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1. 10 2012 17 Protocol Electrophoresis using the ABI PRISM 3100 Avant 3100 Genetic Analyzer For detailed instructions on instrument setup spectral calibration application of the ABI PRISM 3100 Data Collection software version 1 01 or 1 1 and the GeneScan software refer to the ABI PRISM 3100 Avant 3100 Genetic Analyzer User s Manual The system with 4 capillaries is the ABI PRISM 3100 Avant Genetic Analyzer the system with 16 capillaries is the ABI PRISM 3100 Genetic Analyzer The virtual filter set G5 is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 11 Table 11 Materials required for electrophoresis Material Specifications Capillary 36 cm Capillary Array for ABI PRISM 3100 Avant 3100 Genetic Analyzer Polymer 4 Polymer for ABI PRISM 3100 Avant 3100 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA Spectral calibration matrix generation Proper spectral calibration is critical for evaluation of multicolor systems with the ABI PRISM 3100 3100 Avant Genetic Analyzer and should be done before conducting fragment length analysis The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes Spectral calibration comprises the following steps Preparing the spectral
2. Component Volume per sample Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 ul 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed 3 Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 may be used to cool the plate 6 Load the samples on the tray Since injections take place simultaneously on all capillaries 8 or 24 samples must be pipetted onto the plate of multi capillary analyzers If fewer samples are analyzed the empty positions must be filled with 12 ul Hi Di Formamide Investigator IDplex Plus Handbook 10 2012 41 To ensure a reliable allelic assignment multi capillary analyzers inject one allelic ladder for each set of 24 samples 8 capillary instruments One allelic ladder per injections 24 capillary instruments One allelic ladder 1 injection Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Ensure ambient conditions are kept as recommended by the instrument manufacturer Setting up a run If you are using the Investigator IDplex Plus Kit for the first time on an Applied Biosystems 3500 Genetic Analyzer you will first need to set up a number of protocols B Instrument Protocol M Size Stan
3. 330 350 360 32 300 390 9 10 0 9 40 450 1 88868 E 2 30 1500 750 018551 2851179 UUU BB t Ba 3 HAHAA gt Bo 63 EJ 3750 31254 5 175 YA 19 4 03 63 bats Jed et aes 27 G9 03 04 69 09 14 a Eg G3 G3 17 03 n i T0 120 19 140 19 160 19 100 190 20 210 220 20 240 240 2 20 290 240 X0 9 4 8 Figure 3 Electropherogram of the allelic ladder IDplex Plus analyzed on an Applied Biosystems 3500 Genetic Analyzer Allele assignment was performed using Investigator IDproof Software 52 Investigator IDplex Plus Handbook 10 2012 Interpretation of Results Post PCR analysis and automatic allele assignment with suitable analysis software ensure a precise and reliable discrimination of alleles General procedure for the analysis 1 Check the DNA size standard 2 Check the allelic ladder 3 Check the positive control 4 Check the negative control 5 Analyze and interpret the sample data Pull up peaks Pull up peaks may occur if peak heights are outside the linear detection range see Troubleshooting Guide page 54 or if an incorrect matrix was applied They appear at positions of specific peaks in other color channels typically with lower signal intensities Peak heights should not exceed 3000 RFU in order to pre
4. ABI PRISM 3100 Avant 3100 Genetic Analyzer Parameter Settings Analysis Range Data Processing Peak Detection Size Call Range Size Calling Method Split Peak Correction Start 2000 Stop 10 000 Baseline Checked Multi component Checked Smooth options Light Peak Amplitude Thresholds Be Yet G Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 pts Min 60 Max 550 Local Southern Method None The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneScan or GeneMapper ID Software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be 3 times higher than the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis Note For information on the use of the recommended Template Files as analysis parameters refer to the appropriate Investigator Template Files User Guide Genotyper GeneMapper ID or GeneMapper ID X Investigator IDplex Plus Handbook 10 2012 25 Protocol Electrophoresis Using the Applied Biosystems 3130 3130xl Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the ABI PRISM Data Collection Software version 3 0 and the GeneMapper ID Software refer t
5. Click the column header to select the entire column and select Fill Down from the Edit menu to apply the information to the selected samples Confirm by clicking OK 20 Investigator IDplex Plus Handbook 10 2012 8 Link the reaction plate the autosampler tray with the created plate ID and start the run 9 Upon completion of the run check in the Spectral Calibration Result dialog box that all capillaries have successfully passed calibration label A If individual capillaries are labeled X refer to the ABI PRISM 3100 Avant 3100 Genetic Analyzer User s Manual 10 Click OK to confirm completion of the run Checking the matrix 1 Select Display Spectral Calibration from the Tools menu then Dye Set and G5 to review the spectral calibration profile for each capillary 2 The quality value Q value must be greater than 0 95 and the condition number C value must be between 1 and 20 Both values must be within the predetermined range 3 Check for a flat baseline in the matrix samples There should be 5 peaks with heights of 1000 5000 RFU in each matrix sample Note The optimal range is 2000 4000 RFU 4 Check the new matrix with the current samples There should be no pull up peaks between the dye panels B G Y R and O with the new matrix 5 If the calibration failed follow instructions in the section Spectral parameter on page 20 6 If all capillaries have passed the cali
6. calibration standards Loading the standards to the 96 well reaction plate one sample per capillary M Entering the plate composition M Performing a spectral calibration run and checking the matrix 18 Investigator IDplex Plus Handbook 10 2012 Preparing the spectral calibration standards Example for 4 capillaries ABI PRISM 3100 Avant Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 12 Table 12 Setup of formamide and Matrix Standard BT5 mixture for 4 capillaries Component Volume Hi Di Formamide 60 ul Matrix Standard BT5 multi cap 5 ul 2 Load 12 ul of the mixture to 96 well plate e g positions 1 01 Denature for 3 min at 95 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 may be used to cool the plate 59 Example for 16 capillaries PRISM 3100 Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 13 Table 13 Setup of formamide and Matrix Standard BT5 mixture for 16 capillaries Component Volume Hi Di Formamide 204 ul Matrix Standard BT5 multi cap 17 ul 2 Load 12 pl of the mixture to 96 well plate e g positions AT H1 and A2 H2 3 Denature for 3 min at 95 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 may be used to cool the plate a ANT Investigator IDpl
7. click Create 5 The parameters in Table 37 must be entered or selected The DNA Size Standard 550 BTO should be used with the following lengths of fragments 60 80 90 100 120 140 160 180 200 220 240 250 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp Investigator IDplex Plus Handbook 10 2012 43 Table 37 Size standard parameters Parameter Setting Size Standard e g SST BTO 60 500bp Dye Color Orange 6 Click Save to confirm the changes 7 To set up the QC Protocol go to Library and select Analyze followed by QC Protocols and click Create 8 The parameters in Table 38 must be entered or selected Table 38 QC Protocol parameters Parameter Setting Protocol Name e g BTO 550 Size Standard SST BTO 60 500bp from step 4 Sizecaller SizeCaller v1 1 0 9 Goto Analysis Settings followed by Peak Amplitude Threshold and disable Purple Ensure that all other colors are enabled Check the recommended analysis settings in Table 41 All other settings should remain as Default 10 Click Save to confirm the changes 11 To set up an Assay go to Library and select Manage followed by Assays and click Create 12 To analyze Investigator IDplex Plus fragments the parameters in Table 39 must be selected LLLLLLLLLLLLLLLLLLLAAZAAA A i AA mW AU 44 Investigator ID
8. must be generated Table 5 Table 5 The 5 fluorescent labels of BT5 Color Matrix standard Blue B 6 FAM Green C BTG Yellow Y BTY Red R BTR Orange O BTO Investigator IDplex Plus Handbook 10 2012 11 1 Five electrophoresis runs should be conducted for each fluorescent label under the same conditions as for the samples and allelic ladders of the Investigator IDplex Plus Kit in order to generate suitable matrix files Table 6 Table 6 Matrix setup for a single capillary instrument ABI PRISM 310 Genetic Analyzer Matrix sample Component Volume Matrix sample 1 Hi Di Formamide 12 0 ul Matrix standard 6 FAM 1 0 ul Matrix sample 2 Hi Di Formamide 12 0 ul Matrix standard BTG 1 0 ul Matrix sample 3 Hi Di Formamide 12 0 ul Matrix standard BTY 1 0 ul Matrix sample 4 Hi Di Formamide 12 0 ul Matrix standard BTR 1 0 ul Matrix sample 5 Hi Di Formamide 12 0 ul Matrix standard BTO 1 0 ul 2 Denature for 3 min at 95 3 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 may be used to cool the plate 4 Load the samples on the tray 5 Create a Sample Sheet and enter the sample designation Table 7 shows the injection list for the matrix generation E d waelAAAqhm LLLAL 12 Investigator IDplex Plus Handbook 10 2012 Table 7 Injection list for matr
9. to open the matrix folder and select the new matrix Component Volume per sample Hi Di Formamide DNA Size Standard 550 BTO 12 0 ul 0 5 ul EA Alternatively the thermal cycler set to 4 C may be used to cool the plate Investigator IDplex Plus Handbook Load the samples on the tray 10 2012 Open the folder of the respective run and select Add Sample Files Select the sample s in the Sample File column Set up a mixture of formamide and DNA size standard according to Aliquot 12 pl of the mixture to a tube for each sample to be analyzed Add 1 pl PCR product or allelic ladder diluted if necessary Denature for 3 min at 95 C Snap freeze by placing the plate on ice for 3 min 15 Setting up the GeneScan Software Create a Sample Sheet and enter sample designation Table 9 Injection list for the ABI PRISM 310 Genetic Analyzer Component Settings Module File GS STR POP 4 1 ml G5 Matrix File e g Matrix BT5 Size Standard e g SST BTO 60 500bp Injection Time s 55 Injection Voltage kV 15 Run Voltage kV 15 Run Temperature 60 Run Time min 28 Deviating from standard settings the injection time may range between 1 and 10 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time up to 105 may be necessary
10. 0 702 055818 BTG 6 7 8 9 10 11 12 13 14 15 17 0251338 BTG 14 16 17 18 19 20 21 22 23 24 25 26 27 28 0165539 8 9 10 11 12 13 14 15 CSFIPO BTY Or 7 09 T1011 12 13 414 T5 M6 D135317 BTY 5 6 7 8 9 10 11 12 13 14 15 16 gt 922022422122 222287292207 225 2627 2852090530 Ole 99 34 3 2 42 2 44 2 45 2 47 2 50 2 018551 8 9 10 10 2 11 12 13 14 15 16 17 17 2 18 18 2 19 20 21 21 2 22 23 24 25 26 27 28 0851179 TOT DE T2 r3 VAY oy don 9 For information about known microvariants not contained in the Investigator IDplex Plus Allelic Ladder see the National Institute of Standards and Technology NIST web site www cstl nist gov biotech strbase Investigator IDplex Plus Handbook 10 2012 51 18000 0351358 021511 14000 0165539 0135317 FGA 5625 018551 0851179 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300 315 330 345 360 375 390 405 420 435 450 Figure 2 Electropherogram of the Investigator IDplex Plus Kit using 500 pg Control DNA 9948 Analysis was performed on an Applied Biosystems 3500 Genetic Analyzer Allele assignment was performed using Investigator IDproof Software T0 120 10 140 150 160 19 10 20 210 220 29 20 250 mM 20 V 30 320
11. 1PO 10 11 10 12 9 10 FGA 24 26 23 24 21 24 THO1 6 9 3 8 9 3 9 3 9 3 8 9 8 8 8 9 vWA 17 17 17 18 16 16 For further confirmation the table above displays the alleles of the reference DNA purchased from Coriell Cell Repositories CCR as well as 2 reference DNAs purchased from CCR and ATCC up to the standard of Szibor et al 2003 Investigator IDplex Plus Handbook 10 2012 49 Alleles Table 45 shows the alleles of the allelic ladder All analyses were performed using POP 4 polymer Figure 2 and Figure 3 Different analysis instruments DNA size standards or polymers may result in different fragment lengths In addition a visual alignment with the allelic ladder is recommended Scaling Horizontal 70 480 bp Vertical Depending on signal intensity 50 Investigator IDplex Plus Handbook 10 2012 Table 45 Allelic ladder fragments included in the Investigator IDplex Plus Kit Locus Dye label Repeat numbers of allelic ladder Amelogenin 6 FAM X Y THO1 6 FAM 9 10 3 0351358 6 9 10 11 12 13 14 15 16 17 18 19 20 21 vWA 6 FAM la 14 15 16 17 18 10 20 2l 22 24 24 2 25 26 26 2 27 28 28 2 29 29 2 021511 6 30 31 31 2 32 32 2 33 33 2 34 34 2 35 36 36 2 37 TPOX BTG 4 6 8 9 10 11 12 13 15 075820 BTG 5 7 8 9 TO 11 12 13 14 15 0195433 BTG OA 1O Ply 903 2 A 5 25 lO 16 2 7
12. 3 0195433 G08036 AAGG AAAG AAGG 19412 AAGG D21511 AP000433 TCTA 21q21 1 TCA TCTA TCTA CSF1PO X14720 5q33 1 FGA FIBRA M64982 TTTC 4428 2 Sl CTCC TTCC 01 TC11 000269 TCAT 11p15 5 TPOX M68651 AATG 2p25 3 vWA M25858 TCTA TCTG TCTA 3 12p13 31 Investigator IDplex Plus Handbook 10 2012 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate material safety data sheets MSDSs available from the product supplier Hi Di Formamide 25 ml Applied Biosystems cat 4311320 Matrix Standards 5 for single capillary instruments e g ABI PRISM 310 Genetic Analyzer QIAGEN cat no 386113 Matrix Standards BT5 for multi capillary instruments e g ABI PRISM 3100 and Applied Biosystems 3130 and 3500 Genetic Analyzers QIAGEN cat nos 386123 or 386125 Pipets and pipet tips M One ofthe following DNA analyzers ABI PRISM 310 Genetic Analyzer ABI PRISM 3100 Avant 3100 Genetic Analyzer Applied Biosystems 3130 3130xl Genetic Analyzer Applied Biosystems 3500 3500xL Genetic Analyzer One of the following PCR thermal cyclers QIAGEN Rotor Gene Q GeneAmp PCR System 9700 Bio Rad PTC 200 Biometra UNO Thermoblock Eppendorf Mastercycl
13. 3200 Under Points enter the calculated difference between end and start values e g 2300 5 Click OK to calculate the new matrix ilf Make New Matrix Select the Matrix Standard Sample Files Number Of Dyes B 6FAM_080204 fsa Start At 200 G BTG_080204 fsa Start At 000 ii BTY 080204 fsa Start At 000 R BTR_080204 fsa Start At 000 0 BTO_080204 fsa Start At 000 Points 300 Cancel K Matrix sample selection UUr 14 Investigator IDplex Plus Handbook 10 2012 6 Select Save as in the File menu to save the new matrix in the matrix folder Matrix BT5 mtx Reactions B G Y R Josss1 10000 fo20se o3258 ososs 10000 fost19 0 1044 03619 0 5341 10000 o 0160 00477 02082 lt on om amp oO 0 52006 o 0029 60095 10000 New matrix BT5 Checking the matrix 1 M To check the new matrix with current samples select New in the File menu and then select Project Re analyze the samples Note There should no pull up peaks between the dye panels B G Y R O with the new matrix Sample preparation 1 Table 8 Setup of formamide and DNA size standard mixture Table 8 Click Sample and then Install New Matrix
14. AM BTG BTY BTR and BTO Matrix standard 6 FAM BTG BTY BTR and BTO Matrix standard 6 FAM BTG BTY BTR and BTO Software package on CD including installation files for the Desktop Server and Client versions of IDproof Software Free use of the IDproof Desktop version of the software for 30 days after installation Allows the unlimited use of the Desktop version of the software to be installed on a single workstation with a local database Allows for setup of a server that maintains the database and various workstations to access that database Must be purchased in conjunction with the Client Key Larger kit sizes available please inquire 58 Cat no 383615 384013 387016 387116 386113 386123 386125 9020775 389001 389002 389003 Investigator IDplex Plus Handbook 10 2012 Product Investigator IDproof Client Key Investigator IDproof Mixture Software Investigator IDproof Mixture Demo Key Investigator IDproof Mixture Single Key Investigator IDproof Mixture Server Key Investigator IDproof Mixture Client Key Investigator Template Files Contents Must be purchased in conjunction with the Server Key Software package on CD including installation files for the Desktop Server and Client versions of IDproof Mixture Software Free use of the IDproof Mixture Desktop version of the software for 30 days after installation Allows the unlimited use of the Desktop v
15. G BTG Yellow Y BTY Red R BTR Orange O BTO Prior to the spectral calibration a dye set for the Matrix Standard BT5 must be set up 1 To create a new dye set go to Library and select Analyze followed by Dye Sets and click Create 2 Enter a Dye Set Name e g BT5 3 Select Matrix Standard as a chemistry and AnyDye Template as a dye set template 4 Disable Purple in the field Arrange Dyes Ensure that all other colors are enabled 5 Under Calibration Peak Order the colors need to be arranged as follows 5 blue 4 green 3 yellow 2 red and 1 orange 6 Do not alter the Parameter settings 7 Click Save to confirm the changes on Investigator IDplex Plus Handbook 10 2012 37 Create New Dye Set Setup a Dye Set Dye Set 5 Chemistry Matrix Standard M Dye Set Template AnyDye Template Arrange Dyes Dye Selection Reduced Selection Calibration Peak Order 5 4 3 S 2 v Parameters The parameters will be used for instruments configured with 36cm capillary array and polymer POP4 Matrix Condition Number Upper Limit 20 0 Locate Start Point After Scan 300 Before Scan 5000 Limit Scans 20000 Sensitivity 0 1 Minimum Quality Score 0 8 Notes Matrix Std BT5 multi cap Close Setup of dye set BT5 Preparation of the stan
16. October 2012 Investigator IDplex Plus Handbook For multiplex amplification of 13 CODIS core loci plus 0251338 0195433 and Amelogenin QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www giagen com Contents Kit Contents 4 Storage 4 Intended Use 4 Safety Information 5 Introduction 6 Equipment and Reagents to Be Supplied by User 8 Protocols PCR Amplification 9 H Electrophoresis Using the ABI PRISM 310 Genetic Analyzer 11 Electrophoresis using the ABI PRISM 3100 Avant 3100 Genetic Analyzer 18 it Electrophoresis Using the Applied Biosystems 3130 3130xl Genetic Analyzer 26 E Electrophoresis Using the Applied Biosystems 3500 3500xL Genetic Analyzer 36 B Analysis 47 Interpretation of Results 53 Troubleshooting Guide 54 References 56 Ordering Information 57 a Investigator IDplex Plus Handbook 10 2012 3 Kit Contents Investigator IDplex Pl
17. Open the Protocol Manager of the Data Collection Software 3 Click New in the Instrument Protocol window to open the Protocol Editor dialog box and enter the information in Table 27 4 Click OK to exit the Protocol Editor 32 Investigator IDplex Plus Handbook 10 2012 Table 27 Settings in Instrument Protocol Protocol Editor Settings Name Run36 4 5 26min REGULAR Run Module 3kV 10s 500bp Dye Set Any5Dye See Table 26 Run Module 3kV 10s 500bp for the Applied Biosystems 3130 3130xl Genetic Analyzer 5 Before each run it is necessary to create a plate definition In the Plate Manager of the Data Collection Software click New to open the New Plate Dialog box 6 Enter the information in Table 28 Table 28 GeneMapper Plate Editor I Protocol Editor Settings Name e g Plate BT5 Date Application Select GeneMapper Application Plate type 96 Well Owner Name Operator Name 7 Click OK and a new table in the Plate Editor opens automatically Table 29 8 Click the column header to select the entire column Select Fill Down from the Edit menu to apply the information to all selected samples Click OK 9 In the Run Scheduler click Find All and select Link to link the reaction plate on the autosampler tray to the newly created plate record position A or B Investigator IDplex Plus Handbook 10 2012 33 Table 29 GeneMapper Plat
18. Prepare a master mix according to Table 2 The master mix contains all of the components needed for PCR except the template sample DNA and nuclease free water Prepare a volume of master mix 1096 greater than that required for the total number of PCR assays to be performed This should include positive and negative control reactions 3 Mix the master mix thoroughly and dispense appropriate volumes into PCR tubes or the wells of a PCR plate 4 Add template DNA and nuclease free water to the master mix to give a final sample volume of 25 wl 5 Prepare positive and negative controls Positive control Use 5 ul of the Control DNA Negative control Use nuclease free water instead of template DNA in the reaction Investigator IDplex Plus Handbook 10 2012 9 Table 2 Reaction setup Component Volume per reaction Fast Reaction Mix 7 5 ul Primer Mix 2 5 ul Nuclease free water added in step 4 Variable Template DNA added in step 4 Variable Total volume 25 pl 6 Mix reactions thoroughly For optimal results mix the prepared and sealed PCR plate prior to amplification using an Eppendorf Thermomixer Comfort orbital shaker for 5 min at 1200 rpm at room temperature Use constant shaking without pauses 7 Program the thermal cycler according to the manvfacturer s instructions using the conditions outlined in Table 3 Note If using the GeneAmp PCR System 9700 with an Aluminum Block use Std Mode or wit
19. RISM 3100 and Applied Biosystems 3130 3500 Genetic Analyzers 54 Investigator IDplex Plus Handbook 10 2012 Comments and suggestions c Bubbles in the capillary Repeat electrophoresis to confirm results lead to pull up peaks in all color panels spikes that result in allele misnomer d Differences in the run For reliable allelic assignment on multi capillary performance among analyzers a number of allelic ladders should be the capillaries of a run multi capillary analyzer may result in allelic assignment shift Injection file of the allelic ladder is not appropriate a An additional signal Use a different injection file of the allelic ladder can be identified as and check the data of the analyzed sizes from peak of the allelic the Size Standard in bp of the allelic ladder ladder because of Always use the DNA Size Standard 550 for dysfunctions during the electrophoresis If peaks of the allelic ladder are miscalled the ladder cannot be used for the analysis Investigator Human Identification PCR Kits b One peak of the allelic allelic ladder must be loaded onto the ladder is below the analysis instrument at a higher concentration peak detection value than samples to be analyzed 50 200 RFU of the Alternatively allelic ladder data can be analyzed ru method used with a lower peak detection value in Analysis an US IS not Software identified c One peak of the allelic Compare the length of the f
20. a Collection Software click New to open the Run Module Editor dialog box Note Modify the Run Module Default settings from HiDFragmentAnalysis3 POPA 1 to those shown in Table 26 2 Modify the Injection Voltage to 3 kV and the Injection Time to 10 s Table 26 3 Click Save As enter a name for the new Run Module e g 3kV 10s 500bp and confirm by clicking OK 4 Click Close to exit the Run Module Editor ea Investigator IDplex Plus Handbook 10 2012 31 Table 26 Run Module 3kV_10s_500bp for the Applied Biosystems 3130 3130xl Genetic Analyzer Parameter Settings Oven Temperature Default Poly Fill Volume Default Current Stability Default Pre Run Voltage kV Default Pre Run Time s Default Injection Voltage kV 3 0 Injection Time s 10 Voltage Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Voltage kV Default Run Time s 1560 Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time of up to 20 s may be necessary t The run time for Investigator IDplex Plus was modified in order to be able to analyze fragments with lengths of up to 500 bp Starting the run 1 Place the prepared 96 well plate on the autosampler tray 2
21. als required for electrophoresis Material Specifications Capillary 36 cm Array for Applied Biosystems 3500 3500xL Genetic Analyzer Polymer 4 for Applied Biosystems 3500 3500xL Genetic Analyzer Buffer Anode Buffer Container ABC 3500 Series Cathode Buffer Container CBC 3500 Series Spectral calibration matrix generation Before conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO for each analyzer Table 32 The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes IMPORTANT Spectral calibration must be performed for each new capillary array Spectral calibration is comprised of the following steps Preparation of the instrument Preparation of dye set 5 Preparation of the standard calibration plate E Plate assembly and loading the plate in the instrument 36 Investigator IDplex Plus Handbook 10 2012 E Performing a spectral calibration run Checking the matrix Preparation of the instrument Before the spectral calibration process ensure that the spatial calibration has been performed This process is described in detail in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Preparation of dye set BT5 Table 32 The 5 fluorescent labels of BT5 Color Matrix standard Blue B 6 FAM Green
22. anufacturer Setting up the GeneScan Software 1 Edit the default run module in Dye Set G5 once for the first run Select Module Editor to open the dialog box 2 Select the appropriate Run Module as template from the GeneScan table see Table 16 3 Modify the Injection Voltage to 3 kV and the Injection Time to 10 s 4 Click Save As and enter the name of the new module e g 3kV 10s 500bp Confirm by clicking OK 5 Click Close to exit the Run Module Editor 22 Investigator IDplex Plus Handbook 10 2012 Table 16 Run Module 3kV_10s_500bp for the ABI PRISM 3100 Avant 3100 Genetic Analyzer Parameter Setting Run Temperature C Default Cap Fill Volume Default Maximum Current A Default Current Tolerance A Default Run Current A Default Voltage Tolerance kV Default Pre Run Voltage kV Default Pre Run Time s Default Injection Voltage kV 3 0 Injection Time s 10 Run Voltage kV Default Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Time min 26 Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time of up to 20 s may be necessary run time for Investigator IDplex Plus was modified in order to be able to analyze fragme
23. ard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Investigator IDplex Plus Handbook 10 2012 Cat no 381625 381627 381535 381545 381315 381213 380315 380325 383213 57 Product Investigator Argus Y 12 QS Kit 100 Investigator DIPplex Kit 25 Contents Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Investigator Quantification Kits Investigator Quantiplex Kit 200 Investigator Quantiplex HYres Kit Investigator Human Matrix Standard BT5 single cap 5 x 25 Matrix Standard BT5 multi cap 25 Matrix Standard BT5 multi cap 50 Analysis software Investigator IDproof Software Investigator IDproof Demo Key Investigator IDproof Single Key Investigator IDproof Server Key Primer mix IC FQ reaction mix FQ control DNA 71 dilution buffer Primer mix IC YQ reaction mix FQ control DNA Z1 dilution buffer Identification PCR Kit Accessories Matrix standard 6 F
24. ates containing the spectral calibration mixture are placed in the autosampler tray the spectral calibration process can be started l 2 To access the Spectral Calibration screen select Maintenance on the Dashboard of the 3500 Series Data Collection software The number of wells in the spectral calibration plate and their location in the instrument must be specified set Optional Enable Allow Borrowing Click Start Run Investigator IDplex Plus Handbook 10 2012 Select Matrix Standard as a chemistry standard and BT5 for dye 39 Checking the matrix Click a capillary in the table in order to display the results for each capillary spectral data Quality value and Condition Number below the run results table The quality value value of each capillary must be greater than 0 8 and the number range C value must be between 1 and 20 M Check the matrix samples for a flat baseline As shown in the figure there should be 5 peaks with peak heights of about 1000 5000 RFU for each matrix sample Note The optimal range is 2000 4000 RFU Intensity vs Scan Number Calibrated Data E23 E E71 E F1 ET ES ES BE 0 400 800 1200 1600 2000 2400 2800 3200 4000 3000 2000 1000 Intensity vs Scan Number Electropherogram of spectral calibration of the matrix standard BT5 on an Applied Biosystems 3500 Genetic Analyzer When a spectral calibrati
25. bration the last calibration file for Dye Set G5 must be activated manually Click Set Active Spectral Calibration under the Tools menu 7 Rename the calibration file under Set Matrix Name e g BT5_Date of calibration Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 15 Table 15 Setup of formamide and DNA size standard mixture Component Volume per sample Hi Di Formamide 12 ul DNA Size Standard 550 BTO 0 5 ul Investigator IDplex Plus Handbook 10 2012 21 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed 3 Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 C 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 6 Load the samples on the tray Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted onto the plate of multi capillary analyzers If fewer samples are analyzed the empty positions must be filled with 12 ul Hi Di Formamide To ensure a reliable allelic assignment on multi capillary analyzers several ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Ensure ambient conditions are kept as recommended by the instrument m
26. clicking 8 Link the reaction plate on the autosampler tray with the created plate ID position A or B and start the run GA Instruments ga3130 3130 1 Spectral Viewer Intensity vs Scan Number Capillary Data Mon May 25 09 21 23 CEST 2009 sal d R Y G B 01 Dye Set AnySDye Active Calibration for Dye Set AnySDye Matrix used for Capillary 4 4 Mon May 25 09 21 23 CEST 2009 Condition 6 937256 Q Value 0 978649 List of Calibrations for Dye Set AnySDye Mon May 25 09 21 23 CEST 2009 Electropherogram of spectral calibration with matrix standard BT5 on an Applied Biosystems 3130 Genetic Analyzer a ENT Investigator IDplex Plus Handbook 10 2012 29 Checking the matrix 1 The quality value Q value of each capillary must be greater than 0 95 and the condition number range C value must be between 1 and 20 2 Check for a flat baseline in the matrix samples As shown in the figure on the previous page there should be 5 peaks with peak heights of about 1000 5000 RFU in each matrix sample Note The optimal range is 2000 4000 RFU 3 Check the new matrix with the current samples There should be no pull up peaks between the dye panels B G Y R O with the new matrix 4 If calibration failed use the optimized values of the Matrix Standard BT5 and repeat the calibration run 5 If all capillaries have passed the test the last calibration file for the Dye Set Any5Dye is activated auto
27. dard QC Protocol Assay All protocols can be set up via the Dashboard of the 3500 Series Data Collection software 1 To set up the Instrument Protocol go to Library and select Analyze followed by Instrument Protocols and click Create Note Modify the Run Module Default settings from HID36 POP4 as shown in Table 36 2 The parameters in Table 36 must be entered or selected 42 Investigator IDplex Plus Handbook 10 2012 Table 36 Instrument Protocol parameters for the Applied Biosystems 3500 3500xL Genetic Analyzer Protocol Name Oven Temperature C Run Voltage kV PreRun Voltage kV Injection Voltage kV Run Time s PreRun Time s Injection Time s Data Delay s Advanced Options Parameter Setting Application Type HID Capillary Length 36 cm Polymer POP4 Dye Set e g 5 Run Module HID36 4 e g Investigator IDplex Plus Default Default Default 3 0 1300 Default 8 0 Default Default Deviating from the standard settings the injection time may range between 1 and 20 s depending on the type of sample If samples with very high signal intensities are recorded a shorter injection time may be selected For samples with low DNA content an injection time of up to 20 s may be necessary 3 Click Save to confirm the changes 4 To set up the Size Standard go to Library select Analyze followed by Size Standards and
28. dard calibration plate Example for 8 capillaries Applied Biosystems 3500 Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 33 Table 33 Setup of formamide and Matrix Standard BT5 mixture for 8 capillaries Component Volume Hi Di Formamide 90 ul Matrix Standard BT5 multi cap 10 ul 38 Investigator IDplex Plus Handbook 10 2012 o Denature for 3 min at 95 C Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate Load 10 pl of the mixture to a 96 well plate e g positions A1 H1 Example for 24 capillaries Applied Biosystems 3500xL Genetic Analyzer l Table 34 Table 34 Setup of formamide and Matrix Standard BT5 mixture for 24 capillaries Component Volume Hi Di Formamide 225 ul Matrix Standard BT5 multi cap 25 ul 2 Load 10 pl of the mixture to a 96 well plate e g positions A1 H1 A2 H2 and A3 H3 3 Denature for 3 min at 95 C 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate Plate assembly and loading the plate in the instrument The necessary steps are described in detail in the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Performing a spectral calibration run Set up a mixture of formamide and Matrix Standard BT5 according to Once the multiwell pl
29. e Editor 11 Parameter Settings Sample Name Enter the name for the samples Priority e g 100 Default Sample Type Sample or Allelic Ladder Size Standard e g SST BTO_60 500bp Panel e g IDplex Plus Panels Analysis Method e g Analysis HID 3130 Snp Set User defined 1 3 Results Group 1 Select results group Instrument Protocol 1 Run36 POP4 BT5 26min setting described before 10 Start the run 11 During the run view Error Status in the Event Log or examine the quality of the raw data for each capillary in the Capillaries Viewer or the Cap Array Viewer 12 View data as an overview Run History or Cap Array Viewer of the Data Collection Software Run data are saved in the Run Folder of the previously chosen Result Group 34 Investigator IDplex Plus Handbook 10 2012 Analysis parameters analysis method Table 30 lists the recommended analysis parameters Table 30 Recommended settings for the Applied Biosystems 3130 3130xl Genetic Analyzer Parameter Settings Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Point 2000 Stop Point 10 000 Sizing All Sizes Smoothing and Baselining Smoothing Light Baseline Window 51 pts Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds B Xe Ger R O Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 pts Slope Thresholds 0 0 The peak amplit
30. er ep PCR tubes or plates Validity analysis software for human identification products Investigator Human Identification PCR Kits require calibration with an allelic ladder Therefore the software used must be compatible with human identification HID products for forensic applications We recommend Investigator IDproof Investigator IDproof Mixture GeneMapper ID GeneMapper ID X or Genotyper Software The Investigator Template Files facilitate data analysis and are valid with the software mentioned above a 8 Investigator IDplex Plus Handbook 10 2012 Protocol PCR Amplification This protocol is for PCR amplification of STR loci from forensic samples using the Investigator IDplex Plus Kit Important points before starting Set up all reaction mixtures in an area separate from that used for DNA isolation and PCR product analysis post PCR Use disposable tips containing hydrophobic filters to minimize cross contamination risks Things to do before starting M Before opening the tubes containing PCR components vortex and then centrifuge briefly to collect the contents at the bottom of the tubes The recommended amount of DNA under standard conditions is 0 5 ng Internal validations demonstrated robust and balanced results with 0 2 2 ng DNA and reliable results with 0 1 ng DNA Procedure 1 Thaw PCR components and template nucleic acid Mix thoroughly before use 2
31. erforming a spectral calibration run and checking the matrix 26 Investigator IDplex Plus Handbook 10 2012 Preparing the spectral calibration standards Example for 4 capillaries Applied Biosystems 3130 Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 20 Table 20 Setup of formamide and Matrix Standard BT5 mixture for 4 capillaries Component Volume Hi Di Formamide 60 ul Matrix Standard BT5 multi cap 5 ul 2 Load 12 ul of the mixture to 96 well plate e g positions A1 DT1 Denature for 3 min at 95 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 may be used to cool the plate Example for 16 capillaries Applied Biosystems 3130xl Genetic Analyzer 1 Set up a mixture of formamide and Matrix Standard BT5 according to Table 21 Table 21 Setup of formamide and Matrix Standard BT5 mixture for 16 capillaries Component Volume Hi Di Formamide 204 ul Matrix Standard BT5 multi cap 17 ul 2 Load 12 ul of the mixture to 96 well plate e g positions 1 1 and A2 H2 3 Denature for 3 min at 95 4 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 may be used to cool the plate a Investigator IDplex Plus Handbook 10 2012 27 Performing spectral calibration run 1 Place the 96 well plate on the autosampler tray 2 In the Prot
32. ersion of the software to be installed on a single workstation with a local database Allows for setup of a server that maintains the database and various workstations to access that database Must be purchased in conjunction with the Client Key Must be purchased in conjunction with the Server Key All template files for Investigator Human Identification PCR Kits for use with GeneMapper ID GeneMapper ID X and Genotyper Software as well as DIPSorter freeware CD ROM DNA extraction and purification QlAamp DNA Investigator Kit 50 21 DNA Investigator Kit 48 MinElute PCR Purification Kit 50 50 QlAamp MinElute Columns Proteinase K Carrier RNA Buffers Collection Tubes 2 ml Reagent Cartridges DNA Investigator Disposable Filter Tips Disposable Tip Holders Sample Tubes 2 ml Elution Tubes 1 5 ml Buffer G2 Proteinase K Carrier RNA 50 MinElute Spin Columns Buffers Collection Tubes 2 ml Larger kit sizes available please inquire Investigator IDplex Plus Handbook 10 2012 Cat no 389004 9020777 389401 389402 389403 389404 389900 56504 952034 28004 59 For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor re 60 Investigator IDplex Pl
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34. ex Plus Handbook 10 2012 19 Performing a spectral calibration run The parameter file for DyeSetG5 must be modified once to achieve successful calibration with the Data Collection Software version 1 0 1 or 1 1 Spectral parameter 1 To change settings in the parameter file go to the following path D AppliedBio Support Files Data Collection SupportFiles CalibrationData Spectral Calibration ParamFiles 2 Select MtxSTD Genescan_SetG5 to open the PAR file Change Condition Bounds Range to 1 0 20 0 4 If the calibration was unsuccessful also change Sensitivity to 0 1 and Quality to 0 8 5 Select Save As in the File menu and save the parameter file under a new name e g MtxStd Genescan_SetG5_BT5 par Note Always use this parameter file for spectral calibration runs using QIAGEN Matrix Standard BT5 Plate Editor for spectral calibration Place the 96 well plate on the autosampler tray Run the ABI PRISM 3100 Data Collection Software In Plate View click New to open the Plate Editor dialog box Enter a name for the plate Select a Spectral Calibration Select 96 Well as plate type and click Finish pm o Table 14 Plate Editor for spectral calibration Parameter Settings Sample Name Enter name for the matrix samples Dye Set G5 Spectral Run Module Default e g Spect36 POP4 Spectral Parameters MtxStd GeneScan SetG5 BT5 par parameters created before 7
35. h a Silver 96 Well Block or Gold plated Silver 96 Well Block use Max Mode Do not use 9600 Emulation Mode Table 3 Standard cycling protocol recommended for all DNA samples Temperature Time Number of cycles 926 5 96 10 30 cycles 61 C 120s 10 C Hot start to activate DNA polymerase 8 After the cycling protocol is completed store samples at 20 C protected from the light or proceed directly with running the electrophoresis 10 Investigator IDplex Plus Handbook 10 2012 Protocol Electrophoresis Using the ABI PRISM 310 Genetic Analyzer For general instructions on instrument setup matrix generation and application of the GeneScan or GeneMapper ID Software refer to the ABI PRISM 310 Genetic Analyzer User s Manual Electrophoresis using the GeneScan Software is described below The virtual filter set G5 is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 4 Table 4 Materials required for electrophoresis Material Specifications Capillary 47 cm 50 green Polymer POP 4 for ABI PRISM 310 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA Matrix generation Before conducting DNA fragment size analysis with the filter set G5 a matrix with the 5 fluorescent labels 6 FAM BTG BTY BTR and
36. i Int 138 37 56 Investigator IDplex Plus Handbook 10 2012 Ordering Information Product Investigator IDplex Plus Kit 100 Investigator IDplex Plus Kit 400 Related products Investigator Human Investigator ESSplex Plus Kit 100 Investigator ESSplex SE Plus Kit 100 Investigator Nonaplex ESS Kit 100 Investigator HDplex Kit 25 Investigator Triplex AFS QS Kit 100 Investigator Triplex DSF Kit 100 Investigator Argus X 12 Kit 25 Contents Primer mix Fast Reaction Mix including HotStarTaq Plus DNA Polymerase Control DNA allelic ladder IDplex Plus DNA size standard 550 and nuclease free water Primer mix Fast Reaction Mix including HotStarTaq Plus DNA Polymerase Control DNA allelic ladder IDplex Plus DNA size standard 550 BTO and nuclease free water Identification PCR Kits Primer mix Fast Reaction Mix including HotStarTaq Plus DNA Polymerase Control DNA allelic ladder ESSplex Plus DNA size standard 550 BTO and nuclease free water Primer mix Fast Reaction Mix including HotStarTaq Plus DNA Polymerase Control DNA allelic ladder ESSplex SE Plus DNA size standard 550 BTO and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size standard and nuclease free water Primer mix reaction mix DNA Polymerase Control DNA allelic ladder DNA size stand
37. iments or to other applicable guidelines 4 Investigator IDplex Plus Handbook 10 2012 Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www qgiagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtained 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 es Investigator IDplex Plus Handbook 10 2012 5 Introduction The Investigator IDplex Plus Kit is used for multiplex PCR in forensic human identity and paternity testing The kit covers the 13 CODIS Combined DNA Index System core loci 0251338 0195433 and Amelogenin One PCR simultaneously amplifies the 15 polymorphic STR markers 0251338 0351358 055818 075820 0851179 0135317 0165539 018551 0195433 021511 CSFIPO FGA vWA and the gender specific Amelogenin The Investigator IDplex Plus Kit has been developed specifically for rapid and reliable generation of DNA profiles from blood buccal swabs and forensic stains The kit utilizes QIAGEN s fast cycling PCR technology which allows amplification in around 90 minutes and provides highly robust
38. ix generation Parameter Settings Module File GS STR POP 4 1 ml G5 Matrix File None Size Standard None Injection Time s 5 Injection Voltage kV 15 Run Voltage kV 15 Run Temperature C 60 Run Time min 24 Always prepare matrix standards without DNA Size Standard BTO Analysis of the matrix samples Run the GeneScan Software Select New from the File menu and then select Project Open the folder of the current run and select Add Sample Files Select a matrix sample in the Sample File column Click Sample and then Raw Data Check the matrix samples for a flat baseline As shown in the figure next page there should be at least 5 peaks with peak heights of 1000 4000 RFU for each matrix sample Note The optimal range is 2000 4000 RFU pU MUS ce Investigator IDplex Plus Handbook 10 2012 13 W 3200 data points X 55007 Electropherogram with raw data of the matrix standard 6 FAM 7 Select an analysis range with a flat baseline and re inject the matrix sample if necessary 8 Record start and end value data points of the analysis range e g start value 3200 end value 5500 9 Calculate the difference between the end and start values e g 5500 3200 2300 data points Generation of a matrix 1 Select New in the File menu and then select Matrix Import the matrix samples for all dyes Y R and Enter a Start At value e g
39. ized by QIAGEN QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third parties Other than expressly stated licenses QIAGEN makes no warranty that this kit and or its use s do not infringe the rights of third parties This kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated Qu AI ZO The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and or its components For updated license terms see www giagen com 2012 all rights reserved www qiagen com Australia techservice au qiagen com Austria techservice at qiagen com Belgium techservice bnI qiagen com Brazil suportetecnico brasil qiagen com Canada techservice ca qiagen com China techservice cn Oqiagen com Denmark techservice nordic qiagen com Finland techservice nordic qiagen com France techservice fr qiagen com Germany techservice de qiagen com Hong Kong techs
40. matically in the Spectral Viewer Rename the calibration file e g BT5 Date of calibration Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 25 Table 25 Setup of formamide and DNA size standard mixture Component Volume per sample Hi Di Formamide 12 0 ul DNA Size Standard 550 BTO 0 5 ul 2 Aliquot 12 pl of the mixture to a tube for each sample to be analyzed 3 Add 1 pl PCR product or allelic ladder diluted if necessary 4 Denature for 3 min at 95 5 Snap freeze by placing the plate on ice for 3 min Alternatively the thermal cycler set to 4 C may be used to cool the plate 6 Load the samples on the tray 30 Investigator IDplex Plus Handbook 10 2012 Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted onto the plate of multi capillary analyzers If fewer samples are analyzed the empty positions must be filled with 12 ul Hi Di Formamide To ensure a reliable allelic assignment on multi capillary analyzers several ladders should be run Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur especially at low temperatures Ensure ambient conditions are kept as recommended by the instrument manufacturer Setting up the Data Collection Software 1 Edit the Run Module once for the first run In the Module Manager of the Dat
41. nal intensity Reduce the volume of the DNA Size Standard must be increased 550 BTO to peak heights of about 500 RFU Purify the PCR products before starting the analysis We recommend the MinElute PCR Purification Kit for rapid and effective purification see Ordering Information Matrix spectral calibration is not appropriate There are pull up This matrix cannot be used for the analysis peaks between the dye Repeat the matrix generation spectral panels B G Y R O calibration Be sure to carefully follow the correct with the current matrix protocol for the specific analysis instrument spectral calibration Many peaks are labeled as off ladder OL alleles in the samples a DNA Size Standard Click the orange Size Match Editor icon in the 550 BTO was not upper toolbar or the GeneMapper ID or defined or identified GeneMapper ID X Software Mark the orange correctly fragments of all samples Always use the DNA Size Standard 550 included in Investigator Human Identification PCR Kits 2 Signal intensities are Reduce the injection time in increments to a too high If the peak minimum of 1 s reduce the amount of the PCR heights of the samples amplification product for analysis or reduce the are outside the linear quantity of DNA for PCR detection range gt 4000 RFU gt 5000 RFU stutters split peaks and artifacts may be increased 24000 RFU for the ABI PRISM 310 Genetic Analyzer gt 5000 RFU for the ABI P
42. nt door to re initialize the instrument Then click Link Plate for Run In the next screen enter the desired Run Name and click Start Run Analysis parameters analysis method Table 41 lists the recommended analysis parameters in the worksheet Peak Detector Table 41 Recommended settings for the Applied Biosystems 3500 3500xL Genetic Analyzer Parameter Settings Peak Detection Algorithm Ranges Smoothing and Baselining Size Calling Method Peak Detection Advanced Analysis Partial Range Start Point 1000 Stop Point 20 000 Sizing All Sizes Smoothing Light Baseline Window 51 pts Local Southern Method Peak Amplitude Thresholds B ow G R O Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 pts Slope Thresholds 0 0 The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneMapper ID X Software version 1 2 The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times higher than the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis 46 Investigator IDplex Plus Handbook 10 2012 Protocol Analysis For general instructions on automatic sample analysis refer to the appropriate User and or Workflo
43. nts with lengths of up to 500 bp Starting the run Enter a name for the plate Place the prepared 96 well plate on the autosampler tray Run the ABI PRISM 3100 Data Collection Software In Plate View click New to open the Plate Editor dialog box Select GeneScan as the application type Select 96 Well as plate type and click Finish Investigator IDplex Plus Handbook 10 2012 23 Table 17 Settings in Plate Editor Parameter Settings Sample Name Enter name for the matrix samples Dyes O Color Info Ladder or sample Project Name e g 3100 Project Dye Set G5 Run Module 3kV 10s 500bp Analysis Module 1 DefaultAnalysis gsp See Table 16 Run Module 3kV 10s 500bp for the ABI PRISM 3100 Avant 3100 Genetic Analyzer 7 Complete the table in the Plate Editor and click OK 8 Click the column header to highlight the entire column and select Fill Down from the Edit menu to apply the information to the selected samples 9 Link the reaction plate on the autosampler tray to the created plate ID and start the run 10 Upon completion of the run view the data as Color Data in the Array View of the 3100 Data Collection Software or as Analyzed Sample Files under D AppliedBio 3100 DataExtractor ExtractRuns 24 Investigator IDplex Plus Handbook 10 2012 Analysis parameters Table 18 lists the recommended analysis parameters Table 18 Recommended analysis parameters for the
44. o the Applied Biosystems 3130 3130xl Genetic Analyzers Getting Started Guide The system with 4 capillaries is the Applied Biosystems 3130 Genetic Analyzer and the system with 16 capillaries is the Applied Biosystems 3130xl Genetic Analyzer The virtual filter set Any5Dye is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 19 Table 19 Materials required for electrophoresis Material Specifications Capillary 36 cm Capillary Array for Applied Biosystems 3130 3130xl Genetic Analyzer Polymer 4 Polymer for Applied Biosystems 3130 3130xl Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA Spectral calibration matrix generation Before conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the 5 fluorescent labels 6 FAM BTG BTY BTR and BTO for each analyzer The calibration procedure creates a matrix which is used to correct the overlapping of fluorescence emission spectra of the dyes Spectral calibration is comprised of the following steps Preparing the spectral calibration standards M Loading the standards to the 96 well reaction plate one sample per capillary Creating the instrument protocol for spectral calibration Protocol Manager Defining the plate composition in the plate editor Plate Manager E P
45. ocol Manager of the Data Collection Software open the Instrument Protocol window 3 Click New to open the Protocol Editor dialog box 4 Complete the dialog box with information from Table 22 and click Table 22 Instrument protocol for spectral calibration Protocol Editor Settings Name User e g Spectral36 POPA Type SPECTRAL Dye Set Any5Dye Polymer User e g POP4 Array Length User e g 36cm Chemistry Matrix Standard Run Module Default e g Spect36 4 1 Depends on the type of polymer and length of capillary used 5 Click New in the Plate Manager of the Data Collection Software to open the New Plate Dialog box 6 Enter information from Table 23 and click OK A new table in the Plate Editor opens automatically Table 24 Table 23 Plate Editor for spectral calibration 1 New plate dialog Settings Name e g Spectral BT5 date Application Spectral Calibration Plate Type 96 well Owner Name Operator Name 28 Investigator IDplex Plus Handbook 10 2012 Table 24 Plate Editor for spectral calibration Il Parameter Settings Sample Name Enter name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral3 POPA BT5 setting described before 7 Click the column header to select the entire column and select Fill Down from the Edit menu to apply the information to the selected samples Confirm by
46. on is successfully completed the Overall row displays green results If the Overall row displays red results refer to the spectral calibration troubleshooting section of the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide v Capillary Run Data Capillary 1 2 3 4 5 6 7 1 2 3 W Passed Failed Borrowed Not Calibrated Example of successful spectral calibration of the matrix standard BT5 for all capillaries on an Applied Biosystems 3500 Genetic Analyzer 40 Investigator IDplex Plus Handbook 10 2012 For each capillary select and display the spectral and raw data Check that the data meet the following criteria order of the peaks in the spectral profile from left to right read orange red yellow green blue No extraneous peaks appear in the raw data profile Peak morphology the spectral profile shows no gross overlaps dips or other irregularities Separate and distinct peaks should be visible If the data for all capillaries meet the criteria above click Accept Results If any capillary data does not meet the criteria above click Reject Results and refer to the spectral calibration troubleshooting section of the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide Sample preparation 1 Set up a mixture of formamide and DNA size standard according to Table 35 Table 35 Setup of formamide and DNA size standard mixture
47. plex Plus Handbook 10 2012 Table 39 Assay parameters Parameter Setting Assay Name e g Investigator IDplex Plus Color Default Application Type HID Instrument Protocol e g Investigator IDplex Plus from step 1 QC Protocols e g BTO 550 from step 7 13 Click Save to confirm the changes Starting the run 1 In the Dashboard click Create New Plate 2 Go to Define Plate Properties and select Plate Details Select or enter the parameters in Table 40 Table 40 Plate properties Property Setting Name e g Investigator IDplex Plus Number of Wells 96 Plate Type HID Capillary Length 36 cm Polymer POP4 3 Click Assign Plate Contents to confirm the changes 4 Enter the designated sample name in each well containing a sample or allelic ladder This will identify the well positions of each sample for the data collection and processing 5 Choose the correct Assay for the analysis If you followed the steps under Setting up the Run this would be Investigator IDplex Plus from step 11 All named wells on the plate must have an assigned assay 6 Select the wells for which to specify an assay Check the box next to the assay name assign it to the selected wells Investigator IDplex Plus Handbook 10 2012 45 7 Optional Repeat for file name conventions and results group 8 If not already done load the assembled plate to the instrument and close the instrume
48. ragments in bp of ladder is not identified the first allele in one color of the allelic ladder because it is outside with the corresponding value in the categories the expected size range Then compare it with the other alleles of the software in bp L Point alleles are not Point alleles are alleles with at least 1 bp found difference to the next integer allele Check the settings of the analysis method Lower the Peak Window Size value to 11 points Investigator IDplex Plus Handbook 10 2012 55 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor Cited references Bar W et al 1997 DNA recommendations Further report of the DNA Commission of the ISFG regarding the use of short tandem repeat systems Int J Legal Med 110 175 Hill C R Kline M C Coble M D and Butler J M 2008 Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples J Forensic 53 73 Szibor R et al 2003 Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sc
49. results through its inhibitor resistant chemistry The primers are fluorescence labeled with one of the following dyes 6 FAM Amelogenin 01 0351358 021511 E BTG TPOX 075820 0195433 055818 0251338 BTY 0165539 CSFIPO 0135317 FGA BTR 018551 0851179 The recommended amount of DNA under standard conditions is 0 5 ng Internal validations demonstrated robust and balanced results with 0 2 2 ng DNA and reliable results with 0 1 ng DNA The Investigator IDplex Plus Kit was validated using the GeneAmp PCR System 9700 with Gold plated 96 Well Silver Block and the Applied Biosystems 3500 Genetic Analyzer Table 1 shows the STR loci with their chromosomal mapping and repeat motifs which are concordant with the International Society for Forensic Genetics ISFG guidelines for the use of microsatellite markers B r et al 1997 6 Investigator IDplex Plus Handbook 10 2012 Table 1 Locus specific information of the Investigator IDplex Plus Kit GenBank accession Repeat motif of the Chromosomal Locus number reference allele mapping Amelogenin X M55418 E Xp22 1 22 3 Amelogenin Y M55419 11 2 0251338 G08202 TGCC 4 TTCC 2035 0351358 11449919 TCTG TCTA 3 25 3 055818 G08446 5q23 2 D7S820 G08616 7421 11 0851179 G08710 8423 1 23 2 0135317 G09017 TATC 3 13431 1 0165539 607925 16424 1 018551 118333 18421
50. run time for Investigator IDplex Plus was modified in order to be able to analyze fragments with lengths of up to 500 bp 16 Investigator IDplex Plus Handbook 10 2012 Analysis parameters Table 10 lists the recommended analysis parameters Table 10 Recommended analysis parameters for the ABI PRISM 310 Genetic Analyzer Parameter Settings Analysis Range Data Processing Peak Detection Size Call Range Size Calling Method Split Peak Correction Start 2000 Stop 10 000 Baseline Checked Multi component Checked Smooth options Light Peak Amplitude Thresholds B Y G R O Min Peak Half Width 2 pts Polynomial Degree 3 Peak Window Size 11 pts Min 60 Max 550 Local Southern Method None The peak amplitude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneScan or GeneMapper ID Software Thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be 3 times as high as the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis Note For information on the use of the recommended Template Files as analysis parameters refer to the appropriate Investigator Template Files User Guide Genotyper GeneMapper ID or GeneMapper ID X Investigator IDplex Plus Handbook
51. ude threshold cutoff value corresponds to the minimum peak height that will be detected by the GeneMapper ID Software The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be 3 times higher than the background noise of the baseline t Only the setting for Peak Window Size is different to defaults from Applied Biosystems for HID analysis Note For information on the use of the recommended Template Files as analysis parameters refer to the appropriate Investigator Template Files User Guide Genotyper GeneMapper ID or GeneMapper ID X Investigator IDplex Plus Handbook 10 2012 35 Protocol Electrophoresis Using the Applied Biosystems 3500 3500xL Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the Applied Biosystems 3500 Series Data Collection Software version 1 0 and the GeneMapper ID X Software version 1 2 refer to the Applied Biosystems 3500 3500xL Genetic Analyzers User Guide The system with 8 capillaries is the Applied Biosystems 3500 Genetic Analyzer and the system with 24 capillaries is the Applied Biosystems 3500xL Genetic Analyzer The virtual filter set AnyDye is used for combined application of the 5 fluorescent labels 6 FAM BTG BTY and This matrix standard is known as BT5 The materials required for electrophoresis are given in Table 31 Table 31 Materi
52. us Handbook 10 2012 Notes Investigator IDplex Plus Handbook 10 2012 61 Notes 62 Investigator IDplex Plus Handbook 10 2012 Trademarks QIAGEN HotStarTaq Investigator MinElute Rotor Gene QIAGEN Group 3500 6 FAM ABI PRISM Applied Biosystems Avant GeneAmp GeneMapper GeneScan Genotyper Hi Di POP 4 Applera Corporation or its subsidiaries Biometra Biometra medizinische Analytik GmbH Eppendorf Mastercycler Eppendorf AG GenBank US Department of Health and Human Services Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Limited License Agreement for the Investigator IDplex Plus Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms 1 The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product this handbook and additional protocols available at www giagen com Some of these additional protocols have been provided by QIAGEN users for QIAGEN users These protocols have not been thoroughly tested or optim
53. us Kit 100 400 Catalog no 381625 381627 Number of 25 pl reactions 100 400 Fast Reaction Mix 750 ul 2 x 1500 ul Primer Mix IDplex Plus 250 4 x 250 ul Control DNA 9948 200 ul 200 ul DNA size standard 550 BTO 50 ul 200 ul Allelic ladder IDplex Plus 25 ul 4 x 25 ul Nuclease free water 2 1 29 _ 5x1 9 ml Quick Start Protocol 1 1 Contains HotStarTaq Plus DNA Polymerase dNTPs MgCl and bovine serum albumin BSA Storage The Investigator IDplex Plus Kit is shipped on dry ice It should be stored immediately upon receipt at 20 C in a constant temperature freezer Avoid repeated thawing and freezing Primer mix and allelic ladder must be stored protected from the light DNA samples and post PCR reagents allelic ladder and DNA size standard should be stored separately from the PCR reagents Under these conditions the components are stable until the expiration date indicated on the kit Once opened the Investigator IDplex Plus Kit should be stored at 4 82 for a maximum of 2 weeks Intended Use The Investigator IDplex Plus Kit is intended for molecular biology applications in forensic human identity and paternity testing This product is not intended for the diagnosis prevention or treatment of a disease All due care and attention should be exercised in the handling of the products We recommend all users of QIAGEN products to adhere to the NIH guidelines that have been developed for recombinant DNA exper
54. vent pull up peaks Stutter peaks The occurrence of stutter peaks depends on the sequence of the repeat structure and the number of alleles 4 peaks are caused by a loss of a repeat unit during amplification of tetranucleotide STR motifs caused by slippage effects of the Taq DNA Polymerase whereas n 3 peaks appear particularly during amplification of the trinucleotide STR motif 02251045 These peaks should be interpreted using the Investigator Template Files for Genotyper GeneMapper ID and GeneMapper ID X Software Template independent addition of nucleotides Because of its terminal transferase activity the Taq DNA Polymerase may cause incomplete adenylation at the 3 end of the amplified DNA fragments The artifact peak is one base shorter than expected 1 peaks All primers included in the Investigator IDplex Plus Kit are designed to minimize these artifacts Peak height of the artifact correlates with the amount of DNA Laboratories should define their own limits for analysis of the peaks Artifacts Room temperature may influence the performance of PCR products on multi capillary instruments so that shoulder peaks or split peaks occur If shoulder or split peaks appear we recommend injecting the sample again Investigator IDplex Plus Handbook 10 2012 53 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise Comments and suggestions Sample preparation Sample sig
55. w Guides for Investigator IDproof Software Investigator IDproof Mixture Software GeneScan GeneMapper ID or GeneMapper ID X Software Finding the exact lengths of the amplified products depends on the device type the conditions of electrophoresis as well as the DNA size standard used Due to the complexity of some loci determining the size should be based on evenly distributed references The DNA Size Standard 550 BTO should be used with the following lengths of fragments 60 80 90 100 120 140 160 180 200 220 240 250 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp Figure 1 Electropherogram of the DNA Size Standard 550 BTO fragments with lengths in bp Analysis software Allele allocation should be carried out with suitable analysis software e g QIAGEN Investigator IDproof or IDproof Mixture Software or Genotyper GeneMapper ID or GeneMapper ID X Software in combination with the Investigator Template Files available as a download from www qiagen com or on a CD ROM cat no 389900 see Table 42 and Table 43 The recommended Investigator Template File for Genotyper Software is the IDplex A C 5 Investigator IDplex Plus Handbook 10 2012 47 Table 42 Recommended Investigator Template Files for GeneMapper ID File type File name Panels IDplex Plus Panels BinSets IDplex Plus Bins Size standard SST BTO 60 500bp Analysis Method Analysis HID 310 Anal
56. ysis HID 3130 Analysis HID 310 50rfu Analysis HID 3130 50rfu Plot Settings Plots 5dyes Table Settings Table for 2 alleles Table for 10 alleles Panels and BinSets must always be used other template files are optional Table 43 Recommended Investigator Template Files for GeneMapper ID X File type File name Panels IDplex Plus Panels x BinSets IDplex Plus Bins x Stutter IDplex Plus Stutter x Size standard SST BTO 60 500bp Analysis Method Analysis HID 310 Analysis HID 3130 Analysis HID 310 50rfu Analysis HID 3130 50rfu Analysis HID 3500 Plot Settings Plots 5dyes Table Settings 310 Data Analysis 31xx Data Analysis Panels and BinSets must always be used other template files are optional e LLLLLLLLLLLLLLLLLA AZAZA1 17 ELLALLLLAACAAAZAZ UU rocUqooioiobobok c k kE LL XXLLC ALZLLLUAXI 48 Investigator IDplex Plus Handbook 10 2012 Controls The alleles listed in Table 44 represent the Control DNA 9948 included in the Investigator IDplex Plus Kit and DNA from other commercially available standard cell lines Table 44 Allele assignment of the Investigator IDplex Plus Kit Locus CCR 9948 CCR 9947A ATCC K 562 Amelogenin X Y X X X X 0251338 23 23 19 23 17 17 0351358 15 17 14 15 16 16 055818 11 13 11 11 11 12 075820 11 11 10 11 9 11 0851179 12 13 13 13 12 12 0135317 11 11 11 11 8 8 0165539 11 11 11 12 11 12 018551 15 18 15 19 15 16 0195433 13 14 14 15 14 14 2 D21S11 29 30 30 30 29 30 31 CSF

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