition Repeat Sequen
76. ts vary therefore injection time or the amount of sample mixed with loading cocktail may need to be adjusted To modify the injection time in the run module select Instrument Protocol from the Library menu in the data collection software If peak heights are higher than desired use less DNA template in the ampli cation reactions or reduce the number of cycles in the ampli cation program to achieve the desired signal intensity 6 Centrifuge plate brie y to remove air bubbles from the wells 7 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 12 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 5 A Detection of Ampli ed Fragments Using the Applied Biosystems 3500 or 3500xL Genetic Analyzer with POP 4 Polymer continued Instrument Preparation Refer to the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide for the instrument maintenance schedule and instructions to install the capillary array bu ers and polymer pouch and perform a spatial calibration Samples may be analyzed as described in the Applied Biosystems 3500 3500xL Genetic Analyzer User Guide 1 Open the 3500 Data Collection Software The Dashboard screen will launch Figure 2 Ensure that t
77. unning Allelic ladder mix and primer pair mix were not compatible the same as samples Ensure that the allelic ladder mix is from the same kit as the primer pair mix Poor quality formamide Use only Hi Di formamide when analyzing samples Be sure the allelic ladder and samples are from the same instrument run Migration of samples changed slightly over the course of a CE run with many samples This may be due to changes in temperature or the CE column over time Use a di erent injection of allelic ladder to determine sizes Poor injection of allelic ladder Include more than one ladder per instrument run Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 37 www promega com TM392 Revised 7 14 Symptoms Causes and Comments Peak height imbalance The reaction volume was too low This system is optimized for a nal reaction volume of 25 l Decreasing the reaction volume can result in suboptimal performance Miscellaneous balance problems Thaw the 5X Primer Pair Mix and 5X Master Mix completely and vortex for 15 seconds before use Note that the 5X Master Mix will take longer to thaw than the 5X Primer Pair Mix Do not centrifuge the 5X Primer Pair Mix or 5X Master Mix after mixing Calibrate thermal cyclers and pipettes routinely PCR ampli cation mix prepared in Section 4 or 9 B was not mixed well Vortex the PCR ampli
78. ware version 4 0 or GeneMapper ID software version 3 2 or 3 2 1 Applied Biosystems 3130 and 3130xl Genetic Analyzer 4 or 16 capillaries POP 4 36cm GeneMapper software version 4 0 or GeneMapper ID software version 3 2 or 3 2 1 Applied Biosystems 3500 and 3500xL Genetic Analyzer 8 or 24 capillaries POP 4 36cm GeneMapper ID X software version 1 2 or later Promega Corpora on 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 TM392 Revised 7 14 To facilitate analysis of data generated with the GenePrint 10 System we have created the following les for use with GeneMapper software version 4 0 GeneMapper ID software version 3 2 or 3 2 1 and GeneMapper ID X software versions 1 2 and later panels and bins text les ILS_600 xml size standard le GenePrint 10 analysis method le stutter text le for GeneMapper ID X software only Each set of les can be downloaded as a zipped le at www promega com geneprintsupport Three separate zipped les are provided one for GeneMapper software version 4 0 one for GeneMapper ID software versions 3 2 and 3 2 1 and one for GeneMapper ID X software versions 1 2 and later Instructions for importing these les into each software are given below along with instructions on how to use these les to analyze GeneP
79. wn menu to select Allelic Ladder Sample Positive Control or Negative Control as appropriate for the sample Every folder in the project must contain at least one allelic ladder injection that is designated as Allelic Ladder in the Sample Type column for proper genotyping 5 In the Analysis Method column select the GenePrint 10 GeneMapper ID X analysis method imported in Section 6 K 6 In the Panel column select the panels text le that was imported in Section 6 I 7 In the Size Standard column select the size standard that was imported in Section 6 J 8 Select Analyze green arrow button to start data analysis Note By default the software displays the Analysis Requirement Summary Allelic Ladder Analysis Summary and Analysis Summary windows after quality review by the software Ensure that all requirements are met as each window appears If you do not have the Analysis Requirements window activated you may need to do additional manual troubleshooting 30 Promega Corpora on 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 TM392 Revised 7 14 www promega com 6 L Processing Single Source Data with GeneMapper ID X Software Versions 1 2 and Later continued 9 If all analysis requirements are met the Save Project window will open Figure 12 9430TA Figure 12 The Save Project window 10 Ente

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