080627_Mentype Triplex TVD_engl
Contents
1. Fed ce rco Ce e e ev 24 Mentype Triplex TVD December 2007 Protocols for 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 DNA sample volume of 3 0 uL template DNA Determine the number of reactions to be set up positive control and negative control reactions should be included Add one or two reactions to this number to compensate the pipetting error Number of PCR Reactions Volume in uL 1 10 25 100 Nuclease free Water 14 1 141 0 352 5 1410 0 Reaction Mix A 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 uL 0 4 _ 4 0 10 0 40 0 Volume of Master Mix 22 0 220 0 550 0 2200 0 contains Mg dNTP Mix BSA All components should be mixed vortex and centrifuged for about 10 s before preparation of the Master Mix The DNA volume applied to the assay depends on its respective concentration An increase of DNA volume to more than 5 uL is not recommended because potential PCR inhibitors may interfere Adjust the final reaction volume with Nuclease free Water to 25 uL Generally store DNA templates in Nuclease free Water or in diluted TE buffer 10 mM Tris HCI pH 8 0 and 1 mM EDTA e g 0 1x TE buffer The Primer Mixes are adjusted for balanced peak heights with 30 PCR cycles and 0 1 1 0 ng Control DNA XY1 in a reaction volume of 25 u2. bp ABI 310 6 FAM 121 2 125 3 129 4 133 9 138 1 142 1 146 1 149 7 153 7 157 7 161 8 Further Alleles 10 15 2 16 1 18 2 18 3 22 23b 245 25 Allele D21S11 25 26 27 28 29 29 2 30 31 32 32 2 33 33 2 34 34 2 35 37 n a Further Alleles 6 FAM 186 0 242 24 2 24 3 25 2 253 190 0 26 15 26 2 194 2 27 1 27 25 27 3 198 3 28 2 28 3 202 3 29 1 204 3 29 32 206 2 30 1 30 2 30 3 210 2 31 1 212 2 31 3 214 2 32 1 216 2 32 3 218 2 33 1 220 2 33 3 222 2 341 224 2 34 3 226 2 35 19 35 2 35 3 36 15 36 2 36 3 2344 372 383 38 20 39 39 27 40 2 41 20 46 a http Avww uni duesseldorf de WWW MedFak Serology database html b http www cstl nist gov biotech strbase str fact htm c Biotype AG DNA pool d Ayres et al 2002 e Grubwieser et al 2005 Mentype Triplex TVD December 2007 24 6 Interpretation of Results As mentioned above post PCR analysis and automatic allele allocation with suitable analysis software ensure a precise and reliable discrimination of alleles Pull up Peaks If peak heights are outside the linear detection range 23000 RFU or if an incorrect matrix has applied pull up peaks can occur at positions of specific peaks in all colour channels In order to avoid pull up peaks peak heights should not exceed more than 3000 RFU Stutter Peaks Appearance of stutter peaks depends o
3. 5400 n 050128 NED fsa Start At 5400 R 050128 ROX fsa Start At 3400 Points Boc Fig 2 Select Matrix Samples Import matrix samples for all dyes B Y Enter Start At value e g 3400 Enter difference value at Points e g 3000 Select OK to calculate the new matrix as shown in Fig 3 Matrix Biotype D mtx x Reactions B G Y R 1 0000 0 0445 0 0012 Josa72 10000 fosess foo107 Jo 4soo o asse 10000 00456 127 o 1792 0 4864 10000 Fig 3 New Matrix Biotype lt Q Save in the Matrix Folder FILE SAVE AS g Matrix Biotype Matrix Check Please check the new matrix with current samples FILE NEW PROJECT open run folder ADD SAMPLE FILES Select sample s in the column SAMPLE FILE SAMPLE INSTALL NEW MATRIX open matrix folder and select new matrix Re Analyse your samples With the new matrix there should be no pull up peaks between the dye panels B G Y R Mentype Triplex TVD December 2007 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 3 min denaturation at 95 cooling at 4 C for analysis load the samples on the tray Signal Intensities In order to increase the intensity of signals Reduce the vo
4. Cycles 128G 15s 68 60 10 hold Small amounts of DNA might result in allelic dropouts and imbalances of the peaks Furthermore unspecific amplification products could appear With increasing numbers of cycles cross contaminations could be emerged because of minimal amounts of impurities Mentype Triplex TVD December 2007 2 Electrophoresis using the ABI PRISM 377 DNA Sequencer For general instructions on instrument setup matrix generation and application of the GeneScan analysis Software please read the ABI PRISM 377 DNA Sequencer User s Manual Electrophoresis by using the GeneScan Software is described below For the combined application of the four fluorescent dyes 6 FAM HEX NED and ROX also called DS 30 the use of the virtual Filter Set D is allocated Generally Filter Sets A and F are suitable too Prior to any analysis of DNA fragment size a matrix with the appropriate four fluorescent dyes has to be generated for the instrument Appropriate matrix standards can be purchased from Applied Biosystems 2 1 Polyacrylamide Gel 596 Composition Urea 3096 Acrylamide bisacrylamide solution 29 1 10x TBE buffer Water Amount Volume 21 0g 8 4mL 6 0 mL 20 0 mL Filtrate and degas solution 1096 Ammonium persulfate TEMED Use glass plates with a spacing of 36 cm 2 2 Sample Preparation Composition Hi Di Formamide Blue Dextran DNA Size Standard 550 ROX PCR product dilut
5. Polymer 310 Genetic Analyzer POP 4 Buffer 10x Genetic Analyzer Buffer with EDTA 3 1 Matrix Generation Prior to any analysis of DNA fragment size a matrix with the appropriate four fluorescent labels has to be generated Appropriate matrix standards can be purchased from Applied Biosystems Dye Color 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 To generate useful matrix files it is necessary to perform four electrophoresis runs with the matrix samples PCR fragments labelled with 6 FAM HEX NED and ROX The runs have to be done under the same conditions like for the samples and the Allelic Ladders of the Biotype test kit Matrix sample Composition Volume Matrix sample 1 Hi DiTM Formamide 12 5 uL Matrix Standard 6 FAM 1 0 uL Matrix sample 2 Hi DiTM Formamide 12 5 uL Matrix Standard HEX 1 0 uL Matrix sample 3 Hi DiTM Formamide 12 5 uL Matrix Standard NED 1 0 uL Matrix sample 4 Hi DiTM Formamide 12 5 uL Matrix Standard ROX 1 0 pL 3 min denaturation at 95 cooling at 4 C for analysis load the samples on the tray Create a Sample Sheet and enter sample designation Mentype Triplex TVD December 2007 Injection List for Matrix Generation Injection list Module File GS STR POP 4 1 mL D Matrix File NONE Size Standard NONE Inje
6. or 250 U 94187 Hi DiTM Formamide 25 mL Applied Biosystems 4311320 Matrix Standards DS 30 for ABI PRISM 310 Genetic Analyzer Applied Biosystems 401546 and 402996 NED and ABI PRISM 377 DNA Sequencer Matrix Standards DS 30 for ABI PRISM 3100 3130 Applied Biosystems 4345827 and ABI PRISM 3700 3730 Trademarks and Patents Mentype is a registered trademark of Biotype AG JumpStart is a registered trademark of Sigma Aldrich ABI PRISM GeneScan Genotyper GeneMapper and Applied Biosystems are registered trademarks of Applera Corporation or its subsidiaries in the U S and certain other countries 6 FAM HEX NED ROX POP 4 and Hi Di are trademarks of Applera Corporation GeneAmp is a registered trademark of Roche Molecular Systems The PCR is under patent law Patentees are Hoffmann La Roche Inc and F Hoffmann La Roche Roche DyeEx is a registered trademark of Qiagen GenBank is a trademark of National Institute of Health Warning and Safety Instructions The PCR Amplification Kit contains the following potentially hazardous chemical Kit Component Chemical Danger Primer Mix Reaction Mix sodium azide NaN very toxic if swallowed develops toxic and Allelic Ladder gases when it gets in contact with acids For the Material Safety Data Sheet MSDS of all Biotype products please contact us For MSDS of additional reagents to be needed please contact the corresponding manufactures Mentyp
7. 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 former chosen Result Group 4 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 Smoothing and Baselining Smoothing Light Baseline Window 51 pts Local Southern Method Peak Amplitude Thresholds Bie G Min 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 from 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 higher then the background noise of the baseline Size Calling Method Peak Detection Sometimes point alleles i e alleles with at least 1 bp difference to the next integer allele like THO1 allele 9 3 and 10 can not be distinguished For improved peak detection minimize the Peak Window Size further on Mentype Triplex TVD December 2007 20 5 Analysis For general instructio
8. 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 Matrix Standard Run Module Spect36_POP4_1 Select OK to complete the Protocol Editor dialog box Mentype Triplex TVD December 2007 In the Plate Manager of the Data Collection Software click New to open the New Plate Dialog box Plate Editor for Spectral Calibration 1 New Plate Dialog Name e g Spectral DyeSet D Date Application Spectral Calibration Plate Type 96 Well Owner Name Operator Name Select OK to complete the New Plate dialog box new table in the Plate Editor opens automatically For further analyses for spectral calibration use the same plate setting click Import select the xml file and click Open Plate Editor for Spectral Calibration II Column Name Sample Name Type name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral36_POP4_DS30 setting described before For each of the columns click the column header to select entire column then select Edit Fill Down to apply the information to all of the selected samples and enter OK In Run Scheduler click Find All select link to link up the reaction plate on the autosampler with the newly created plate record position A or B and start the run View Pass Fail Status after the run in the Event Log and open the Spectral Viewer t
9. L If more DNA template is introduced higher peaks will be expected for small PCR fragments and relatively low peaks for larger fragments Reduce the amount of template DNA to correct this imbalance Positive Control For the positive amplification control dilute the Control DNA XY1 to 0 1 1 0 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 containing the PCR Master Mix Mentype Triplex TVD December 2007 1 2 PCR Amplification Parameter In order to activate the Taq DNA Polymerase and to prevent the formation of non specific amplification products perform a hot start PCR reaction The number of cycles depends on the amount of DNA For all samples 30 cycles are recommended For stains with small amounts of genomic DNA 34 cycles are recommended optionally in order to achieve optimal signal intensities Standard Method Recommended for all DNA Samples Temperature Time 94 4 min hot start for Activation of the JumpStart Taq DNA Polymerase 94 305 58 C 120s 30 Cycles 72 C 15s 68 60 10 hold Optional Recommended for Stains with small Amounts of DNA Temperature Time 94 4 min hot start for Activation of the JumpStart Taq DNA Polymerase 94 305 58 C 120s 34
10. Mentype Triplex TVD pce ampitication Product Description Mentype Triplex TVD is a complement to the Mentype PCR Amplification Kits for fast and reliable DNA genotyping of blood buccal swabs and forensic stains In one PCR reaction the three polymorphic tetranucleotide Short Tandem Repeat STR loci THO1 TC11 vWA and 021511 are amplified simultaneously One primer of each locus is fluorescence labelled with 6 FAM Mentype Triplex TVD is preferentially employed for low copy and degraded DNA samples which do not give distinct results with complex multiplexes The detection limit of Mentype Triplex TVD PCR Amplification Kit is about 100 pg genomic DNA The use of 0 1 1 0 ng DNA is recommended Validation and evaluation of the test kit have been performed for the GeneAmp 9700 thermal cycler ABI 310 Genetic Analyzer and ABI PRISM 3100 3130 Genetic Analyzer Table 1 Locus Specific Information of Mentype Triplex TVD Locus GenBank Repeat Motif Reference Allele Accession of the Reference Allele Allele Range 021511 AP000433 TCTAL TCTG s TCTAJ TA TCTA 29 24 46 TCA TCTA TCCATA THO1 TC11 D00269 9 3 14 WA M25858 TCTA TCTG TCTA 18 10 25 The repeat motifs shown in Table 1 are concordant with the International Society for Forensic Genetics ISFG guidelines for the use of microsatellite markers Bar et al 1997 Table 2 Chromosomal Mapping of Mentype Triplex TVD L
11. ction s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 24 prepare Matrix Standards always without DNA Size Standard ROX Analysis of the Matrix Samples Open GeneScan or GeneMapper ID Software FILE NEW PROJECT open current run folder ADD SAMPLE FILES Click a single matrix sample in the column SAMPLE FILE Select SAMPLE RAW DATA Review the matrix samples for a flat baseline beyond the primer peak There should be at least five peaks with peak heights about 400 4000 Y in every matrix sample optimal range 1000 3000 as shown in Fig 1 il GeneScan 3 7 6 FAM fsa EJ Edit Project Sample Settings View Windows Help x ieu lo 500 1000 1500 2000 2500 3000 3500 4000 400 5000 5500 5000 8100 3400 Data Points X Kira TIZ TR ver l Fig 1 Electropherogram with Raw Data of the Matrix Standard 6 Select analysis range with flat baseline Re inject the matrix sample if necessary Note start and end value Data Points of the analysis range e g start value 3400 end value 6400 Calculate the difference value e g 6400 3400 3000 Data Points Mentype Triplex TVD December 2007 Generation of a New Matrix FILE NEW MATRIX as shown in Fig 2 W Make New Matrix Select the Matrix Standard Sample Files Number Of Dyes B 050128 FAM fsa Start At 5400 050128 HEX fsa Start At
12. e Triplex TVD December 2007 Gontent 1 PGR AmplifiCall0l Ax o Dt Kal Dm E oed 5 1 1 Master Mix Preparation 5 1 2 PCR Amplification Parameter d a ecd hacer rre eo E eu dete d ttes 6 2 Electrophoresis using the ABI PRISM 377 DNA 5 7 2 1 Polyacrylamide Gel 5 Dota E Bud 7 2 2 Sample 7 2 3 Setting for GeneScan Software irridet 8 3 Electrophoresis using the ABI PRISM 310 Genetic Analyzer 9 3 1 Matrix t co baa t rt 9 3 2 Sample Preparation 12 3 3 Setting for GeneScan Software ieeeeeeeeeeeeee 12 3 4 Analysis 1 13 4 Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer 14 4 1 Spectral Calibration Matrix Generation 14 4 2 Sample Preparation 17 4 3 Setting for GeneMapper ID Software 17 4 4 Analysis Parameter Analysis Method 19 n eW 20 5 1 md dt peu 21 5 2 Lengths of Fragments and Alleles 21 Interpretation of 5
13. e corresponds to the minimum peak height that will be detected from 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 higher then the background noise of the baseline Sometimes point alleles i e alleles with at least 1 bp difference to the next integer allele like THO1 allele 9 3 and 10 can not be distinguished For improved peak detection minimize the Peak Window Size further on Mentype Triplex TVD December 2007 4 Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer For detailed instructions on instrument setup spectral calibration or application of the ABI PRISM Data Collection Software and the GeneMapper ID Software refer to the ABI PRISM 3130 3130 Genetic Analyzers Getting Started Guide Electrophoresis by using the GeneMapper ID Software is described below The 4 Capillary System is named ABI 3130 before ABI 3100 Avant the 16 Capillary System is named ABI 3130x before ABI 3100 For the combined application of the four fluorescent labels 6 FAM HEX NED and ROX also called DS 30 the use of the Dye Set D is allocated Material Capillary 3130 capillary array 36 cm Polymer 3130 POP 4 polymer Buffer 10x Genetic Analyzer Buffer with EDTA 4 1 Spectral Calibration Matrix Generation Prior to any analysis of DNA fragment size a spectral cal
14. e of short tandem repeat systems nt J Legal Med 110 175 176 Gill P Brenner CH Buckleton JS Carracedo A Krawczak M Mayr WR Morling N Prinz M Schneider PM Weir BS 2006 DNA Commission of the International Society of Forensic Genetics ISFG Recommendations on the interpretation of mixtures Forensic Sci Int Jul 13 160 2 3 90 101 Griffiths RA Barber MD Johnson PE Gillbard SM Haywood MD Smith CD Arnold J Burke T Urquhart AJ Gill P 1998 New reference allelic ladders to improve allelic designation in a multiplex STR system nt J Legal Med 111 267 272 Grubwieser P Muhlmann R Niederst tter H Pavlic M Parson W 2005 Unusual variant alleles in commonly used short tandem repeat loci nt J Legal Med 2005 Jan 5 Szibor R Edelmann J Hering S Plate 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 Triplex TVD December 2007 25 26 Notes Mentype Triplex TVD December 2007 Notes Mentype Triplex TVD December 2007 27 28 Notes Mentype Triplex TVD December 2007
15. ed if necessary or Allelic Ladder 3 min denaturation at 95 C cooling at 4 C apply 1 5 uL sample to the gel Mentype Triplex TVD 350 uL 15 uL Volume 1 5 uL 0 5 uL 1 0 uL December 2007 2 3 Setting for GeneScan Software Plate Check Module D PreRun Module GS PR 36D 1200 Run Module GS Run 36D 1200 Matrix D 6 FAM HEX NED ROX Standard SST550 ROX Programming of the Run Module for Biotype Test Kits 6 FAM HEX NED ROX Open program 377 Collection Open File New GeneScan Run Select the module GS Run 36D 1200 in the run window Click at the sheet symbol in the run window Use the following settings Parameter Setting Voltage 3000 V Current 50 0 mA Power 150 W Save the module in the field Collection time 2 7 h Save Copy Gel temperature 51 C Laser power 40 0 mW Mentype Triplex TVD December 2007 3 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 please read the ABI PRISM 310 Genetic Analyzer User s Manual Electrophoresis by using the GeneScan Software is described below For the combined application of the four fluorescent labels 6 FAM HEX NED and ROX also called DS 30 the use of the virtual Filter Set D is allocated Generally Filter Sets A and F are suitable too Material Capillary 47 cm 50 green
16. ibration with the four florescent labels 6 FAM HEX NED and ROX has to be generated for each analyzer The spectral calibration creates a matrix to correct the overlapping of fluorescence emission spectra of the dyes Performing a spectral calibration can be divided into the following tasks Choosing and setting up the Spectral Calibration Standards Loading the standards on the 96 well reaction plate per capillary one sample Creating the instrument protocol for performing spectral calibration Protocol Manager Define the plate assembly within the plate editor Plate Manager Performing a spectral calibration run and reviewing data Mentype Triplex TVD December 2007 Setting up the Spectral Calibration Standards Example for 4 Capillaries ABI 3130 Composition Volume Hi Di Formamide 47 5 uL Matrix Standard 2 5 UL 3 min denaturation at 95 cooling at 4 C for analysis load 10 uL of the Matrix Standard into a 96 well reaction plate well A1 D1 Example for 16 Capillaries ABI 31 30xI Composition Volume Hi Di Formamide 190 uL Matrix Standard 10 0 uL 3 min denaturation at 95 C cooling at 4 C for analysis load 10 uL of the Matrix Standard into a 96 well reaction plate well A1 H1 and A2 H2 Performing Spectral Calibration Run nsert the 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection Software click New in the window Instrument Protocol to open the Protocol
17. ified in order to analyze lengths of fragments up to 280 bp Click Save As and enter the name of the new module g 15min 50 400bp and enter OK Click Close to exit the Module Editor Previous to every run it is necessary to compile the plate as follows In the Plate Manager of the Data Collection Software click New to open the New Plate Dialog box GeneMapper Plate Editor I New Plate Dialog Name e g Plate_DyeSet D_Date Application select GeneMapper Application Plate Type 96 Well Owner Name Operator Name Select OK to complete the New Plate dialog box A new table in the Plate Editor opens automatically Mentype Triplex TVD December 2007 GeneMapper Plate Editor Il Column Name Sample Name Priority Sample Type Size Standard Panel Analysis Method Snp Set User defined 1 3 Results Group 1 Instrument Protocol 1 Type name for the samples e 0 100 Default Sample or Allelic Ladder e g SST550_50 400bp 0 Biotype Panels v1 choose test kit g Analysis HID 3130 select results group Run36_POP4_DyeSet D setting typed before For each of the columns click the column header to select entire column then select Edit Fill Down to apply the information to all of the selected samples and enter OK In Run Scheduler click Find All select link to link up the reaction plate on the autosampler with the newly created plate record position A or B and start the run During
18. lume of the DNA Size Standard 550 the peaks of the Size Standard should be about 500 relative fluorescent units RFU Purify the PCR products with DyeEx 2 0 Spin Kit Qiagen 63204 3 3 Setting for GeneScan Software Create a Sample sheet and enter sample designation Injection List Module File GS STR POP 4 1 mL D Matrix File e g Matrix Biotype Size Standard e g SST550_50 400bp Injection s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 24 Apart 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 to be 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 Triplex TVD was modified in order to analyze lengths of fragments up to 280 bp Mentype Triplex TVD December 2007 3 4 Analysis Parameter The recommended analysis parameters are Analysis Range Start 2000 Stop 10000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds bz YN G R Min Peak Half Width 2 pts Polynorminal Degree 3 Peak Window Size 11 pts Size Call Range Min 50 Max 550 Size Calling Method Local Southern Method Split Peak Correction None The peak amplitude threshold Cutoff valu
19. n the sequence of the repeat structure and on the number of alleles n 4 peaks are due to a loss of a repeat unit during amplification of tetranucleotide STR motives caused by slippage effects of the DNA polymerase Interpretation of those peaks should be done in accordance with the Template Files of the Genotyper and GeneMapper ID Software Template independent Nucleotide Addition The terminal transferase activity of Taq DNA polymerase leads to the addition of an adenosine residue preferentially at the 3 end of amplified DNA fragments Incomplete extension is responsible for double peaks resulting from the absence of the terminal adenosine residue The artefact peak is one base shorter than expected 1 peaks All Biotype primers are designed to minimize these artefacts In addition the final extension step of 68 C for 60 minutes is included to the PCR protocol in order to reduce formation of artefacts Peak height of the artefact correlates with the amount of DNA Every laboratory should determine their own thresholds for analysis of the peaks Mentype Triplex TVD December 2007 References Ayres KL Chaseling J Balding DJ 2002 Implications for DNA identification arising from an analysis of Australian forensic database Forensic Sci Int 129 90 98 W Brinkmann B Budowle B Carracedo A Gill P Lincoln P Mayr W Olaisen B 1997 DNA recommendations Further report of the DNA Commission of the ISFH regarding the us
20. ng for GeneMapper ID Software Performing Run nsert the 96 well plate prepare on the autosampler tray In the Protocol Manager of the Data Collection Software click New in the window Instrument Protocol to open the Protocol Editor dialog box Instrument Protocol Protocol Editor Name Run36_POP4_DyeSet D Type REGULAR Run Module HiDFragmentAnalysis36 POP4 1 Dye Set D for detailed description see Setting of the Run Module on the next page Select OK to complete the Protocol Editor dialog box Mentype Triplex TVD December 2007 Previous to the first run it is necessary to edit the Run Module as follows In the Module Manager of the Data Collection Software click New to open the Run Module Editor dialog box Run Module 15min 50 400bp Run Modul Editor Value Oven Temperature 60 Poly Fill Volume 4840 Current Stability UA 5 PreRun Voltage kV 15 PreRun Time s 180 Injection Voltage kV 3 0 Injection Time s 5 Voltage Number of Steps 40 Voltage Step Interval 15 Data Delay Time s 1 Run Voltage kV 15 0 Run Time s 900 Apart from 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 to be recorded a shorter injection time may be selected For samples with low DNA content an injection time up to 20 s may be necessary Depending on the analysis conditions the Run Time for Mentype Triplex TVD was mod
21. ns on automatic sample analysing please read the GeneScar or GeneMapper ID Software User s Manual The determination of the exact lengths of the amplified products depends on the type of device on the conditions of electrophoresis as well as on the DNA Size Standard used Due to the complexity of some STR loci the size determination should be based on evenly distributed points of references Thus please use the DNA Size Standard 550 ROX with the following lengths of fragments 50 60 70 80 90 100 120 140 160 180 190 200 220 240 260 280 300 320 340 360 380 400 425 450 475 500 525 and 550 bp B 8 100 120 140 160 180 200 220 24 260 280 300 520 340 300 380 400 420 440 460 450 500 520 54 DNA Size Standard 550 fsa 17 Red 600 400 200 70 00 100 00 n 0 00 8000 140 00 180 00 220 00 260 00 300 00 30 00 000 428 00 450 00 475 00 500 00 526 00 60 00 80 00 160 00 190 00 240 00 320 00 360 00 000 550 00 200 00 120 00 Fig 5 Electropherogram of the DNA Size Standard 550 ROX Lengths of Fragments in bp Allele designation of analyzed samples should be carried out either manually or with suitable analysis software e 0 GeneMapper ID or Genoty
22. o review and evaluate the spectral calibration profile for each capillary Info Raw Data EPT Data Matrix Standard DS 30 Biotype fsa 0 1000 2000 3000 4000 5000 6000 9000 80004 70004 60004 50007 Fig 4 Electropherogram with Raw Data of the Matrix Standard for 05 30 Note If all capillaries passed the test activate this Spectral Calibration file for Dye Set D in the Spectral Viewer Mentype Triplex TVD December 2007 4 2 Sample Preparation Composition Volume Hi DiTM 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 3 min denaturation at 95 cooling at 4 C for analysis load the samples on the tray Because injections proceed simultaneously for all capillaries four samples must be pipette for analysis on 4 capillary analysers If less then four samples are analysed fill up the empty positions on the plate with 12 uL Hi Di M Formamide For reliable allelic assignment on 4 capillary analysers one Allelic Ladder per capillary should be run The room temperature might influence the running performance of PCR products that might result in split peaks especially at low temperatures or an altered run velocity of DNA fragments Please pay attention to keep conditions recommended by the instrument manufacturer 4 3 Setti
23. ocus Chromosomal Mapping D21811 21g21 1 THO1 11p15 5pter 12 13 31 Gontent Mentype Triplex TVD PCR Amplification Kit 100 Reactions Nuclease free Water Reaction Mix A Primer Mix Control DNA XY1 2 ng uL DNA Size Standard 550 ROX Allelic Ladder Ordering Information Mentype Triplex TVD 25 Mentype Triplex TVD 100 Mentype Triplex TVD 400 Storage Reactions Reactions Reactions 3 0 mL 500 uL 250 uL 10 uL 50 uL 10 uL Cat No 41 03313 0025 Cat No 41 03313 0100 Cat No 41 03313 0400 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 The content of Biotype test kits is subjected to an intensive quality assurance of the Biotype AG The quality of test kits is controlled continuously in order to document the unrestricted usability For questions regarding the quality assurance please feel free to contact us Mentype Triplex TVD December 2007 Additional Required Reagents In order to use the Biotype PCR Amplification Kit additional reagents are needed We strongly recommend the application of the following products Reagent Supplier Order Number JumpStart Taq DNA Polymerase NEUE hot start 2 5 U uL 50 U
24. per Software in combination with the Mentype Triplex TVD Template File from Biotype AG Template Files can be received as free downloads from our homepage www biotype de or as CD ROM on request Mentype Triplex TVD December 2007 21 5 1 Controls The Control DNA XY1 of the PCR Amplification Kit and those DNA commonly available from standard cell lines represent the following alleles Table 3 Allele Determinations of Mentype Triplex TVD Control ATCC CCR CCR CCR DNA XY1 K 562 9947A 9948 3657 D21811 27 28 29 30 31 30 30 29 30 28 29 THO1 6 933 9 3 9 3 8 9 3 6 9 3 719 3 vWA 15 18 16 16 17 18 17 17 14 19 For further confirmation the table above displays the alleles of the reference DNA K 562 purchased from ATCC http atcc org Produtcs PurifiedDNA cfm celllines as well as three reference DNA purchased from Coriell Cell Repositories CCR http locus umdnj edu nigms that is up to standard of Szibor et al 2003 5 2 Lengths of Fragments and Alleles Table 4 displays the values for all lengths of fragments of individual alleles that refer to the DNA Size Standard 550 ROX All analyses have been performed on an ABI PRISM 310 Genetic Analyzer with POP 4 polymer Different analysis instruments DNA Size Standards or polymers might result in different lengths of fragment These data could serve as approximate allele sizes for the creation of a new analysis template In order to balance instrument specific deviations fu
25. rther fine tuning of the equipment should be done by measuring sample fragments of known lengths In addition a visual alignment with the Allelic Ladder is recommended Scaling Horizontal 55 285 bp Vertical Depending on signal intensity Mentype Triplex TVD December 2007 QAL Xejdu 200 99 9990 gt Il 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 a 5 Triplex TvD ladder fsa 1Blue ladder c 3000 2000 1000 EEE 25 18 10 6 19 Triplex TYD_XY1 fsa 21 Blue 1500 a 1000 500 15 E 55 550 RO Triplex TvD XY1 fsa 21 Red 600 400 200 Saeed Cep Fig 6 Electropherogram of the Mentype Triplex TVD using 100 pg Control DNA XY1 B and the Allelic Ladder A Analysis was done on an ABI PRISM 310 Genetic Analyzer using the DNA Size Standard 550 ROX Allele assignment was performed using the Genotyper Software and the Mentype Triplex TVD Template File de 23 Table 4 Fragment Lengths of the Allelic Ladder Mentype Triplex TVD analysed on an ABI PRISM 310 Genetic Analyzer Allele THO1 5 co nN OD 10 3 13 3 Size bp ABI 310 6 FAM 72 8 76 6 80 6 85 0 89 3 92 3 93 3 96 3 105 5 108 7 Further Alleles 3 45 5 3 6 15 63 7 3 8 3 91 11 120 14 Allele i i i i L y N c A gt N _ O Size
Download Pdf Manuals
Related Search