COrDIS Plus - PCRdiagnostics.eu
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1. LC20 3_B01_002 3 fsa LC20 3 COrbIs 20 L L D351358 H01 D125391 D1S1656 1051248 D2251045 100 200 30 400 1600 1400 1200 1000 800 600 400 200 0 a neee nas 2E se EH Ey eebe b SEE boy psfie Del bol 22ks E3 mienku mema bs bo bs bg ba fas pes pals bs fig 17 3 LC20 3_B01_002 3 fsa Lc20 3 cOrbis 20 i i 25441 75820 135317 GA 100 200 300 400 3000 2000 1000 o L ud L L pe Oe NS A eee i AN AAAA lehoina is SEEEN LE apum hs i helo A212 afoefas 2 27 fs kalsa E 3 u pd ha fas ofeo af22f23 25 TE mapanas as LC20 3_B01_002 3 fsa Lc20 3 COrDIS 20 w i POX 18551 Di 65539 851179 CSF1PO 55818 100 200 300 400 2000 1e007 1600 1400 1200 1000 600 600 4007 200 o2 a rt IILI aeaea Lo I Cs ai I aa aama AELE Heaks bisak bal Ed ainm Pho fa hs fs Pho fal hs GE be bans mamak De Bebe bi e bs by fy LC20 3_B01_002 3 fsa Lce20 3 coris 20 E E AAA D21511 5E33 100 200 wo 400 1600 1400 1200 1000 600 600 400 200 o eeddlal T LH M idi L i AMAA AMi A ULALA NA ALE LA NAULA I eleko babal shel Del Boe eleki E she fhe faofaafaa alfos 2 fos a fee afs0fz 2 2 26 E E fa e u W By fs E 63 3 b bd bed ed eaea Ea Bo Bafs ma Bsa Figure 3 COrDIS Plus Allelic Ladder The analysis was performed at ABI PRISM Genetic Anal2. Hi DiTM Formamide 160 ul Dissolved MXS 5 16 ul Add 10 ul of the mixture per well to a 96 well microplate at positions AO1 HO1 and A02 H02 Spin briefly the microplate to remove all bubbles from the bottom of the wells Spectral calibration After correct plate assembly place the 96 well microplate with denatured matrix standards onto autosampler Step A Creating a Spectral Instrument Protocol Open Protocol Manager of the Data Collection Software Go to Instrument Protocol and click New for opening of the Protocol Editor Select the following parameters Instrument Protocol Protocol Editor Name e g Spectral36_POP4_MXS5 Type SPECTRAL Dye Set any5 Polymer POP4 Array Length 36 Chemistry Matrix Standard Run Module Spect36_POP4_1 Click OK and close Protocol Editor Step B Creating the Plate Record Go to Plate Manager of the Data Collection Software and click New the Plate Dialog window opens Plate Editor for Spectral Calibration New Plate Dialog Name e g Spectral_anyS_MXS_date Page 9 of 18 COrDIS Pluseee s User manual Application Spectral Calibration Plate Type 96 Well Owner Name Operator Name Click OK a new table of the Plate Editor opens Plate Editor Add to position AOI Sample Name Name of Matrix Probes Priority e g 100 Instrument Protocol Spectral36_POP4_MXSS5 as specified earlier Highlight the entire well AO1 and select in the Edit window Fill Down Special The software fills the a
3. High amounts of DNA in reaction setup are also reason for occurrence of Split Peaks A portion of amplification product is 1 base pair smaller than the main peak The reason is insufficient adenylation by polymerase so that not all amplification products become an Adenosine added to its 3 end 7 References Bar W Brinkmann B Budowle B Carracedo A Gill P Lincoln P Mayr W and Olaisen B 1997 DNA recommendations Further report of the DNA Commission of the ISFG regarding the use of short tandem repeat systems Int J Legal Med 110 175 176 Coble M D and Butler J M 2005 Characterization of new miniSTR loci to aid analysis of degraded DNA J Forensic Sci 50 43 53 Gill P Fereday L Morling N Schneider P M 2006 The evolution of DNA databases recommendations for new European loci Forensic Sci Int 156 242 244 Gill P Fereday L Morling N and Schneider P M 2006 Letter to the Editor New multiplexes for Europe Amendments and clarification of strategic development Forensic Sci Int 163 155 157 Hill C R Kline M C Coble M D Butlerm J M 2008 Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples J Forensic Sci 53 1 73 80 Page 17 of 18 COrDIS Pluseee s User manual Huckenbeck W Kuntze K and Scheil H G 1997 The distribution of the human DNA PCR polymorphisms Verlag Dr K ster 1 Auflage Berlin ISBN 3 89574 300 3 see
4. Material not provided with the Kit but supplied by GORDIZ Matrix Standard MXS_5 for ABI 3130 and ABI 3500 MXS_3130 Matrix Standard Set MXS_1I for ABI 310 please inquire Genotyper Macro or GeneMapper Macro please inquire Page 5 of 18 COrDIS Pluseee s User manual Supplemental Material not provided with the Kit to be supplied by the user Chemical Company Order Number Hi Di Formamide Applied Biosystems P N 4311320 10 x Genetic Analyzer Buffer Applied Biosystems P N 402824 Polymer POP 4 Applied Biosystems P N 402838 2 Solubilization of dried Components 2 1 Allelic Ladder Immediately after delivery the tube with allelic ladder should be separated from other kit components and stored protected from light in a separate place post amplification area of your lab To get a working solution add 20 ul of deionised water provided with this kit to the tube with dried allelic ladder yellow cap mix thoroughly and spin down for a few seconds Once the allelic ladder is suspended it should be stored in the dark at 4 C Do not freeze repeatedly For electrophoresis 1 ul of ladder has to be added to appropriate formamide sizing standard mixture 2 2 Control DNA Add 20 ul of deionised water provided with this kit to the tube with dried control DNA green cap mix thoroughly and spin down for a few seconds For PCR reaction setup 1 ul of control DNA has to be added to the reaction tube This amount corresponds to 500 pg
5. 10 ul of the cocktail to each well of the test plate Then add 1 ul of PCR product or Allelic Ladder Finally cover the plate with a septa pad according to the user s manual for the ABI PRISM Genetic Analyzer No temperature denaturation is needed for COrDIS Plus Centrifuge the plate briefly to remove any remaining air bubbles Place the sample plate onto analysis tray and start the run Page 11 of 18 COrDIS Pluseee s User manual 4 5 Starting run For correct instrument preparation and starting run please refer to the instrument user s manual For optimal performance the correct setup of Matrix see section 4 1 Run Module see section 4 2 and Instrument Protocol see section 4 3 must be used Step A Open Plate Editor Before starting a run each samples to be analyzed must be assigned to a position at an analysis plate Go to Plate Manager of the Data Collection Software and click New A new Plate Dialog opens New Plate Dialog Name e g COrDIS_date Application choose GeneMapper Application Plate Type 96 Well Owner Name Operator Name Click OK and a new Table at the Plate Editor opens Step B Table Settings Sample Name Name of the sample Priority usually 100 lower numbers will be analyzed first Sample Type Sample Allelic Ladder Positive Control Neg Control Size Standard S450 Panel COrDIS_Panels Analysis Method e g Analysis_HID_3130 User defined 1 3 SNP Set Results Group choose appropriate Resu
6. 12p13 31 TCTA TCTG 4 TCTA 3 Amelogenin X M55418 X Xp22 1 22 3 Amelogenin Y M55419 Y Yp11 2 In Table 1 the information regarding STR loci of the COrDIS Plus kit is summarized The Repeating Unit designation of the reference sequences was performed according to the recommendations of the International Society for Forensic Genetics ISFG B r et al 1997 Amelogenin is not an STR but displays specific PCR products for X and Y chromosomes 1 2 Ordering Information and Kit Components This kit can be obtained either as strips 8 reactions with single caps or micro plates 96 reactions e 8x 0 2 ml strips each reaction tube with single cap Product CO DIS Plus 25 ul format e 96x 0 2 ml plate Product CO DIS Plus 25 ul format Size 12 x 8 reactions 96 rxn Size 1 x 96 reactions 96 rxn Order Number C20 108S Order Number C20 196P Page 4 of 18 COrDIS PluSeee s User manual Kit Components e g 12 x 8 reactions 12 strips each with 8 x 0 2 ml reaction tubes each with single cap Activation Solution blue lid 0 5 ml Deionised Water white lid 2x1 7 ml Allelic Ladder dried red lid 1 tube 20 Applications Control DNA dried green lid 1 tube 40 Applications Sizing Standard S450 dried orange label 1 tube 120 Applications 1 3 Storage Conditions The kit contains all necessary chemical components for successful PCR including polymerase Reaction mix is aliquoted and supplied in dry form T
7. COrDIS PluSece e User manual COrDIS Plus Multiplex analysis of 19 STR loci plus Amelogenin User manual Page 1 of 18 COrDIS Pluseee s User manual Table of Content 1 Product IfOrMatiOn ois sscesccccaiessceseiacasesscaieaaudsouneasssucatcaucdevewcasessseexescaie 3 t 1 Prod ct Deseripuon ccein ennienni nii a aiias 3 1 2 Ordering Information and Kit Components sssseessssssrreerreresserrrrerrrrrrreee 5 1 3 Storage Conditi NS isser ene anr E EE EEE ORE EEEE EE EEE E EE EEEE 5 14 Supplem ntal Maternal scssssecircetgsci horer riene ri Sonis tiaa E RNE EEEE E RAEE E EAEE TRER 6 2 Solubilization of dried Components sccsscccssccsccccccccssccccccesscessccessees 7 2 1 Allele Ladder areri e E E E eases een ania eee ee 7 220 Contro DNA iai et Serer AEAEE E TEER A ERE EE 7 2 3 The Sizing Standard S450 isiccisssesciaeecavsncsaraiveassbecaieecavsued ancedeavenaradieeers 7 3 PCR Amplificati sssserssssessriseressserors erases tkurr eras oiTa rE ES EU EEEE 8 3 1 R action Setpro na A RETE R E AEE OEE AAA EAEE 8 3 2 Amplification Parameters sssessereessersssssesereeeesreerssssessoreeesersssseesersess 8 4 Electrophoresis on ABI PRISM 3130 3130x1 Genetic Analyzer ssssooeoesoe 9 4 1 Generation of a Matrix Spectral Calibration cc ecee ence eee eee eee eeneeenees 9 AD R n Conditions j 2icceiadieesticeeiene deciedcncade innia a A a EEES 12 4 3 Create an Instrument PROLOG ici sascesaeea
8. K1 DNA Label 81 84 93 147 152 196 204 264 275 324 338 390 400 440 84 134 137 181 188 236 238 391 66 113 124 200 208 256 262 314 317 361 369 413 94 155 158 228 233 403 X X 15 17 6 9 3 21 23 14 17 3 15 15 15 15 14 14 10 12 11 11 20 22 2 8 9 14 16 12 13 10 10 9 11 9 12 16 18 30 2 30 2 242 292 blue blue blue blue blue blue blue blue green green green green yellow yellow yellow yellow yellow yellow red red red Table 2 The allele range of STRs and the expected size ranges are shown for each dye panel Page 14 of 18 COrDI S Pluse eo e User manual 5 3 Amplification of Control DNA e sf 3 3 3 o a ts i WD io ic F N pa i e e 8 8 By in A a I iS lt gt in e 2 2 a by a at el T 4 A s al he z e or 2 F e gt c 2 ece oo c gt S a 8 a amp 5 a Figure 2 COrDIS Plus A template of 500 pg Control DNA was amplified The analysis was performed at ABI PRISM 3130 Genetic Analyzer by using the Size Standard S450 For allele designation the GeneMapper ID Software in combination with COrDIS Plus template file were used The expected alleles of the Control DNA are shown in Table 2 at section 5 2 Page 15 of 18 COrDIS Pluseee s 5 4 User manual Allelic Ladder
9. also web based references Web based references on STR loci http www cstl nist gov biotech strbase www uni duesseldorf de www MedFak Serology database html population data Trade Marks and Patents ABI PRISM GeneScan Genotyper GeneMapper and Applied Biosystems are registered trademarks of Applied Biosystems Inc POP 4 and HiDi Formamide are trademarks of Applied Biosystems Inc GeneAmp is a registered trademark of Roche Molecular Systems and F Hoffmann La Roche GeneBank is a registered trademark of the National Institute of Health Page 18 of 18
10. as well as in all European countries using ESS European Standard Set markers including those with SE33 as database marker Due to the highest discrimination power among all commercial kits available it is ideally suited for forensic applications paternity or parentage testing and complex relationship testing The development and validation of this kit was performed with GeneAmp 9700 Thermocycler and ABI PRISM 3130 3130XL Genetic Analyzer Page 3 of 18 COrDIS Pluseee s User manual Table 1 Information on STR loci Marker GenBank GenBank Chromos Repeating Unit of Accsession _ Allele Localization GenBank Allel D1S1656 NC_000001 9 17 1q42 TAGA 6 TGA TAGA TAGG TG D2S441 ALO79112 12 2p14 TCTA 12 D3S1358 NT_005997 18 3p21 31 TCTA TCTG 2 TCTA s D5S818 ACO008512 1l 5q23 2 AGAT 11 D7S820 AC004848 13 7q21 11 GATA 13 D8S1179 AF216671 13 8q24 13 TCTA 3 D10S1248 AL391869 13 10q26 3 GGAA 13 D12S391 G08921 19 3 12p13 2 AGAT GAT AGAT AGAC AGAT D13S317 AL353628 11 13q31 1 TATC 1 D16S539 AC024591 11 16q24 1 GATA D18S51 AP001534 18 18q21 33 AGAA s D21S11 AP000433 29 21q21 1 TCTA TCTG TCTA TA TCTA TCA TCTA TCCATA TCTA D22S1045 ALO022314 17 22q12 3 ATT i4 ACT ATT CSF1PO X14720 12 5q33 1 AGAT 2 FGA M64982 21 4q31 3 TTTC TTTTTTCT CTTT 3 CTCC TTCC 2 SE33 V00481 26 2 6ql4 AAAG AA AAAG 7 THO1 D00269 9 11p15 5 TCAT o TPOX M68651 11 2p25 3 AATG 11 VWA M25858 18
11. ce to 25 ul by addition of appropriate volume of deionized water The amount of DNA to be added depends on its concentration For routine applications optimal final DNA concentration is 0 5 2 ng per reaction The maximum volume of DNA to be added is 20 ul Kit Component Volume per reaction Activator Solution Sul Template DNA 0 2 2 ng up to 20 ul Deionized Water to a final volume of 25 ul Please keep in mind that when adding more than 10 ul of DNA solution the possible presence of residual inhibiting substances can lead to negative effects on PCR sensitivity On the other hand the kit has a very high tolerance to many inhibiting substances Therefore usually higher amounts of DNA can be applied without problems when the extracted DNA is present in a buffer with low EDTA content eg TE buffer Otherwise due to chelation by EDTA available magnesium concentration of the reaction mix can become suboptimal Mix the final reaction volume 5 8 times thoroughly with a 20 ul pipette tip until the suspension becomes completely clear and spin down for a few seconds Since the reaction components are to be dissolved immediately before amplification proper mixing is crucial for optimal performance For every reaction setup should be run in parallel additionally one positive 1 ul of control DNA provided by the kit and one negative controls deionized water instead of DNA 3 2 Amplification Parameters The following PCR parameters are r
12. ecommended as standard procedure for all DNA probes A ramp of 0 3 C s should be used during heating from 59 C to 72 C Due to the high complexity of amplifying 18 primer pairs simultaneously in one reaction this ramping is crucial for optimal performance If ramping speed can not be programmed directly we recommend check correct ramping time by using a timer Under low copy conditions lt 100 pg of DNA 2 more PCR cycles can be added Generally we do not recommend more than 34 cycles in total It should be taken in mind that allelic drop out and heterozygotic disbalances become more frequent in this case Page 7 of 18 COrDIS Pluseee s User manual PCR parameters 94 C 3 min initial denaturation 98 C 30s 59 C 120s 4 cycles 72 C 90 s 94 C 30s 59 C 120s 6 cycles 72 C 90 s 90 C 30s 59 C 120s 20 cycles 72 C 75s 68 C 5min 15 C_ to the end After completion of PCR store the amplification products refrigerated at 4 C 8 C and protected from light If the amplified samples are to be stored for more than a week freezing at 20 C is recommended 4 Electrophoresis on ABI PRISM 3130 3130xl Genetic Analyzer For running the analyzer spectral calibration procedure proper use of ABI PRISM Data Collection Software as well as GeneMapper Software please follow the instructions given at manual ABI PRISM 3130 3130XL Getting Started Guide When using a different DNA analyzer please refer to the corres
13. gher signal intensities see section 4 2 A post PCR clean up of the amplified products to remove residual primers and salts is another possible method In this case the necessary amount of Sizing Standard S450 in the formamide mixture also should be reduced 5 Data Analysis 5 1 The Sizing Standard S450 The sizing standard S450 provided is labelled with orange fluorescent dye It contains 24 DNA fragments of 60 70 80 90 100 120 140 160 180 200 220 230 240 260 280 300 320 340 360 380 400 420 440 and 450 bases in length 1400 l alis fie J nis R ae e oe A all ai L L i eres APES Ae leo olro B400 leoo s00 ol G20 Bsoo 2e00 o s0 0 90 0 20 0 Boo 300 0 320 0 340 0 360 0 380 0 s000 420 0 440 100 0 ad a 0 B400 230 0 Figure 1 Electropherogram of 450 Size distribution of the labelled fragments Page 13 of 18 COrDIS Pluseee s 5 2 Allele and size ranges of STR markers Marker Range Range bp _ Alleles Colour Amelogenin X Amelogenin Y D3S1358 THO1 D12S391 D1S1656 D10S1248 D22S1045 D2S441 D7S820 D13S317 FGA TPOX D18S51 D16S539 D8S1179 CSF1PO D5S818 VWA D21S11 SE33 User manual Allele X Y 8 21 3 14 13 28 9 21 8 21 8 19 8 19 5 16 5 17 12 2 51 2 4 16 7 27 4 16 7 20 5 16 6 18 10 25 24 41 2 4 2 50 2 Marker Size M
14. herefore the kit can be shipped at the ambient temperature Labelled primers Size Standard S450 and Allelic Ladder are light sensitive and must be stored protected from the light Customers can store the reaction mix for a several months at room temperature without any loss of activity For long term storage the reaction tubes should be placed in a refrigerator at 4 C 8 C Do not freeze It can damage the dried polymerase Water and Activator solution should be stored refrigerated at 4 C 8 C For Control DNA storage at 4 C 8 C or long term storage at 20 C is recommended after dissolving Immediately after delivery the tube with Allelic Ladder should be separated from other kit components and stored protected from the light in a separate place at room temperature post amplification area of your lab Once the Allelic Ladder has been dissolved it should be stored in the dark refrigerated at 4 C 8 C Safety Precautions Some of the reagents of the Kit contain NaN and are potentially hazardous and should be handled accordingly Always wear gloves and avoid inhalation and skin contact Quality Assurance The quality of all kit components was verified and controlled during manufacturing The final dry kit was regularly tested over time to ensure high sensitivity for at least 18 months If there are any questions regarding our quality assurance program don t hesitate to contact us 1 4 Supplemental Material Supplemental
15. lts Group Instrument Protocol Run36_POP4_MXS5 The easiest way is to type all Sample Names first Then add to the first sample the data for table settings shown Then go to the first sample and highlight the entire well beginning at priority to the right and go down to the last sample while holding the mouse pressed Select in the Edit window Fill Down The software fills the appropriate settings to all highlighted positions After this change at the correct positions the Sample Type to Allelic Ladder Positive Control Negative Control Step C Starting Run and Run Information Go to Run Schedule and click to Find All Find the plate click the plate name and go to link After this the run can be started The run can be viewed during electrophoresis at Capillaries Viewer or Cap Array Viewer In Event Log information about errors can be viewed Error Status An overview about all relevant run data is presented at Run History of the Data Collection Software The run sample data are located at the corresponding Run Folder in the Results Group that was chosen in Step B of section 4 5 Page 12 of 18 COrDI 5 Pluse core User manual 4 6 Improving signal intensities If the STR peaks are too weak there are some after PCR procedures available for further improvement Performing an additional run with increased Injection Time up to 10 sec or with increased Injection Voltage up to 10kV is usually the simplest and fastest way to achieve hi
16. nssawncgarceervountgereeeyseweetedaainenasetaveuces 12 4 4 Sample Preparation and Loading 0 cece cece cence ence eee ee ene eee eneeeaeeneenes 12 4 5 Starting Run and Run Informations icesccscccesiescaidersecveetecsdvscsacasadeasaeienesesdes 13 4 6 Improving signal MENS ES 5 1 concesenschuoneneenseteneieeuebasnsceesreusaapneniyena beg esess 13 5 D ta AnalySiScsrescesscssoercesesecciceres sssr stonnen n eE nE Een EES 15 5 1 The Sizing Standard S450 oeseri deny eowseexw cus diauareeeweee ees 15 5 2 Allele and size ranges of STR markerSs 0 cece cece eee eee eee cece nett eeneeeaeenees 15 5 3 Amplification of Control DNAs ccsccaccencseearnceuesetonrnncadeasearqeamieetederscaieaess 16 S54 Allelic Ladder aerian aree pas eee eee ee 17 5 5 Measured Lengths of Amplified PCR Products ABI Prism 3130 0008 18 6 Results Interpretation Amplification Artifacts ccccceecesccsscesceseoes 22 6 1 PUP OAKS cierren nie aE E EEE AEREE a OAOA RETE E 22 6 2 Stutter Peaks atid Split PEAKS ate wevssseensidnnsyssncenseverzesaagsavaediseeteduvereeiansases 22 7 Troubleshooting ssisisesssssessossscssssssisisosssssessossesrsssssssssossssnessossessessse sins 22 8 Referentes sssriirscessssssssssrssreerssrssosty eris s rsrs reos St ESPs TENENTE E EEN STEE EE CSSS TES 23 Page 2 of 18 COrDIS Pluseee s User manual 1 Product information 1 1 Product Description CO DIS Plus is a short tandem repeat STR m
17. odule has to be edited Go to Module Manager of Data Collection Software and click New The Run Module Editor will be opened Make sure that the following Run Paremeters are set Run Module Parameter Setting Oven Temperature 60 Poly Fill Volume 4840 Page 10 of 18 COrDIS Pluseee s User manual Current Stability 5 PreRun Voltage 15 PreRun Time 180 Injection Voltage 3 Injection Time 5 Voltage Number of Steps 40 Voltage Step Interval 15 Data Delay Time 1 Run Voltage 15 0 Run Time 1600 Click Save As and select an appropriate name for this module eg COrDIS Plus_450bp After this click OK and leave the module by clicking Close 4 3 Create an Instrument Protocol Go to Protocol Manager of the Data Collection Software At the Instrument Protocol window click New to open the Protocol Editor The following parameters have to be set Protocol Editor Name Run36_POP4_MXS5 Type REGULAR Run Module COrDIS Plus_450bp Dye Set Any5Dye parameters see at 3 2 Click OK to leave Protocol Editor 4 4 Sample Preparation and Loading For sample preparation prepare a run mix by combining Hi DiTM Formamide and Sizing Standard S450 dissolved as described in 2 3 as follows Components per analysis tube Amount Hi Di Formamide 10 0 ul Size Standard 450 1 0 ul Keep in mind that always a complete number of 4 or 16 wells have to be filled with formamide Do not forget to add at least one well with Allelic Ladder After mixing add
18. of DNA Alternatively for convenience the control DNA can be resuspended in more than 20 ul of distilled water In this case the volume of DNA to be added to the reaction tube has to be increased to achieve a final concentration of 500 pg DNA Once the control DNA is suspended it should be stored in the dark at 4 C 8 C 2 3 The Sizing Standard S450 The sizing standard S450 provided with COrDIS Plus kit is a dried mix of different fragments labelled with orange fluorescent dye It is ideally suited for analysis of COrDIS Plus when using 5 dye matrix generated with matrix standard MXS 5 The sizing standard S450 contains 24 DNA fragments of 60 70 80 90 100 120 140 160 180 200 220 230 240 260 280 300 320 340 360 380 400 420 440 and 450 bases in length Before use add 120 ul of deionised water to the yellow labelled tube and mix thoroughly after 5 minutes incubation The diluted size standard can be stored refrigerated at 4 C 8 C for several weeks For analysis on sequencers add 1 ul of S450 to each well containing formamide and PCR products as well Page 6 of 18 COrDIS Pluseee s User manual 3 PCR Amplification 3 1 Reaction setup The final reaction volume is 25 ul Before addition 5x concentrated Activator solution should be mixed thoroughly Add 5 ul of the Activator to each reaction tube and incubate at room temperature for 2 minutes After this add DNA and compensate the remaining volume differen
19. ponding application guide of the manufacturer For detection of the 5 dyes of this kit use dye set any5 dyes Material Capillary 3130 Capillary Array 36 cm Polymer 3130 POP 4 Polymer Buffer 10 x Genetic Analyzer Buffer with EDTA 4 1 Generation of a Matrix Spectral Calibration The use of COrDIS Plus needs the application of a 5 colour spectral matrix The correct matrix can be generated using Matrix Standard MXS_5 not provided with the kit order number MXS_5 It contains a mixture of 5 different fragments each labelled with a different single dye These labels are corresponding to the dyes of the STR amplification products and the sizing standard 450 as well For preparation of a Matrix Standard MXS_5 working solution add 50 ul of deionised water to the tube containing dried MXS_5 purple label and incubate at room temperature for 5 min Then mix the solution thoroughly and spin briefly in a microcentrifuge The dissolved solution can be stored refrigerated in the dark and is stable for at least 6 months at 5 C 8 C Page 8 of 18 COrDIS Pluseee s User manual Matrix Standard Setup for Spectral Calibration ABI 3130 4 capillaries Hi Di Formamide 40 ul Dissolved MXS 5 4 ul Add 10 ul of the mixture per well to a 96 well microplate at positions AO1 D0O1 Spin briefly the microplate to remove all bubbles from the bottom of the wells Matrix Standard Setup for Spectral Calibration ABI 3130XL 16 capillaries
20. ppropriate number of wells for a single run e g AO1 to A04 ABI 3130 4 capillaries or A01 to H02 ABI 3130XL 16 capillaries Click OK to finish and to leave Plate Editor Step C Running Spectral Calibration Go to Run Scheduler Plate View and click Find All Select the appropriate Plate Name e g Spectral_any5_MXS_date Link this plate and start the run Step D Evaluation of the Spectral Matrix After completing Spectral Run view the pass or fail status of each capillary Open Instrument Status and go to Event Log In the Event Messages the status of each capillary is displayed Each capillary should have a Q value not less than 0 95 The peak height should be at least 1 000 rfu but less than 5 000 rfu optimal range is between 2000 and 4000 rfu Additionally at the Spectral Viewer the spectral calibration profile for each capillary can be evaluated Correct calibration must be achieved for at least 3 out of 4 capillaries or 12 out of 16 capillaries respectively When using MXS 5 as the matrix standard at the spectral calibration profile the correct peak order is blue green yellow red orange beginning from the left Finally save the run data by clicking Rename and rename the run eg AnyS_MXS_date and click OK You should be aware that for each filter set always the latest calibration run is active If you wish to use another matrix it must be activated before 4 2 Run Conditions Before performing first run a Run M
21. ultiplex assay with 19 STR markers and Amelogenin for gender determination It combines all 13 CODIS loci D3S1358 D5S818 D7S820 D8S1179 D13S317 D16S539 D18S51 D21S11 CSFIPO FGA THO1 TPOX and VWA 5 new core loci recommended for extension of European national databases D1S1656 D2S441 D10S11248 D12S391 and D22S1405 as well as the most powerful in discrimination STR locus SE33 PCR primers have been designed to fit into a single amplification reaction with PCR products less than 440 bp taking in account all known alleles so far PCR products are labelled with blue green yellow and red fluorescent colours by using a sizing standard labelled in a fifth orange colour Full STR profiles can be generated when at least 200 pg of non degraded genomic DNA are used for PCR The kit is provided as lyophilised in 0 2 ml PCR reaction tubes or plates and can be stored protected from the light at room temperature for at least one year without any loss of sensitivity The reaction components should be activated simply by addition of 5 ul of activator solution to each tube The final reaction volume is 25 ul so that theoretically up to 20 ul of DNA solution can be added per reaction Due to its extremely high tolerance to inhibiting substances such high amounts of DNA can be amplified successfully CO DIS Plus has the potential to be applied for national database DNA typing in countries that are using CODIS Combined DNA Index System markers
22. yzer by using the Size Standard S450 For allele designation the GeneMapper ID Software in combination with COrDIS Plus template file were used Page 16 of 18 COrDIS Pluseee s User manual 6 Results Interpretation Amplification Artifacts 6 1 Pull up Peaks A Pull up or bleedthrough can occur when the matrix can not compensate high amplification signals higher than 4 000 5 000 rfu Typically there are seen peaks exactly at the same size position of the main peak in the neighbour colour panels but with lower signal intensities comparing to the main peak Another reason can be the application of a wrong matrix during Data Collection 6 2 Stutter Peaks and Split Peaks Occurrence of stutter peaks is typical for STRs but not for Amelogenin locus Stutters are observed at one repeat unit shorter than the true peak n 1 and are slippage artefacts of polymerase during amplification Usually for alleles of one particular STR the relative height of stutter peak increases with allelic number STR locus SE33 has a typical composed stutter consisting of n 1 stutter 4 base pairs shorter and a second stutter signal exactly at the middle of distance between n 1 stutter and main peak 2 base pairs shorter than main peak The height of this intermediate stutter peak is about half of the N 1 stutter If too high amounts of DNA are used for amplification additional stutter peaks can be observed at n 2 and or n 1 positions of the main peak
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