Mentype® Argus Y-MHQS
Contents
1. Denaturation for 3 min at 95 C Cool down to 4 C For analysis load the samples on the tray Because injections take place simultaneously on all capillaries four samples must be pipetted when using 4 capillary analysers If less than four samples are analysed fill up the empty positions on the plate with 12 uL Hi Di Formamide One allelic ladder should be run per capillary in order to ensure reliable allelic assignment on 4 capillary analysers Room temperature can influence the performance of PCR products on multi capillary units so split peaks may occur especially at low temperatures Pay attention to keeping ambient conditions as recommended by the instrument manufacturer Signal intensities Options to increase the signal intensity Reduce the volume of the DNA Size Standard 550 ROX to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis Mentype Argus December 2008 3 3 Setting up the GeneMapper ID software Edit the Run Module as follows for the first run In the Module Manager of the Data Collection software click on New to open the Run Module Editor dialog box Run Module 20min_400bp Parameter Value Oven Temperature C 60 Poly Fill Volume 4840 Current Stability uA 5 PreRun Voltage kV 15 PreRun Time s 180 injection Voltage kV 3 njection Time s 5 Voltage Number of Steps 40 Voltage Step Int2. Analysis Range Start 2000 Stop 10000 Data Processing Baseline Checked ulticomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds GEURE in Peak Half Width 2 pts Polynorminal Degree 3 Peak Window Size 11 pts Size Call Range in 50 550 Size Calling Method Local Southern Method Split Peak Correction lone 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 three times as high as the background noise of the baseline Point alleles i e alleles with at least 1 bp difference to the next integer allele may occasionally not be distinguished For improved peak detection minimise the Peak Window Size further Mentype Argus December 2008 3 Electrophoresis using the ABI PRISM 3130 3130 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 to the ABI PRISM 9 3130 3130xl Genetic Analyzers Getting Started Guide Electrophoresis on an ABI PRISM 3130 Genetic Analyser by using the GeneMapper ID software is described below The system with 4 capillaries is named ABI 3130
3. 2 117 122 24 217 243 13 290 3 121 25 221 25 2 13 2 292 4 126 26 225 14 294 14 2 14 3 5 130 27 230 28 29 15 298 6 134 17 18 15 2 300 15 3 16 300 162 163 17 305 171 17 2 173 18 309 18 1 19 313 20 317 21 321 22 23 24 28 Mentype Argus December 2008 Table 5 Fragment lengths of the allelic ladder Mentype Argus analysed on an ABI PRISM 310 Genetic Analyzer green panel Marker allele Size bp Wan Marker allele Size bp Marker allele Size bp DYS391 HEX DYS389 I HEX DYS389 II HEX 8 106 6 7 11 245 8 9 10 10 2 27 363 23 24 25 26 9 110 9 3 12 249 28 367 10 114 13 253 29 371 11 119 14 257 30 375 12 123 15 261 16 17 31 379 13 127 14 32 383 DYS392 HEX 33 387 34 35 DYS19 HEX 7 292 6 8 9 11 78 10 10 301 12 82 11 304 11 1 13 86 12 307 14 91 14 3 13 310 15 95 14 313 16 99 15 316 16 17 203 18 207 19 rounded to integer The off ladder alleles of Biotype s DNA pool are allocated with the actual Biotype template files for GeneMapper ID or Genotyper software For further alleles see amongst others http www cstl nist gov biotech strbase str fact htm or http www yhrd org Mentype Argus December 2008 5 Interpretation of results As mentioned above post PCR analysis and automatic allele assignment with suitable analysis software ensure a precise and reliable discrimination of alleles Pull up peaks Pull up peaks may occur if peak heights are o
4. control dilute the Control DNA XY1 to 0 5 ng in the appropriate volume Instead of the template DNA pipette the diluted Control DNA into a reaction tube containing the PCR master mix Negative control For the negative amplification control pipette nuclease free water instead of template DNA into a reaction tube which contains the PCR master mix Mentype Argus December 2008 1 2 PCR amplification parameter Perform a hot start PCR in order to activate the Taq DNA Polymerase and to prevent the formation of non specific amplification products The number of cycles depends on the amount of DNA 30 cycles are recommended for all samples 34 cycles are recommended optionally in order to achieve optimal signal intensities for stains with small amounts of genomic DNA Standard method Recommended for all DNA samples Temperature Time 94 C 4 min hot start for activation of the JumpStart Taq DNA Polymerase 94 C 30s 58 C 120s 30 cycles 72 C 758 68 C 60 min 10 C Optional Recommended for stains with small amounts of genomic DNA Temperature Time 94 C 4 min hot start for activation of the JumpStart Taq DNA Polymerase 94 C 30s 58 C 120s 34 cycles 72 C 758 68 C 60 min 10 hold Small amounts of DNA may result in allelic dropouts and imbalances of the peaks Furthermore unspecific amplification products could appear With increasing numbers of cycles there is t
5. the matrix sample if necessary Note down start and end value data points of the analysis range e g start value 3400 end value 6400 Calculate the difference e g 6400 3400 3000 data points Mentype Argus December 2008 Generation of a new matrix File gt New Matrix Select the Matrix Standard Sample Files Number Of Dyes 050128_FAM fsa Statat 8 050128 HEX tsa Statat 050128 Stat 050128 Statat Points 13001 Fig 3 Matrix sample selection Import matrix samples for all dyes B G Y R Enter a Start At value e g 3400 Enter the calculated difference under Points e g 3000 Click on OK to calculate the new matrix Matrix Biotype D mtx x Reactions B G v R 00415 1 00012 B G 0 8472 1 0000 6863 6 0107 af 4509 4886 1 0000 0 0456 R 0 1273 0 1792 0 4964 1 0006 Fig 4 New matrix 05 30 Save the matrix in the matrix folder File Save as e g Matrix 05 30 Matrix check Check the new matrix with current samples File New Project open folder of the respective run Add Sample Files Select sample s in the Sample File column Sample Install New Matrix open matrix folder and select new matrix Re analyse your samples There should be no pull up peaks between the dye panels B G Y R with the new matrix Mentype Argus December 200
6. well A1 D1 Example for 16 Capillaries ABI 3130xl Composition Volume Hi Di Formamide 190 uL Matrix standard DS 30 10 0 uL Denaturation for 3 min at 95 C Cool down to 4 C For analysis load 10 uL of the matrix standard into a 96 well reaction plate well A1 H1 and A2 H2 Performing a spectral calibration run nsert the 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection Software click New the window Instrument Protocol to open the Protocol Editor dialog box Instrument Protocol for spectral calibration Protocol Editor Name e g Spectral36_POP4_DS30 Type SPECTRAL Dye Set D Polymer POP4 Array Length 36 Chemistry atrix Standard Run Module Spect36_POP4_1 Select OK to complete the Protocol Editor dialog box In the Plate Manager of the Data Collection software click New to open the New Plate Dialog box Plate Editor for spectral calibration I New Plate Dialog Name e g Spectral_DS 30 Application Spectral Calibration Plate Type 96 Well Owner Name Operator Name EA Select OK A new table in the Plate Editor opens automatically Mentype Argus December 2008 Plate Editor for spectral calibration II Column Sample Name Type name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral36_POP4_DS30 setting described before Click into the column header to select the entire column select Edit Fill Down to apply the informa
7. 0 425 450 475 500 525 and 550 bp T T T T T T T T T T T T T T T T T T T T T T T T T 8D 80 100 120 140 160 180 200 220 240 280 280 300 320 340 300 380 400 420 440 480 480 500 520 540 Size Standard 550 fsa 17 Red p500 Eann 00 20 00 80 00 140 00 wooo rao soo soo 75 00 460 00 475 00 500 00 525 00 50 00 90 00 150 00 190 00 240 00 260 00 320 00 380 00 400 00 550 00 Fano 100200 200 00 120 00 Fig 6 Electropherogram of the DNA Size Standard 550 ROX fragments with lengths in bp Note The basic template files for the DNA Size Standard 550 ROX has to be adjusted to 400 bp within the GeneMapper ID software The new template could be saved as SST ROX_50 400bp and used for further analyses As mentioned before Mentype Argus Y MH contains an internal PCR control Quality Sensor which provides helpful information on the efficiency of the PCR and on presence of PCR inhibitors A 6 FAM labelled 81 bp fragment is amplified independently of the DNA The PCR control assay without DNA shows only the sensor fragment Fig 7 and indicates successful polymerase chain reaction Ne me Pages 03 dim r E Fig 7 Elec
8. 8 2 2 Sample preparation Composition Volume Hi Di Formamide 12 0 uL DNA Size Standard 550 ROX 0 5 pL prepare 12 uL of the mix formamide DNA size standard for all samples add 1 uL PCR product diluted if necessary or allelic ladder Denaturation for 3 min at 95 C Cool down to 4 C For analysis load the samples on the tray Signal intensities Options to increase the signal intensity Reduce the volume of the DNA Size Standard 550 ROX to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis 2 3 Setting up the GeneScan software Create a Sample sheet and enter sample designation Injection list File GS STR POP 4 1 mL D atrix File e g Matrix Biotype Size Standard e g SST ROX_50 400bp Injection s 5 njection kV 15 0 Run 15 0 Run 60 Run Time min 26 Deviating from standard settings the injection time may range between 1 and 10 s depending on the type of sample If blood 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 10 s may be necessary Depending on the analysis conditions the run time for Mentype Argus was modified in order to analyse fragments with lengths of up to 400 bp Mentype Argus December 2008 2 4 Analysis parameter The recommended analysis parameters are
9. Chromosomal Mapping of Mentype Argus Locus Chromosomal mapping DYS19 Yp11 2 DYS385 Yq11 222 DYS389 11 21 DYS389 II 11 21 DYS390 Yq11 221 DYS391 Yq11 21 DYS392 Yq11 222 DYS393 Yp11 31 Content Mentype Argus PCR Amplification Kit 100 Reactions Nuclease free water 3 0 mL Reaction mix B 500 uL Primer mix 250 uL Control DNA XY1 2 ng uL 10 uL Control DNA XX28 100 ng uL 10 uL DNA Size Standard 550 ROX 50 uL Allelic ladder 10 uL Ordering information Mentype Argus 25 Reactions Cat No 42 09112 0025 Mentype Argus Y MH 100 Reactions Cat No 42 09112 0100 Mentype Argus 400 Reactions Cat No 42 09112 0400 Storage Store all components at 20 C and avoid repeated thawing and freezing Primer mix and allelic ladder must be stored protected from light The DNA samples and post PCR reagents allelic ladder and DNA size standard should be stored separately from the PCR reagents The expiry date is indicated on the kit cover Quality assurance All contents of Biotype test kits undergo an intensive quality assurance process at Biotype AG The quality of the test kits is permanently monitored in order to ensure unrestricted usability Please contact us if you have any questions regarding quality assurance Mentype Argus December 2008 Additionally required reagents Additional reagents are needed in order to be able to use the Biotype PCR Amplific
10. MSDS for all Biotype products which are available on request Please contact the respective manufacturers for copies of the MSDS for any additionally needed reagents Mentype Argus December 2008 Content 1 Electrophoresis using the ABI PRISM 310 Genetic Analyzer Electrophoresis using the ABI PRISM 3130 31 30x Genetic Analyzer POR aimplifiCatiori ura re tree dient pound er p Vet tends 1 1 Master mix preparation 1 2 PCR amplification parameter 2 1 Matrix generation senes 2 2 Sample preparation 1 2 3 Setting up the GeneScan software ssses 1 2 4 Analysis parameter seems 1 3 1 Spectral calibration matrix generation see 1 3 2 Sample preparati0 i url ayak kasika 1 3 3 Setting up the GeneMapper ID software 3 4 Analysis parameter analysis method WANA S S A y na o eh Dr Ht rt RE s 4 1 Biotype template files nen 4 200 Hln Ha e rto nere tes 4 3 Lengths of fragments and alleles Interpretation of results Mentype Argus December 2008 Protocols for PCR amplification electrophoresis and analysis 1 PCR amplification 1 1 Master mix preparation The table below shows the volumes of all PCR reagents per 25 uL reaction volume including a sample volume of 3 0 UL tem
11. Mentype Argus ampitcation Product description The Mentype Argus PCR Amplification Kit is a multiplex application for the nine Y chromosomal Short Tandem Repeat STR loci of the Minimal Haplotype MH standard The primers of DYS19 DYS385ab DYS389 I DYS389 II DYS390 DYS391 DYS392 and DYS393 are fluorescence labelled with 6 FAM or HEX The test kit was developed specifically for fast and reliable generation of male DNA profiles from mixtures of male and female DNA up to a ratio of 1 6000 so that separation of sperm from female cells or differential lysis is not required The detection limit of the Mentype Argus PCR Amplification Kit is up to 100 pg genomic male DNA However it is recommended to use 0 1 1 0 ng male DNA As special feature Mentype Argus Y MH contains an internal PCR control Quality Sensor QS which provides helpful information on the efficiency of the PCR and on the presence of PCR inhibitors The Mentype Argus Y MH was developed according to the recommendations of the International Forensic Y User Group http www yhrd org index html This consortium has evaluated a core set of a highly informative Y STR the so called Minimal Haplotype which was recommended for court Generation of DNA profiles using Mentype Argus corresponds to the guidelines of the International Society for Forensic Genetics Gill et al 2001 a and b Gusmao et al 2005a All import
12. acterization of the N 3 Stutter Product in the Trinucleotide Repeat Locus DYS392 J Forensic Sci 51 5 1069 1073 Mentype Argus December 2008 Rodig H Grum M Grimmecke HD 2007 Population study and evaluation of 20 Y chromosome STR loci in Germans nt J Legal Med 121 1 24 27 Stein B Willuweit S Nagy M Vogt PH Roewer L 2005 AZF deletions of the Y chromosome and failed amplification of commonly used Y STRs 217 Congress of the International Society for Forensic Genetics Ponta Delgada Portugal Szibor Edelmann J Hering S Plate 1 Wittig H Roewer L Wiegand P Cali F Romano V Michael M 2003 Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sci Int 138 37 43 Mentype Argus December 2008 Notes Mentype Argus December 2008 Notes Mentype Argus December 2008
13. ant population genetic data could be calculated with the GenoProof Software The test kit was validated and evaluated using the GeneAmp 9700 thermal cycler ABI PRISM 310 Genetic Analyzer and ABI PRISM 3100 3130 Genetic Analyzer Table 1 Locus specific information of Mentype Argus Y MH STR Locus GenBank Repeat motif Reference Allele accession of the reference allele allele range DYS19 017019 AGA TAGG 5 9 19 DYS385 AC022486 1 6 28 DYS389 I 004617 CTG TCTA s 2 9 17 DYS389 II 004617 CTG s 29 24 35 DYS390 AC011289 TCTA 24 12 17 29 DYS391 011302 1 5 16 DYS392 011745 AThi3 3 4 20 DYS393 AC006152 AGAT 2 7 18 Table 1 shows the STR loci with their repeat motifs and alleles The most frequent alleles for European populations are included in the allelic ladder Allele ranges include all known alleles of YHRD http www yhrd org as at 12 2008 and of the current literature DYS393 2 8 Mb DYS19 9 2 Mb DYS391 DYS389 l Il DYS390 DYS385 DYS392 21 5 Mb Fig 1 The ideogram of the Y chromosome describes the physical localisation of the STR loci which can be analysed with Mentype Argus The positions of the STR loci are shown in Mb http www ncbi nIm nih gov genome guide human as at 08 2008 Mentype Argus December 2008 Table 2
14. ate Type 96 Well Owner Name Operator Name 2 Click OK A new table in the Pate Editor opens automatically GeneMapper Plate Editor II Column Sample Name Type name for the samples Priority e g 100 Default Sample Type Sample or Allelic Ladder Size Standard e g SST ROX 50 400bp Panel e g Biotype Panels v2 choose test kit Analysis Method e g Analysis HID 3130 Snp Set User defined 1 3 Results Group 1 select results group Instrument Protocol 1 Run36_POP4_DS 30 setting described earlier Click into the column header to select the entire column select Edit Fill Down to apply the information to all selected samples and click on OK In the Run Scheduler click on Find All select Link to link the reaction plate on the autosampler up with the newly created plate record position A or B and start the run 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 View data as overview in 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 Mentype Argus December 2008 3 4 Analysis parameter analysis method The recommended settings in the worksheet Peak Detector are Peak Detection Algorithm Advanced Ranges Analysis Partial Range Start Pt 2000 Stop Pt 10000 Sizing All Sizes S
15. ation Kit The use of the following products is strongly recommended Reagent Supplier Order number JumpStart DNA Polymerase hot start 2 5 U uL 50 U oder 250 U Sigma Aldrich DA184 Hi Di Formamide 25 mL Applied Biosystems 4311320 Matrix Standards DS 30 for ABI PRISM 310 Genetic Analyzer Applied Biosystems 401546 und 402996 NED Matrix Standards DS 30 m for ABI PRISM 3100 3130 3730 Applied Biosystems Trademarks and patents Mentype is a registered trademark of Biotype AG GenoProof is a registered trademark of Qualitype AG JumpStart is a registered trademark of Sigma Aldrich ABI PRISM GeneScan Genotyper GeneMapper and Applied Biosystems are registered trademarks of Applied Biosystems Inc or its subsidiaries in the U S and certain other countries 6 FAM HEX NED ROX POP 4 and Hi Di are trademarks of Applied Biosystems Inc GeneAmp is a registered trademark of Roche Molecular Systems The PCR is covered by patents Patentees are Hoffmann La Roche Inc and F Hoffmann La Roche Roche GenBank is a trademark of National Institute of Health Warnings and safety instructions The PCR Amplification Kit contains the following potentially hazardous chemicals Kit component Chemical Hazards Primer mix reaction mix Sodium azide Very toxic if swallowed develops toxic and allelic ladder gases when it gets in contact with acids Observe the Material Safety Data Sheets
16. erval 15 Data Delay Time s 1 Run Voltage kV 15 0 Run Time s 1200 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 Depending on the analysis conditions the run time for Mentype Argus was modified in order to be able to analyse fragments with lengths of up to 400 bp Click on Save As enter the name of the new module e g 20min 4006 and confirm with OK Click on Close to exit the Run Module Editor Starting the run Place the prepared 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection software click on New in the Instrument Protocol window to open the Protocol Editor dialog box Instrument Protocol Protocol Editor Name e g Run36_POP4_DS 30 Type REGULAR Run Module HIDFragmentAnalysis36_POP4_1 Dye Set D parameter see above Click on OK to exit the Protocol Editor Mentype Argus December 2008 Prior to each run it is necessary to create plate definition as follows In the Plate Manager of the Data Collection software click on New to open the New Plate Dialog box GeneMapper Plate Editor I New Plate Dialog Name e g Plate_DS 30_Date Application select GeneMapper Application Pl
17. fferent 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 85 405 bp with Quality Sensor 75 405 bp Vertical Depending on signal intensity Mentype Argus December 2008 Figure 8 80 100 120 140 160 180 200 220 240 260 280 320 380 420 Argus YMH QS XY1 500pg fsa Blue jos Dvssoa _ oce is Argus YMH QS XY1 500pg fsa Green 2000 DYS391 DYS19 DYS389 j DYS392 DYS389 I n 8 Argus YMH QS XY1 500pg fsa Red 800 600 400 200 0 00 100 00 120 00 140 00 160 00 180 00 200 00 240 00 260 00 200 00 520 00 340 00 560 00 380 00 400 00 425 00 280 00 zm DNA Size Standard 550 ROX B s0 100 140 180 190 200 220 240 300 320 340 40 0 Argus YMH QS DNAM x fsa Blue 900 Qs DYS393 DYS390 DYS385 E Argus YMH QS DNAMx fsa Green 600 400 DYS391 DYS19 DYS389 DYS392 0 389 1 1 1 13 Argus YMH QS DNAMx fsa Red E 800 600 400 200 elem 120 00 140 00 160 00 160 00 200 00 240 00 260 00 300 00 520 00 540 00 380 00 38000 400 00 425 00 20 00 5 DNA Size Standard 550 ROX Fig 8 Electropherogram of the Mentype Argus Y MH using 500 pg Control DNA XY1 A 100 pg male Control DNA XY1 mixed wi
18. former ABI 3100 Avant and the system with 16 capillaries is named ABI 3130xl former ABI 3100 The virtual filter set D shall be used for combined application of the four fluorescent labels 6 FAM HEX NED and ROX also called DS 30 Material Capillary 36 cm Capillary Array for 3130 3130xl Polymer POP 4 Polymer for 3130 Buffer 10x Genetic Analyzer Buffer with EDTA 3 1 Spectral calibration matrix generation Prior to conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the four fluorescent labels 6 FAM HEX NED and ROX 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 comprises the following steps Preparation the spectral calibration standards 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 Performing a spectral calibration run and checking the matrix Mentype Argus December 2008 Setting up the spectral calibration standards Example for 4 capillaries ABI 3130 Composition Volume Hi Di Formamide 47 5 uL Matrix standard DS 30 2 5 uL Denaturation for 3 min at 95 C Cool down to 4 C For analysis load 10 uL of the matrix standard into a 96 well reaction plate
19. he Mentype Argus Y MH test kit varies between 0 613x10 and 5 296x10 Gusmao et al 20056 Allele frequencies of the European Minimal Haplotype Standard are collected and published by the Biotype AG Rodig et al 2007 They can be received as download from our homepage www biotype de Furthermore there is free access to the Y Chromosome Haplotype Reference Database from the International Forensic Y User Group http www yhrd org index html in order to calculate frequencies oneself Mentype Argus December 2008 4 1 Biotype template files Allele allocation should be carried out with a suitable analysis software e g GeneMapper ID or Genotyper software in combination with the Mentype Argus Y MH template files from Biotype AG Template files are available from our homepage or as CD ROM on request Recommended Biotype templates for GeneMapper ID software are Panels Biotype_Panels_v2 choose kit or higher versions BinSets Biotype_Bins_v2 or higher versions Size Standard SST BTO_50 500bp adjust up to 400bp adjustment described earlier Analysis Method Analysis_HID_310 Analysis_HID_3130 Plot Settings Plots_Blue Plots_Green Plots_Yellow Plots_Red Plots_4dyes Table Settings Table for 2 alleles Table for 10 alleles Panels and BinSets always have to be used whereas the other template files are optional Recommended Biotype template files for Genotyper software are Argus_YMH QS_
20. he risk of cross contamination caused by minimal amounts of impurities Mentype Argus December 2008 2 Electrophoresis using the ABI PRISM 310 Genetic Analyzer For general instructions on instrument setup matrix generation and application of the GeneScan GeneMapper ID software refer to the AB PRISM 310 Genetic Analyzer User s Manual Electrophoresis using the GeneScan software is described below The virtual filter set D shall be used for combined application of the four fluorescent labels 6 FAM HEX NED and ROX also called DS 30 Generally Filter Sets A and F are suitable too Material Capillary 47 cm 50 um green Polymer 4 for 310 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA 2 1 Matrix generation Prior to conducting DNA fragment size analysis with the filter set D a matrix with the four fluorescent labels 6 FAM HEX NED and ROX must be generated The suitable matrix standard DS 30 is available from Applied Biosystems Colour Matrix standard Order number Blue B 6 FAM Applied Biosystems 401546 Green G HEX Applied Biosystems 401546 Yellow Y NED Applied Biosystems 402996 Red R ROX Applied Biosystems 401546 Four electrophoresis runs shall be conducted one for each fluorescent label 6 FAM HEX NED and ROX under the same conditions as for the samples and allelic ladders of the Biotype test kit to generate
21. moothing and Baselining Smoothing Light Baseline Window 51 pts Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds Ben Fu G R in Peak Half Width 2 pts Polynominal 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 software The thresholds are usually 50 200 RFU and should be determined individually by the laboratory Recommendation The minimal peak height should be three times as high as the background noise of the baseline Point alleles i e alleles with at least 1 bp difference to the next integer allele may occasionally not be distinguished For improved peak detection minimise the Peak Window Size further Mentype Argus December 2008 4 Analysis For general instructions on automatic sample analysing refer to the GeneScan GeneMapper ID Software User s Manual 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 ROX shall thus be used with the following lengths of fragments 50 60 70 80 90 100 120 140 160 180 190 200 220 240 260 280 300 320 340 360 380 40
22. ng MA de Knijff P Kayser M Krawczak M Mayr WR Morling N Olaisen B Pascali V Prinz M Roewer L Schneider PM Sajantila A Tyler Smith C 2001b DNA Commission of the International Society of Forensic Genetics recommendations on forensic analysis using Y chromosome STRs Forensic Sci Int 124 1 5 10 Gusm o L Butler JM Carracedo A Gill P Kayser M Mayr WR Morling N Prinz M Roewer L Tyler Smith C Schneider PM 2005a DNA Commission of the International Society of Forensic Genetics ISFG an update of the recommendations on the use of Y STRs in forensic analysis Int J Legal Med 119 1 10 Gusmao L Sanchez Diz P Calafell F Martin P Alonso CA Alvarez Fernandez F Alves C Borjas Fajardo L Bozzo WR Bravo ML Builes JJ Capilla J Carvalho M Castillo C Catanesi Cl Corach D Di Lonardo AM Espinheira R Fagundes de Carvalho E Farfan MJ Figueiredo HP Gomes 1 Lojo MM Marino M Pinheiro MF Pontes ML Prieto V Ramos Luis E Riancho JA Souza Goes AC Santapa Sumita DR Vallejo G Vidal Rioja L Vide MC Vieira da Silva Cl Whittle MR Zabala W Zarrabeitia MT Alonso A Carracedo A Amorim A 2005b Mutation rates at Y chromosome specific microsatellites Hum Mutat 26 6 520 8 Kittler R Erler A Brauer S Stoneking M Kayser M 2003 Apparent intrachromosomal exchange on the human Y chromosome explained by population history Eur J Hum Genet 11 4 304 14 Mulero JJ Chang CW Hennessy LK 2006 Char
23. plate DNA The number of reactions to be set up shall be determined taking into account positive and negative control reactions Add one or two reactions to this number to compensate the pipetting error Number of PCR samples Data in uL 1 10 25 100 Nuclease free water 14 1 141 0 352 5 1410 0 Reaction mix B 5 0 50 0 125 0 500 0 Primer mix 2 5 25 0 62 5 250 0 Taq DNA Polymerase hot start 2 5 U L 0 4 4 0 10 0 40 0 Volume of master mix 22 0 220 0 550 0 2200 0 contains dNTP Mix BSA All components should be mixed vortex and centrifuged for about 10 s before preparing the master mix The DNA volume applied to the assay depends on its concentration A volume of up to 5 uL may be necessary for DNA trace templates DNA volumes of more than 5 uL are not recommended because potential PCR inhibitors may interfere with the process Fill up the final reaction volume to 25 uL with nuclease free water Generally DNA templates shall be stored in nuclease free water or in diluted TE buffer 10 mM Tris pH 8 0 and 1 mM EDTA e g 0 1x TE buffer The primer mixes are adjusted for balanced peak heights at 30 PCR cycles and 0 5 ng Control DNA XY1 in a reaction volume of 25 uL If more DNA template is introduced higher peaks can be expected for small PCR fragments and relatively low peaks for large fragments Reduce the amount of DNA template to correct this imbalance Positive control For the positive amplification
24. suitable matrix files Matrix sample Composition Volume sania Hi Di Formamide 2 0 uL p atrix standard 6 FAM 1 0 uL atrix sample 2 Hi Di Formamide 2 0 uL p atrix standard HEX 1 0 uL atrix sample 3 Hi Di Formamide 2 0 uL p atrix standard NED 1 0 uL atrix sample 4 Hi Di Formamide 2 0 uL p atrix standard ROX 1 0 uL Denaturation for 3 min at 95 C Cool down to 4 C For analysis load the samples on the tray Create a Sample Sheet and enter sample designation Mentype Argus December 2008 Injection list for matrix generation Injection list File GS STR POP 4 1 mL D atrix File NONE Size Standard NONE Injection 5 5 Injection 15 0 Run kV 15 0 Run 60 Run Time min 24 prepare matrix standards always without DNA Size Standard ROX Analysis of the matrix samples Run the GeneScan software File New Project open folder of current run Add Sample Files Select a matrix sample in the Sample File column Sample Raw Data Check the matrix samples regarding a flat baseline As shown in the figure below there should be at least five peaks with peak heights about 400 4000 Y axis for each matrix sample optimal range 1000 3000 Ee Ld Seng Ves Widow Heb l2J xi 3400 Data Points X Fig 2 Electropherogram with raw data of the matrix standard 6 FAM Select analysis range with flat baseline and re inject
25. th 100 ng female Control DNA XX28 B The Quality Sensor QS is shown at 81 bp Analysis was performed on an ABI PRISM 310 Genetic Analyzer with the DNA Size Standard 550 ROX Allele assignment was performed using the Genotyper software and the Mentype Argus Y MH5 template file Mentype Argus December 2008 Figure 9 Soi 8U pue aJ8M JOS adfousy au Husn 1 sem juawublsse JezAjeuv 209090 OLE J SItd Igv ue uo goH N A snBay Jappe y Jo 6 614 Join dh ib 00 0001 0091 000 usa 5 HA A 5 ot 221 lest E 02 s let o sz g 6 3 a a oz E ET LER 00 0001 009 0002 Jeppe ng ESJ J3PPE 5 S8 5 A 0625 A bt oze 002 osz 092 ozz ooz 081 091 00 085 09 01 001 December 2008 Mentype Argus Y MHO Table 4 Fragment lengths of the allelic ladder Mentype Argus analysed on an ABI PRISM 310 Genetic Analyzer blue panel Marker allele Size bp xe Marker allele Size bp Marker allele Size bp Former PCR control 6 FAM DYS390 6 DYS385 6 Quality Sensor 81 20 201 17 18 19 9 274 7 8 21 205 10 278 0 1 DYS393 6 22 209 11 282 1 8910 23 213 12 286 22
26. tion to all selected samples and click on OK In the Run Scheduler click on Find All select Link to link the reaction plate on the autosampler up with the newly created plate record position A or B and start the run 4 GA Inctnaner n gel RY 9 B Fig 5 Electropherogram of spectral calibration with matrix standard DS 30 Matrix check 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 Check the matrix samples for a flat baseline As shown in Fig 5 there should be four peaks with peak heights of about 1000 5000 Y axis in each matrix sample optimal range 2000 4000 Check the new matrix with your current samples There should be no pull up peaks between the dye panels B G Y R O with the new matrix If calibration was not successful use the optimised values and repeat the calibration run If all capillaries have passed the test the last calibration file for the Dye Set D is activated automatically in the Spectral Viewer Rename the calibration file e g DS 30 Date of calibration using the respective button Mentype Argus December 2008 3 2 Sample preparation Composition Volume Hi Di Formamide 12 0 uL DNA Size Standard 550 ROX 0 5 uL prepare 12 uL of the mix formamide DNA size standard for all samples add 1 uL PCR product diluted if necessary or allelic ladder
27. tropherogram of the 6 FAM labelled PCR control fragment Quality Sensor Fragment length in bp signal intensities in peak height Mentype Argus December 2008 Special features In general the electropherogram displays a single peak for each Y STR locus However locus DYS385 produces two peaks of different or same length These two fragments originate from duplicated and inversed copies of one Y chromosomal locus The primers provided in the test kit simultaneously co amplify the two homologous loci For separate amplification see Kittler et al 2003 Concerning locus DYS385 it must be stressed that alleles 14 3 15 3 16 3 17 3 and 19 3 represent the alleles 15 16 17 18 and 20 respectively with one thymidine deletion between the primer binding site and the repeat region F redi et al 1999 This deletion may serve as an additional distinctive feature for differentiation in forensic casework If more than one peak is obtained in the electropherogram for one or several markers this does not necessarily hint at mixed samples Duplications or triplications of STR markers also result in such an effect and have already been observed for DYS385 and DYS19 Butler et al 2005 Rarely single systems can fail too because of Y chromosomal deletions as known in azoospermic patients as already described for DYS385 and DYS392 Stein et al 2005 Mutation rates for intermediate or infrequent alleles of the Y STR system of t
28. ty Sensor which provides helpful information on the efficiency of the PCR and on presence of PCR inhibitors see Fig 7 Complete sensor failure indicates total inhibition of the PCR or errors in the assay If the sensor signal is amplified in presence of DNA either in the negative control or in the positive control the PCR is not inhibited Samples with sufficient DNA and without inhibiting substances result in the DNA profile according to the kit and the sensor fragment Reduced sensor peak heights in forensic samples indicate partial PCR inhibition If only the Quality Sensor is amplified the sample contains very little only female or degraded DNA Mentype Argus December 2008 References Butler JM Decker AE Kline MC Vallone PM 2005 Chromosomal duplications along the Y chromosome and their potential impact on Y STR interpretation J Forensic Sci 50 4 1 7 F redi S Woller J Padar 2 Angyal M 1999 Y STR haplotyping in two Hungarian populations nt J Legal Med 113 1 38 42 Gill P Brenner C Brinkmann B Budowle B Carracedo A Jobling MA de Knijff P Kayser M Krawczak M Mayr WR Morling N Olaisen B Pascali V Prinz M Roewer L Schneider PM Sajantila A Tyler Smith C 2001a DNA commission of the International Society of Forensic Genetics recommendations on forensic analysis using Y chromosome STRs nt J Legal Med 114 6 305 9 Gill P Brenner C Brinkmann B Budowle B Carracedo A Jobli
29. utside the linear detection range gt 3000 RFU or if an incorrect matrix was applied They appear at positions of specific peaks in other colour channels typically with lower signal intensities Peak heights should not exceed 3000 RFU in order to prevent pull up peaks Stutter peaks The occurrence of stutter peaks depends on the sequence of the repeat structure and the number of alleles n 4 peaks are caused by a loss of a repeat unit during amplification of tetranucleotide STR motives caused by slippage effects of the Taq DNA Polymerase whereas n 3 and n 3 peaks appear particularly during amplification of the trinucleotide STR motif DYS392 Mulero et al 2006 Those peaks should be interpreted in accordance with the template files of the Genotyper and GeneMapper ID software Template independent addition of nucleotides Because of its terminal transferase activity the Taq DNA Polymerase tends to add an adenosine radical at the 3 end of the amplified DNA fragments The artefact peak is one base shorter than expected 1 peaks All Biotype primers are designed to minimise these artefacts Artefact formation is further reduced by the final extension step of the PCR protocol at 68 C for 60 minutes Peak height of the artefact correlates with the amount of DNA Laboratories should define their own limits for analysis of the peaks Quality Sensor to check the PCR results Mentype Argus Y MH contains an internal PCR check Quali
30. v2c or higher versions 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 Analyse and interpret the sample data Mentype Argus December 2008 4 2 Controls The Control DNA XY1 of the test kit and other commercially available DNA from standard cell lines represent the following alleles Table 3 Allele determinations of Mentype Argus S Control DNA Control DNA ATCC CCR CCR CCR XY1 XX28 K 562 9947 9948 3657 DYS19 13 14 13 DYS385ab 16 17 11 14 16 19 DYS389 13 13 12 DYS389 II 30 5 31 29 DYS390 24 E 24 24 DYS391 1 10 10 DYS392 1 2 13 11 DYS393 13 5 5 13 13 PCR fragments are amplified with the Control DNA XX28 but may be used to generate mixtures with the Control DNA XY1 The reference DNA K 562 is availble from ATCC http www atcc org Products PurifiedDNA cfm celllines For further confirmation the table above displays the Y chromosomal alleles of two reference DNAs purchased from Coriell Cell Repositories CCR http locus umdnj edu nigms that is up to the standard of Szibor et al 2003 4 3 Lengths of fragments and alleles Table 4 io table 5 show the fragment lengths of individual alleles that refer to the DNA Size Standard 550 All analyses have been performed on ABI PRISM 310 3130 Genetic Analyzer with POP 4 polymer Di
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