Mentype Argus X-12_engl
Contents
1. Mentype Argus X 12 October 2009 Performing a spectral calibration run First of all the parameter file for DyeSetGb must be modified once to achieve successful calibration with the Data Collection software version 1 0 1 or 1 1 Spectral parameter To change settings in the parameter file go to the following path D AppliedBio Support Files Data Collection Support Files CalibrationData Spectral Calibration ParamFiles Select MtxStd Genescan_SetG5 to open the PAR file Change Condition Bounds Range to 1 0 20 0 If calibration was not successful also change Sensitivity to 0 1 and Quality to 0 8 in a second step Select File Save As to save the parameter file under a new name e g MtxStd Genescan_SetG5_Biotype par Always use this parameter file for spectral calibration runs using Biotype matrix standards BT5 Plate Editor for spectral calibration I 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 of the plate Select Spectral Calibration Select 96 Well as plate type and click on Finish Plate Editor for spectral calibration Il Parameter Set up Sample Name Enter name for the matrix samples Dye Set Spectral Run Module Default e g Spect36 POP4 Spectral Parameters MtxStd GeneScan SetG5 Biotype par parameters created before Click into the column header to select2. Forensic Sci Int Genetics Dec 3 1 37 41 Mentype Argus X 12 October 2009
3. Mentype Argus X 12 October 2009 29 30 Figure 8 400 DXS10079 300 200 DX 10146 DX 8378 1 1 100 Fig 8 Electropherogram of the allelic ladder Mentype Argus X 12 analysed on an ABI PRISM 310 Genetic Analyzer Allele assignment was performed using the GeneMapper ID Software and the Mentype Argus X 12 template file Mentype Argus X 12 October 2009 31 Table 4 Fragment lengths of the allelic ladder Mentype Argus X 12 analysed on an ABI PRISM 310 3130 Genetic Analyzer blue panel Marker Allele Size bpj Marker Allele Size bp uod Marker Allele Size bp Fae Amelogenin 6 FAM DXS8378 6 DXS10134 6 FAM x 77 9 158 8 28 295 Y 80 10 162 29 299 11 166 30 303 DXS10103 6 12 170 31 307 31 1 15 109 13 174 32 311 32 1 16 114 14 178 33 315 33 1 17 118 15 182 34 319 18 122 35 324 35 3 19 126 DXS7132 6 36 328 20 130 10 216 37 332 372 973 21 135 11 220 38 336 38 2 12 224 38 3 339 13 228 39 3 343 39 392 14 232 40 3 347 40 15 2 413 351 41 16 240 42 3 355 17 244 43 3 359 443 363 Mentype Argus X 12 October 2009 32 Table 5 Fragment lengths of the allelic ladder Mentype Argus X 12 analysed on an ABI PRISM 310 3130 Genetic Analyzer green panel Marker Allele Size bp DXS10074 BTG 4 104 7 116 8 121 9 125 10 129 11 133 12 137 13 141 14 145 15 149 16 152 16 2 154 17 156 18 160 19 164 20 168 21 172 Further alleles DXS10101 2
4. TTCC s T TCC TTTC CTCCCTTCC ITC TCCC TICTICTITC TCC CTTT CTTC CTTT io T GGAA 4 AAGA 2 AAAG Na AAGG 2 AGAT Reference allele 35 23 26 22 12 is the common repeat structure for variations see NIST and Szibor et al 2009 Table 1 shows the STR loci with their repeat motifs and alleles that are concordant with Allele range 8 20 8 19 1 15 4 21 14 25 24 38 15 21 28 46 1 13 39 2 24 46 2 13 3 38 1 6 19 the International Society for Forensic Genetics ISFG guidelines for the use of microsatellite markers B r et al 1997 Allele ranges include all known alleles of the current literature as at 10 2009 Table 2 Chromosomal mapping of Mentype Argus X 12 Locus Amelogenin X Amelogenin Y DXS7132 DXS7423 DXS8378 DXS10074 DXS10079 DXS10101 DXS10103 DXS10134 DXS10135 DXS10146 DXS10148 HPRTB Chromosomal mapping Xp22 1 22 3 Yp11 2 Xq11 2 Xq28 Xp22 31 Mentype Argus X 12 October 2009 Content Mentype Argus X 12 PCR Amplification Kit 100 reactions 3 0 mL 500 uL 250 uL Nuclease free water Reaction mix A Primer mix Control DNA XX28 2 ng uL DNA Size Standard 550 BTO Allelic ladder Ordering information Mentype Argus X 12 25 Mentype Argus X 12 100 Mentype Argus X 12 400 Storage Reactions Reactions Reactions Cat Cat Cat No 10 uL 50 uL 25 uL 43 13110 0025
5. Template DNA Sometimes the measured value of the DNA concentration varies depending on the used quantification method so that it may necessary to adjust the optimal DNA amount Mentype Argus X 12 October 2009 2 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 For critical stains 100 pg DNA it is recommended to increase the number of PCR cycles to 32 cycles i e from 25 to 27 in the second PCR step Standard method Recommended for all DNA samples Temperature Time 94 4 min hot start for activation of the JumpStart Tag DNA Polymerase 96 C 30 s 63 C 120s 5 cycles 72 C 758 94 30s 60 120s 25 cycles 72 C 758 68 60 10 old Optional Recommended for stains with small amounts of DNA Temperature Time 94 4 min hot start for activation of the JumpStart Tag DNA Polymerase 96 C 30s 63 120s 5 72 758 94 305 60 120s 27 cycles 72 C 758 68 60 10 oo hold Too 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 the risk of cross contamination caused by minimal amounts o
6. 310 Analysis HID 3130 Analysis HID 310 5Orfu Analysis HID 3130 50 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 X12 v0 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 X 12 October 2009 28 6 2 Controls The Control DNA XX28 of the test kit and other commercially available DNA from standard cell lines represent the following alleles Table 3 Allele assignment of Mentype Argus X 12 STR Locus Kontroll DNA ATCC CCR CCR CCR XX28 K 562 9947A 9948 3657 Amelogenin X X X X X X X Y X Y DXS7132 13 14 13713 12 12 13 12 DXS7423 14 15 17 17 14 15 14 13 DXS8378 10 12 10 10 10 11 1 12 DXS10074 18 20 17 17 16 19 18 7 DXS10079 18 21 17 17 20 23 19 19 DXS10101 28 2 28 2 31 31 30 31 32 29 2 DXS10103 16 19 17 17 17 17 18 20 DXS10134 36 38 3 32 32 35 36 34 34 DXS10135 16 30 27 27 211 27 22 25 DXS10146 26 46 2 29 29 28 28 29 27 DXS10148 18 23 1 23 1 23 1 22 1 23 1 23 23 1 HPRTB 12 13 13 13 14 14 14 13 For further confirmation the table above displays the X chromosomal alleles
7. AnySDye Mon May 25 09 21 23 CEST 2008 Fig 5 Electropherogram of spectral calibration with matrix standard BT5 on an ABI 3130 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 the figure above there should be five 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 f calibration was not successful use the optimised values and repeat the calibration run f all capillaries have passed the test the last calibration file for the Dye Set AnybDye is activated automatically in the Spectral Viewer Rename the calibration file e g 5 Date of calibration using the respective button Mentype Argus X 12 October 2009 22 5 2 Sample preparation Component Volume Hi Di Formamide 12 0 uL DNA Size Standard 550 BTO 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 Denaturation for 3 min at 95 C Cool down to 4 C For analysis load the samples on the tray Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted on the plate of multi capillary
8. 43 13110 0100 43 13110 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 Additionally required reagents Additional reagents are needed in order to use the Biotype PCR Amplification Kit Reagent JumpStart Tag DNA Polymerase hot start 2 5 U uL 50 U or 250 U Hi Di Formamide 25 mL Matrix Standards BT5 single capillary instruments 25 Matrix Standards BT5 ABI multi capillary instruments 25 Matrix Standards BT5 multi capillary instruments 50 Supplier Sigma Aldrich Applied Biosystems Biotype AG Biotype AG Biotype AG Mentype Argus X 12 Order number D4184 4311320 00 10411 0025 00 10421 0025 00 10421 0050 October 2009 Warnings and safety instructions The PCR Amplification Kit contains the following potentially hazardous chemicals Kit component Chemical Hazards Primer mix reaction mix Sodium azide NaN Very toxic if swallowed develops toxic and allelic ladder gases when it gets in contact with acids Observe the Material Safety Data Sheets 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 Quality
9. Med 112 192 194 Nei M Roychoudhury AK 1974 Sampling variances of heterozygosity and genetic distance Genetics 76 379 390 Rodig H Kloep F WeiBbach L Augustin C Edelmann J Hering S Szibor R Gotz F Brabetz W Evaluation of 7 X chromosomal Short Tandem Repeat loci located within the Xq26 region Forensic Sci Int Genet doi 10 10167 fsigen 2009 08 010 Szibor R Edelmann J Hering S Plate Wittig H Roewer L Wiegand P Cali F Romano V Michael M 2003a Cell line DNA typing in forensic genetics the necessity of reliable standards Forensic Sci Int 138 37 43 Szibor R Edelmann J Zarrabeitia MT 2003b Sequence structure and population data of the X linked markers 0 57423 and DXS8377 clarification of conflicting statements published by two working groups Forensic Sci Int 134 72 73 Szibor R Krawczak M Hering S Edelmann J Kuhlisch E Krause D 2003c Use of X linked markers for forensic purposes nt J Legal Med 117 67 74 Szibor R Hering S Edelmann J 2006 A new Web site compiling forensic chromosome X research is now online nt J Legal Med 120 4 252 4 Szibor Edelmann J Hering S Gomes Gusmao L 2009 Nomenclatur discrepancies in the HPRTB short tandem repeat Int J Legal Med DOI 10 1007 800414 008 0314 7 Published online 10 January 2009 Tillmar A Mostad P Egeland T Lindblom B Holmlund G Montelius K 2008 Analysis of linkage and linkage disequilibrium for eight X STR markers
10. 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 X 12 October 2009 26 6 Analysis For general instructions on automatic sample analysis refer to the GeneScan or 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 BTO shall thus 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 Fig 6 Electropherogram of the DNA Size Standard 550 fragments with lengths in bp Note The basic template files for the DNA Size Standard 550 BTO has to be
11. a 79 1 kb region at Xp28 Forensic Sci Int Genetics DOI 10 1016 j fsigen 2007 08 001 Gomes Prinz M Pereira Meyers C Mikulasovich RS Amorim A Carracedo A Gusmao L 2007 Genetic analysis of three US population groups using an X chromosomal STR decaplex nt J Legal Med 121 198 203 Hering S Augustin C Edelmann J Heidel M Dressler J Rodig H Kuhlisch E Szibor 2006 DXS10079 DXS10074 and DXS10075 are STRs located within a 280 kb region of Xq12 and provide stable haplotypes useful for solving of complex kinship cases nt J Legal Med 120 6 337 45 Kishida T Wang W Fukuda M Tamaki Y 1997 Duplex PCR of the Y 27H39 and HPRT loci with reference to Japanese population data on the HPRT locus Jon J Legal Med 51 67 69 Krawczak M 2007 Kinship testing with X chromosomal markers Mathematical and statistical issues Forensic Sci Int Genetics Jun 1 2 111 4 Kr ger J Fuhrmann W Lichte KH Steffens C 1968 Zur Verwendung der sauren Erythrocytenphosphatase bei der Vaterschaftsbegutachtung Dtsch Z Gerichtl Med 64 127 146 Mentype Argus X 12 October 2009 40 Machado FB Medina Acosta E 2009 Genetic map of human X linked microsatellites used in forensic practice Forensic Sci Int Genet volume 3 3 202 204 Mertens G Gielis M Mommers N Mularoni A Lamartine J Heylen H Muylle L Vandenberghe A 1999 Mutation of the repeat number of the HPRTB locus and structure of rare intermediate alleles nt J Legal
12. analysers If fewer samples are analysed the empty positions must be filled with 12 uL Hi Di M Formamide To ensure a reliable allelic assignment on multi capillary analysers a number of allelic ladders should be run independently from sample number 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 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 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis Mentype Argus X 12 October 2009 23 5 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 3kV 10s 400bp Paramater Set up Oven Temperature Default Poly Fill Volume Default Current Stability uA Default PreRun Voltage kV Default PreRun Time s Default njection Voltage kV 3 0 njection Time s 10 Voltage Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Voltage kV Default Run Time s 1200 Deviating from the standard settings the injection time may range between 1 an
13. of the reference DNA 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 20030 6 3 Lengths of fragments and alleles Table 4 to table 7 show the fragment lengths of individual alleles that refer to the DNA Size Standard 550 All analyses have been performed on an ABI PRISM 310 3130 Genetic Analyzer with POP 4 polymer 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 5 405 bp Vertical Depending on signal intensity Mentype Argus X 12 October 2009 Figure 7 400 400 400 400 m m 22 gt lt c DXS10079 300 300 300 300 DXS10135 co gt 92 gt lt O DXS10101 T 92 gt lt O 200 200 200 200 N ce Cc N gt lt O 100 100 100 100 2 2 2 2 2 2 2 2 2 2 2 Si Si m Si o Si 6000 4000 4000 Fig 7 Electropherogram of Mentype Argus X 12 using 350 pg male Control DNA XX28 Analysis was performed on an ABI PRISM 3130 Genetic Analyzer Allele assignment was performed using the GeneMapper ID software and the Mentype Argus X 12 template file
14. the entire column select Edit Fill Down to apply the information of the selected samples and confirm with OK Link your reaction plate on the autosampler tray with the created plate ID and start run On completion of the run check in the Spectral Calibration Result dialog box if all capillaries have successfully passed calibration label A If individual capillaries are labelled X refer to ABI PRISM Genetic Analyzer User s Manual Click on OK to confirm completion of the run Mentype Argus X 12 October 2009 Matrix check Select Tools Display Spectral Calibration Dye Set G5 to review the spectral calibration profile for each capillary 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 previously determined range Check the matrix samples for a flat baseline There should be five 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 try to change the Sensitivity and Quality values in the parameter file as described above If all capillaries have passed the calibration the last calibration file for Dye Set G5 must be activated manually under Tools Set Active Spectral Calibration Rename th
15. 15 9 uL Reaction mix A 5 0 uL Primer mix 2 5 uL DNA Polymerase hot start 2 5 U uL 0 6 uL Volume of master mix 24 0 uL contains Mg dNTPs 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 35 ng Control DNA XX28 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 control dilute the Control DNA XX28 to 0 35 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
16. 2 analysed on an ABI PRISM 310 3130 Genetic Analyzer red panel Marker allele Size bp Further alleles Marker allele Size bp Further alleles HPRTB BTR DXS10148 BTR 9 148 8 13 3 207 10 152 18 224 11 156 19 228 12 160 20 232 13 164 21 236 14 168 22 1 241 15 172 23 1 245 23 16 176 24 1 249 24 17 180 25 1 253 26 1 257 27 1 261 28 1 265 29 1 269 30 1 273 31 276 38 1 305 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 Mentype Argus X 12 October 2009 35 7 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 outside the linear detection range 23000 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
17. 2 Sample preparation Component Volume Hi Di Formamide 12 0 uL DNA Size Standard 550 BTO 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 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 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis 3 3 Setting up the GeneScan software Create a Sample Sheet and enter a sample designation Injection list Parameter Set up Module File GS STR POP 4 1 mL G5 Matrix File e g Matrix BTS Size Standard e g SST BTO_60 400bp Injection s 5 Injection kV 15 0 Run kV 15 0 Run C 60 Run Time min 26 Deviating from the 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 X 12 was modified in order to be able to analyse fragments with lengths of up to 400 bp Mentype Argus X 12 October 2009 3 4 Analysis parameter The recommended analysis parameters
18. 4 24 2 25 25 2 26 26 2 27 27 2 14 3 28 28 2 29 29 2 30 30 2 19 3 31 32 32 2 33 33 2 34 34 2 35 Mentype Argus X 12 Marker Allele Size bp BTG 216 218 220 222 224 226 229 231 233 235 237 239 241 243 245 247 249 251 253 255 257 259 261 Further alleles DXS10135 13 14 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 81 32 33 34 35 36 36 372 39 2 October 2009 Marker Allele Size bp BTG 273 276 283 287 291 295 299 303 307 311 315 319 323 327 331 334 338 342 346 348 354 358 362 368 375 Further alleles 15 16 1 17 1 18 1 19 1 20 1 21 1 22 1 23 1 24 1 24 2 25 1 26 1 28 1 29 1 32 1 33 2 34 1 35 1 35 2 33 Table 6 Fragment lengths of the allelic ladder Mentype Argus X 12 analysed on an ABI PRISM 310 3130 Genetic Analyzer yellow panel Marker Allele Size bp E Marker Allele Size bp hes Marker Allele Size bp DXS7423 BTY DXS10146 BTY DXS10079 BTY 13 154 12 24 188 14 290 14 158 25 192 15 294 15 162 26 196 16 298 16 166 27 199 273 17 302 17 170 28 203 18 306 18 174 29 207 19 310 30 211 20 314 31 215 21 318 32 219 22 322 32 2 221 23 326 33 223 25 333 332 225 34 227 342 229 35 2 233 39 2 248 40 2 252 412 256 42 2 260 43 2 264 442 267 45 2 271 46 2 275 Mentype Argus X 12 October 2009 34 Table 7 Fragment lengths of the allelic ladder Mentype Argus X 1
19. Mentype Argus X 12 pcr ampitication Kit Product description The Mentype Argus X 12 PCR Amplification Kit is a multiplex application for twelve X chromosomal Short Tandem Repeat STR loci that supplements the Mentype Argus and Mentype Argus Y 12 5 test kit for kinship and paternity testing especially in complicated deficiency cases Furthermore the use of gonosomal STRs for the analysis of DNA traces in the forensic field is strongly rising The Mentype Argus X 12 contains the primers of Amelogenin AM for gender determination DXS7132 DXS7423 DXS8378 DXS10074 DXS10079 DXS10101 DXS10103 DXS10134 DXS10135 DXS10146 DXS10148 and HPRTB Three markers belong to one of the four coupling groups of the X chromosome Fig 1 so that three markers of each group have to be handled as haplotype for genotyping The primers are fluorescence labelled with 6 FAM BTG BTR and BTY Primer sequences of the Mentype Argus X 12 are different to that of the Mentype Argus X 8 in order to avoid allelic overlapping Furthermore the changing results in more robustness and sensitivity Because of the new primer design for the locus HPRTB a dinucleotide deletion AG 48 49 bp downstream of the repeat unit Mertens et al 1999 Gomes et al 2007 will not be detected any more so that just the real repeat units will be shown If necessary point alleles like 11 2 12 2 that were detected by the primers of the Mentype Argus X 8 or Argus X UL s
20. STR motives caused by slippage effects of the Taq DNA Polymerase Interpretation of those peaks should be done 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 Artefacts 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 Mentype Argus X 12 October 2009 36 8 Usage of the X chromosomal STRs and their characteristics In principle the guidelines of the ISFG www isfg org are valid for working with X chromosomal STRs too However the following special characteristics should be pointed out Typing of X chromosomal markers is only permissible when the phenotypic and genetic sex is identical The genetic sex can easily be determined by the analysis of the Amelogenin locus If the p
21. V daughter Here the MEC values according to the formula of Kr ger et al 1968 shall be applied whereas only unlinked X STR markers of the Mentype Argus X 12 can be used For markers which are in coupling equilibrium haplotype frequencies must be involved for calculation Siblings and distant relationships The X chromosomal analysis without parental samples can not be used as relationship test in the following cases Different putative fathers could be father of the child Half brothers with same father and different mothers Brothers and half brothers with same mother statistically they have 50 of the characteristics in common thus the test is comparable to a test with autosomal markers Whenever distant relationship should be analysed genotyping of X chromosomal markers is very useful because the usage of coupled markers allows ChrX haplotyping Three markers of each linkage group from Mentype Argus X 12 have to be handled as haplotype for genotyping For further details see Szibor et al 2003c for calculation see the online data base ChrX STR org http www chrx str org Usage of X chromosomal markers for forensic traces In most cases the use of autosomal markers for profiling is much better because all autosomal loci have two alleles Whenever female DNA traces have to be analysed in a male background e g female traces under nails of a man X chromosomal markers are advantaged The likelihood that one all
22. adjusted to 400 bp within the GeneMapper ID software The new template could be saved as e g SST BTO_60 400bp and used for further analyses Special feature Primer sequences of the Mentype Argus X 12 are different to that of the Mentype Argus X 8 in order to avoid allelic overlapping Furthermore the changings result in more robustness and sensitivity Because of the new primer design for the locus HPRTB a dinucleotide deletion AG 48 49 bp downstream of the repeat unit Mertens et al 1999 Gomes et al 2007 will not be detected any more so that just the real repeat units will be shown If necessary point alleles like 11 2 12 2 that were detected by the primers of the Mentype Argus X 8 or Argus X UL should be rounded up the next full allele for comparing the results generated with the Mentype Argus X 12 primers Mentype Argus X 12 October 2009 27 6 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 X 12 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 60 500bp adjust up 4000 adjustment described before Analysis Method Analysis HID
23. are Analysis Range Start 2000 Stop 10000 Data Processing Baseline Checked Multicomponent Checked Smooth Options Light Peak Detection Peak Amplitude Thresholds B XS G R 0 Min Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Size Call Range Min 60 Max 550 Size Calling Method Local Southern Method Split Peak Correction 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 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 X 12 October 2009 4 Electrophoresis using the ABI PRISM 3100 Avant 3100 Genetic Analyzer For detailed instructions on instrument setup spectral calibration or 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 named ABI 3100 Avant and the system with 16 capillaries is named ABI 3100 The virtual filter set 65 shall be used for combined appl
24. assurance All kit components undergo an intensive quality assurance process at Biotype AG The quality of the test kits is permanently monitored in order to ensure unrestricted usability If you have any questions regarding quality assurance please do not hesitate to contact us 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 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 Mentype Argus X 12 October 2009 Protocols for PCR amplification electrophoresis and analysis 2 PCR amplification 2 1 Master mix preparation The table below shows the volumes of all PCR reagents per 25 uL reaction volume including a sample volume of 1 0 uL template 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 Component Volume Nuclease free water
25. d 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 X 12 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 3kV 10s 400bp 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 Set up Name Run36 POP4 BT5 20min Type REGULAR Run Module 3kV 10s 400bp Dye Set AnysDye parameter see above Click on OK to exit the Protocol Editor Mentype Argus X 12 October 2009 24 Prior to each run it is necessary to create a 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 Set up Name e g Plate BT5 Date Application Select GeneMapper Application Plate Type 96 Well Owner Name Operator Name Click on OK A new table in the Plate Editor opens automatically GeneMapper Plate Editor II Parameter Set
26. e calibration file under Set Matrix Name e g BT5 Date of calibration 4 2 Sample preparation Component Volume Hi Di Formamide 12 0 uL DNA Size Standard 550 BTO 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 Denaturation for 3 min at 95 C Cool down to 4 C For analysis load the samples on the tray Since injections take place simultaneously on all capillaries 4 or 16 samples must be pipetted on the plate of multi capillary analysers If fewer samples are analysed the empty positions must be filled with 12 uL Hi Di M Formamide To ensure a reliable allelic assignment on multi capillary analysers a number of allelic ladders should be run independently from sample number 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 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 BTO to peak heights of about 500 relative fluorescent units RFU Purify the PCR products before starting the analysis Mentype Argus X 12 October 2009 4 3 Setting up the GeneScan software Edit the default run module in Dye Set G5 once for the first run Select Module Edito
27. ect GeneScan Select 96 Well as plate type and click on Finish Mentype Argus X 12 October 2009 Plate Editor Parameter Set up Sample Name Enter name for the samples Dyes 0 Colour Info Ladder or sample Project Name e g 3100 Project Dye Set G5 Run Module 3kV 10s 400bp Analysis Module 1 DefaultAnalysis gsp parameter see above Complete the table in the Plate Editor and click on OK Click into the column header to select the entire column and select Edit Fill Down to apply the information of the selected samples Link your reaction plate on the autosampler tray with the created plate ID and start the run On completion of the run view data as Color Data in Array View of the 3100 Data Collection software or as Analyzed Sample Files under D AppliedBio 3100 DataExtractor ExtractRuns 4 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 We G 0 Min Peak Half Width 2 pts Polynominal Degree 3 Peak Window Size 11 pts Size Call Range Min 60 Max 550 Size Calling Method Local Southern Method Split Peak Correction 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 a
28. ele of the trace is covered by the male background is only half in comparison to autosomal tests Note International DNA databases usually do not include markers of the X chromosome Mentype Argus X 12 October 2009 39 9 References Bar 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 use of short tandem repeat systems Forensic Sci Int 87 181 184 Becker D Rodig Augustin C Edelmann G tz Hering S Szibor Brabetz W 2008 Population genetic evaluation of eight X chromosomal short tandem repeat loci using Mentype Argus X 8 PCR amplification kit Forensic Sci Int Genetics 2 69 74 Botstein D White RI Skolnick M Davis RW 1980 Construction of a genetic linkage map in man using restriction fragment length polymorphisms Am J Hum Genet 32 314 331 Desmarais D Zhong Y Chakraborty R Perreault C Busque L 1998 Development of a highly polymorphic STR marker for identity testing purposes at the human androgen receptor gene HUMARA J Forensic Sci 43 1046 1049 Edelmann J Hering S Michael M Lessig R Deichsler D Meier Sundhausen Roewer L Plate 1 Szibor 2001 16 X chromosome STR loci frequency data from a german population Forensic Sci Int 124 215 218 Edelmann J Hering S Augustin C Szibor R 2007 Characterisation of STR markers DXS10146 DXS10134 and DXS10147 located within
29. f impurities Mentype Argus X 12 October 2009 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 refer to the ABI PRISM 310 Genetic Analyzer User s Manual Electrophoresis using the GeneScan software is described below The virtual filter set G5 shall be used for combined application of the five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 47 cm 50 um green Polymer 4 for 310 Genetic Analyzer Buffer 10x Genetic Analyzer Buffer with EDTA 3 1 Matrix generation Prior to conducting DNA fragment size analysis with the filter set G5 a matrix with the five fluorescent labels 6 FAM BTG BTY BTR and BTO must be generated Colour Matrix standard Blue B 6 FAM Green G BIG Yellow Y BTY Red R BIR Orange 0 BTO Five electrophoresis runs shall be conducted one for each fluorescent label 6 FAM BTG BTY BTR and BTO under the same conditions as for the samples and allelic ladders of the Biotype test kit to generate suitable matrix files 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 p Ma
30. 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 X 12 October 2009 20 Setting up the spectral calibration standards Example for 4 capillaries ABI 3130 Component Volume Hi Di Formamide 60 0 uL Matrix standard 5 5 0 uL Load 12 uL of the mix to a 96 well reaction plate e g position A1 D1 Denaturation for 3 min at 95 C Cool down to 4 C Example for 16 capillaries ABl 3130xl Component Volume Hi Di Formamide 204 0 uL Matrix standard BT5 17 0 uL Load 12 uL of the mix to a 96 well reaction plate e g position A1 H1 and A2 H2 Denaturation for 3 min at 95 C Cool down to 4 C Performing a spectral calibration run Place the 96 well plate on the autosampler tray In the Protocol Manager of the Data Collection software click on New in Instrument Protocol to open the Protocol Editor dialog box Instrument Protocol for spectral calibration Protocol Editor Set up Name User e g Spectral36_POP4_BT5 Type SPECTRAL Dye Set AnybDye Polymer User e g POP4 Array Length User e g 36cm Chemistry Ma
31. henotypic sex differs from the genotypic sex results from ChrX typing could not be used Following the national laws this information has to be handled strictly confidential and should only be forwarded to the analysed person on request Generation of X chromosomal profiles for kinship testing should be taken into account whenever autosomal markers do not give reliable or unequivocal results mainly in deficiency cases In some instances e g for complex kinship and paternity testing markers located on the sex chromosomes may turn out to be more informative than their autosomal counterparts and the use of sex chromosomal markers may therefore substantially improve the efficiency of a kinship test This arises from the fact that the mean exclusion chance MEC of a X chromosomal marker for calculation see below is consistently larger than that of an autosomal marker with the same allele frequencies Calculation of population genetic data The Forensic ChrX Research Group initiated the online data base ChrX STR org http www chrx str org that calculates population genetic data on the basis of X chromosmal allele frequencies Szibor et al 2006 The formula for calculation of the Polymorphism Information Content PIC was published by Botstein et al 1980 the one for the Expected Heterocygosity HET by Nei and Roychoudhury 1974 Both can be used for autosomal or X chromosomal marker Kr ger et al 1968 introduced the fo
32. hould be rounded up the next full allele for comparing the results generated with the Mentype Argus X 12 primers The detection limit of the Mentype Argus X 12 PCR Amplification Kit is 100 pg genomic DNA The optimal range under standard conditions is 0 2 0 5 ng DNA However internal validations demonstrated reliable results with 0 1 ng DNA The Forensic ChrX Research Group initiated the online data base ChrX STR org http www chrx str org that calculates population genetic data on basis of X chromosmal allele frequencies Szibor et al 2006 Additionally all important population genetic data can 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 Ep22 33 Amelogenin X bae DXS10148 9 Mb linkage group 1 Spei DXS10135 22 2 0 58378 22 LS 22 12 EpZ2 11 spB21 3 sp21 2 mp21 1 Beli t 11 5 11 25 11 22 411 1 11 2 DXS7132 bale DXS10079 66 Mb 4413 1 DXS10074 m413 2 5 321 1 Mazi linkage group 2 eqz1 31 921 32 sqzi 33 sq 1 ai EQq223 E324 linkage group 3 Ha26 1 DXS10103 nazen HPRTB 133 Mb E425 3 427 1 DXS10101 mqi 4327 3 DXS10146 mare DXS10134 149 Mb linkage group 4 DXS7423 A425 Fig 1 The ideogram of the X chromosome describes the physical l
33. ication of the five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 36 cm Capillary Array for 3100 Avant 3100 Polymer POP 4 Polymer for 3100 Buffer 10x Genetic Analyzer Buffer with EDTA 4 1 Spectral calibration matrix generation Proper spectral calibration is critical to evaluate multicolour systems with the ABI PRISM 3100 Avant 3100 Genetic Analyzer and shall be done prior to 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 Preparation of the spectral calibration standards Loading the standards to the 96 well reaction plate one sample per capillary Entering the plate composition Performing a spectral calibration run and checking the matrix Setting up the spectral calibration standards Example for 4 capillaries ABI 3100 Avant Component Volume Hi Di Formamide 60 0 uL Matrix standard 5 5 0 uL Load 12 uL of the mix to a 96 well reaction plate e g position A1 D1 Denaturation for 3 min at 95 C Cool down to 4 C Example for 16 capillaries ABI 3100 Component Volume Hi Di Formamide 204 0 uL Matrix standard 5 17 0 uL Load 12 uL of the mix to a 96 well reaction plate e g position A1 H1 and A2 H2 Denaturation for 3 min at 95 C Cool down to 4 C
34. is parameter analysis method 25 RM 26 6 1 Biotype template TSS ro etr Bre cr bete e re 27 a d po ei a S aita ae eb cec UA Ga e iot 28 6 3 Lengths of fragments and 28 7 Interpretation of results eden eda b oe tede 35 8 Usage of the X chromosomal STRs and their characteristics 36 SEITE OD ard dea edt aate ne ssl ced Poele oe Esa 39 Mentype Argus X 12 October 2009 1 Description of Mentype Argus X 12 Table 1 Locus specific information of Mentype Argus X 12 Locus Amelogenin X Amelogenin Y DXS7132 DXS7423 DXS8378 DXS10074 DXS10079 DXS10101 DXS10103 DXS10134 DXS10135 DXS10146 DXS10148 HPRTB GenBank accession M55418 M55419 G08111 AC109994 G08098 AL356358 AL049564 AC004383 BV680555 AL034384 AC003684 034384 684 26434 Repeat motif of the reference allele TCTA is TCCA TCTGTCCT TCCA o AAGA 4 TGAAAGAG AGAG AAAG GAAAGAAG GAAA GAAA AAGA AMAG AAAAAGAA AAAG s CTGA CAGA TAGA GAAA s GAGA GAA AA GAGA GAAA GAGA GACAGA GAMA s GAAA AAA GAAA 5 TAAGA GAAAG GAMA zo
35. nd 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 X 12 October 2009 5 Electrophoresis using the ABI PRISM 3130 3130 1 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 3130xI Genetic Analyzers Getting Started Guide The system with 4 capillaries is named ABI 3130 and the system with 16 capillaries is named ABI 3130xl The virtual filter set Any5Dye shall be used for the combined application of the five fluorescent labels 6 FAM BTG BTY BTR and BTO the matrix standard will be called BT5 hereinafter Material Capillary 36 cm Capillary Array for 3130 83130xl Polymer POP 4 Polymer for 3130 Buffer 10x Genetic Analyzer Buffer with EDTA 5 1 Spectral calibration matrix generation Prior to conducting DNA fragment size analysis it is necessary to perform a spectral calibration with the five 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
36. ocalisation of the STR loci which can be analysed with Mentype Argus X 12 Distances from the p telomere are shown in Mb http www ncbi nlm nih gov genome guide human as at 10 2009 Mentype Argus X 12 October 2009 Content 1 Description of Mentype n o 4 2 POSITI TOR DIOE ddr spa eU AAR rns 7 2 1 Master mix preparation 7 2 2 PCR amplification parameter P Pn v rs eoa 8 3 Electrophoresis using the ABI PRISM 310 Genetic Analyzer 9 De M trix CG GLAM det ot teer tede ent 9 3 2 Sample preparation 12 3 3 Setting up the GeneScan software ss 12 3 4 Analysis soa ate br sedes Pista prr a cdi d vereda e rte 13 4 Electrophoresis using the ABI PRISM 3100 Avant 3100 Genetic Analyzer 14 4 1 Spectral calibration matrix generation 14 A Sample a t oet d EON On ee P Eoo d 16 4 3 Setting up the GeneScan software u s 17 4 4 Analysis parameter 18 5 Electrophoresis using the ABI PRISM 3130 3130xl Genetic Analyzer 19 5 1 Spectral calibration matrix generation 19 5 2 Sample Eres Pas bentes 22 5 3 Setting up the GeneMapper ID software 23 5 4 Analys
37. pe AG Linkage groups In contrast to ealier findings Szibor 20030 recent studies showed Tillmar et al 2008 Machado 2009 that markers of linkage groups three HPRTB DXS10101 and four DXS10134 DXS7423 cannot be regarded as unlinked The possibility of linkage between these markers should be considered in calculation and interpretation of relationship tests in deficiency cases If necessary both probabilities should be calculated from the linkage groups 1 2 3 and linkage groups 1 2 4 For use and calculation of X chromosomal markers see Krawczak M 2007 and http inkage rockefeller edu Kinship tests and paternity cases with ChrX STRs Note X chromosomal markers are particularly informative for inferring parent offspring relationships that involve at least one female i e mother daughter mother son and father daughter duos For paternity cases only testing between father and daughter is possible since fathers leave their only X chromosome to their daughter and sons get the X chromosome from their mother Mentype Argus X 12 October 2009 38 Deficiency cases If the putative father PV descends from the putative grandmother PGM without any doubt his X chromosomal characteristics originate from the PGM Therefore she PGM can be analysed X chromosomally instead of him PV The calculation of the PGM likelihood with X chromosomal markers PGM daughter may be performed like the calculation with autosomal markers P
38. r to open the dialog box Select the appropriate Run Module as template from the GeneScan table Modify the Injection Voltage to 3 kV and the Injection Time to 10 s Run Module 3kV 10s 400bp Parameter Set up Run Temperature 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 njection Voltage kV 3 0 njection Time s 10 Run Voltage kV Default Number of Steps Default Voltage Step Interval Default Data Delay Time s Default Run Time min 20 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 X 12 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 3kV 10s 400bp 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 Run the ABI PRISM 3100 Data Collection software In Plate View click on New to open the Plate Editor dialog box Enter a name of the plate Sel
39. rmula for the Mean Exclusion Chance MECxriiger which was developed for either autosomal marker or for trios With exception of deficient cases MECxjiiger is not suitable for X chromosomal marker Here the paternal grandmother can be analysed instead of the putative father Kishida et al 1997 devised the MECkxishiga for X chromosomal marker in consideration of trios including a daughter In comparison with MEOxy ige MECxishida more complex which highlights the fact that in trios involving a daughter X STRs are more efficient then autosomal markers Finally Desmarais et al 1998 introduced formulae for the Mean Exclusion Chance of ChrX markers in trios involving daughters as well as in father daughter duos without information about the maternal genotype MECpesmarais is equivalent to MECxishida whereas MECpesmarais Duo also be used for maternity testing of mother son duos The formula for calculation of the Power of Discrimination was published by Desmarais et al 1998 Mentype Argus X 12 October 2009 37 PIc21 Xf 2Y Y fr i 1 1 1 1 5 1 jal MEC tiger 2 f 1 f Dif 1 FP Dig fff f f 1 6 6 MECkishida i f Lt Zep f Lig fi fj f 1 1 fj MECpesmarais 1 i fe 2 f Zig for MECossaaspu m Xf PDiemae 1 2 X fi 9 fj 1 2i f All important population genetic data could be calculated with the GenoProof software from Quality
40. t value 3200 end value 5500 Calculate the difference e g 5500 3200 2300 data points Mentype Argus X 12 October 2009 Generation of a new matrix File New Matrix x Select the Matrix Standard Sample Files Number Df Dyes 5 v B 6FAM_080204 fsa Stat t 5200 G BTG_080204 fsa Stat t 5000 y BTY_080204 fsa Stat t 3000 R BTR 080204 Stat At 2000 O BTO 080204a Stat t 3000 Points 2300 Fig 3 Matrix sample selection Import matrix samples for all dyes B Y Enter a Start At value e g 3200 Enter the calculated difference under Points e g 2300 Click on OK to calculate the new matrix Matrix BT5 mtx Reactions B G Y R 0 osso 10000 o2056 259 00017 Jo 4se7 10000 0 0029 o 9a44 03619 05311 10000 00095 o 150 o0477 2082 10000 Fig 4 New matrix BT5 o I lt Save the matrix the matrix folder File Save as e g Matrix BT5 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 O with the new matrix Mentype Argus X 12 October 2009 3
41. trix Standard Run Module Default e g Spect36 POP4 1 Depends on the type of polymer and length of capillary used Click on OK to leave the Protocol Editor dialog box In the Plate Manager of the Data Collection software click on New to open the New Plate Dialog box Plate Editor for spectral calibration 1 New Plate Dialog Set up Name e g Spectral BT5 date Application Spectral Calibration Plate Type 96 Well Owner Name Operator Name Click on OK A new table in the Plate Editor will open automatically Mentype Argus X 12 October 2009 21 Plate Editor for spectral calibration Il Parameter Set up Sample Name Enter name for the matrix samples Priority e g 100 Instrument Protocol 1 Spectral36_POP4_BT5 setting described before 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 GA Instruments ga3130 3130 1 Spectral Viewer 3000 Intensity vs Scan Number Capillary Data Mon May 25 09 21 23 CEST 2009 0 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
42. trix standard BTY 1 0 uL Matrix sample 4 Hi Di Formamide 12 0 uL p Matrix standard BTR 1 0 uL Matrix sample 5 Hi Di Formamide 12 0 uL R Matrix standard BTO 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 a sample designation Mentype Argus X 12 October 2009 Injection list for matrix generation Parameter Set up Module File GS STR POP 4 1 mL G5 Matrix File NONE Size Standard NONE Injection 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 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 1000 4000 Y axis for each matrix sample optimal range 2000 4000 1 1 3200 Data Points X a T Y bf Fig 2 Electropherogram with raw data of the matrix standard 6 FAM Select an analysis range with flat baseline and re inject the matrix sample if necessary Note down start and end value data points of the analysis range e g star
43. up Sample Name Enter a name of the samples Priority e g 100 Default Sample Type Sample or allelic ladder Size Standard e g SST BTO 60 400bp Panel e g X12 Panels v0 Analysis Method e g Analysis HID 3130 Snp Set User defined 1 3 Results Group 1 select results group Instrument Protocol 1 Run36 POPA BT5 20min setting described before 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 n 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 X 12 October 2009 25 5 4 Analysis parameter analysis method The recommended settings in the worksheet the 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 Size Calling Method Local Southern Method Peak Detection Peak Amplitude Thresholds Bite G R 0 Min Peak Half
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