ATZLabs_3ZOMY_QF_PCR_Product
Contents
1. BI Pite Technologies n Your Molecular amp Cell Technology Partner haat V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 25 J Culture of Excellence Email customerservice lifetechindia com 24 Web www lifetechindia com 8 2 PCR Artefacts Stutter peaks Large difference in size between two alleles Are detected as extra peaks that are one repeat smaller than the actual STR allele The Stutter peaks may be included stutter peak area Is typically less than 15 of in the ratio calculation the corresponding STR peak area Are detected as extra peaks that are one base smaller than the full length A peak PCR product due to incomplete addition of the 3 A residue PCR is a competitive reaction Where alleles are separated by a large number of repeats the shorter allele can be preferentially amplified leading to an imbalance between the two alleles A peaks may be included in the ratio calculation 8 3 Electrophoretic Artifacts Extra peaks visible in one or all channels Low signal Missing peaks in one or all channels Larger loci showing much more product than smaller loci Pull up or bleedthrough between dye channels Dye blobs may appear in the sample analysis range Failed analysis due to low sizing quality Electrooherogram data is of poor quality Unexpected electrophoretic spikes l BI Biological Industries Culture of Excelle2. S gt l ndustrie Vite Technologies BI 3Z0MY KI For In Vitro Diagnostic Use Cat No 20 900 50 Kit Components The Kit includes reagents for 50 tests 2 x 25 using the 5 Chromosome PCR Mix and 25 1 x 25 additional tests each for Chromosome 21 PCR Mix and Sex Chromosome PCR Mix 5 Chromosome PCR Mix Yellow Cap 20 900 50 part 1 X2 500ul Chromosome 21 PCR Mix White Cap 20 900 50 part 2 X1 500ul Sex Chromosome PCR Mix Red Cap 20 900 50 part 3 X1 500ul PCR Enzyme Orange Cap 20 900 50 part 4 X4 25ul Read More t L S0 9 699 Table of Contents 1 Product Description and Principles of the Procedure 3 Product Components and Storage Conditions 6 2 1 Kit Components 6 2 2 Reagents Required But Not Supplied 6 2 3 Storage and Handling Requirements 6 2 4 Symbols used on Labels 7 3 Before You Begin 8 3 1 Precautions 8 3 2 Spectral Calibration 8 4 PCR Sample Preparation and Amplification Setup 9 4 1 DNA Extraction 9 4 2 Working Solution Preparation 9 4 3 Amplification Setup 9 4 4 Thermal Cycling Protocol 10 5 Capillary Electrophoresis Sample Preparation and Instrument Setup 11 5 1 Sample Preparation 11 5 2 Running Parameters 11 5 3 Instrument Setup 11 6 Data Analysis 17 6 1 Importing New Panel and Bin Settings in the Panel Manager 17 6 2 Importing GeneMapper Analysis Settings in the GeneMapper Manager 17 6 3 Performing Data Analysis 18 6 4 Reviewing QF PCR Data 18 6 5 Using the GeneM
3. Setup Refer to the respective ABI Genetic Analyzer User Manual for instructions on maintenance and handling Prior to running the 3ZOMY Kit the instrument must be spectrally calibrated to support detection of the 5 Color Dye Set See Section 3 2 for details 5 1 Sample Preparation a Prepare a loading solution by combining and mixing 2ul of the Size Standard 3ZOMY size standard BI Cat 20 920 00 or GeneScan 600 LIZ Size Standard Life Technologies Cat 4366589 with 100ul of Hi Di Formamide sufficient mix for 6 wells tubes b Vortex for 15 seconds c Dispense 15ul of the loading solution into the required number of wells in a 96 well plate or into individual tubes ABI310 to be placed on the Genetic Analyzer d Add 1 5ul of the sample PCR product to the corresponding well tube containing loading solution e Seal the plate tubes 5 2 Running Parameters Instrument parameters Run parameters Capillary Length Run Temperature Injection Voltage Run Voltage Run Time ABI 310 POP 4 47cm 60 C 15 kV 15 kV 40 min ABI 3100 3130 POP 4 POP 7 36 cm 60 C 1 5 kV 15 kV 1500 s POP 7 50 cm 60 C 1 6 kV 19 5 kV 1500 s The amount of PCR product injected into the capillaries can be adjusted by increasing decreasing the injection time and or injection voltage 5 3 Instrument Setup The following instructions are for an ABI 3130xl Genetic Analyzer with 3ZOMYKit T
4. Hi Spatial Calibration vi El capillary Viewer Link Plate Name Application Status Size HB capiarray Viewer A 3ZOMY_6 Chrs GeneMapper H Spectral Calibration BP lReextraction amp 3130xL amp E4Instrument Status EYEPT Chart E event Log Hil Spatial Run Schedule S plate view Eh Run View E capillaries Viewer HB capiArray Viewer Gi Spectral Viewer Ah Manual Control Service Log pending 96 lt j gt System Status gt Plate 3SZOMY_5_Chrs has been linked to Bay 0 The Process Plates dialog box appears Click OK to start the run Process Plates Q You are about to start processing plates Life Technologies Your Molecular amp Cell Technology Partner Ph 91 11 42208000 Fax 91 11 42208444 Email customerservice lifetechindia com Web www lifetechindia com CE No Current Run wre Q a ISO 13485 2003 P V3 April 2014 16 6 Data Analysis One of the following software programs is required for data analysis GeneMapper Applied Biosystems or GeneMarker Softgenetics GeneMapper GeneMarker settings for the 3ZOMY Kit and size marker must be imported before analysis of raw data The latest versions can be obtained by request from Bls technical support team 6 1 Importing New Panel and Bin Settings in the Panel Manager a p ze Open the Panel Manager by selecting Tools and Panel Manager in the GeneMapper main menu or by clicking on the icon I
5. chromosome as in the case of trisomy SS a Figure 3 Trisomic marker area ratio 1 1 1 Figure 4 Trisomic marker area ratio 1 2 BI Pite Technologies FD Your Molecular amp Cell Technology Partner haat V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 J Culture of Excellence Email customerservice lifetechindia com 3 Web www lifetechindia com 3ZOMY Chromosome Marker Overview ence latin sta e Diastase reena oons Red presos iamar aoas Red Chromosome 21 Daise zines Jasa Rea Dxvsere xp2258 Yprise 1520 rea XHPRT Xq26 2 q26 3 265 308 Red oo Chromosomes X and Y Table 1 3ZOMY genetic markers for 5 Chromosome PCR Mix Pite Technologies de Biological Industries ph 91 11 42208000 Fax 91 11 42208444 Culture of Excellence Email customerservice lifetechindia com Web www lifetechindia com CE wre Q a ISO 13485 2003 foxsaiso xeon eer areor Red Ter as renon Red V3 April 2014 4 D2181435 219213 3 150 208 208 Blue ee a 290 D21S1411 21q22 3 245 345 D21S1444 21q22 13 450 505 Chromosome 21 362 420 D21S2055 21q22 2 385 488 Blue D21S1409 160 220 Green D21S1280 21q22 11 295 470 Table 2 3ZOMY genetic markers for Chromosome 21 PCR Mix AMELX AMELY AMELX AMELY Xp22 2 Yp11 2 Xp22 2 Yp11 2 X 104 Y 110 Xq21 31 C Dxszso xarra a257 Pea SOE e 31 mse pea Xq23 2 118 X 114 Table 3 3ZOMY genetic marker
6. 0 15 kvolts Pre_Run_Time lt 180 1 1000 sec Injection_Voltage 25 1 15 kvolts Injection_Time 20 4 600 sec Voltage_Nurnber_Of_Steps vl 1 100 nk Voltage_Step_Interval 60 sec Data_Delay_Time 3600 sec Run_Voltage 16 Kvolts Run_Time 1500 300 14000 sec ha h e Click OK l BI Your Molecular amp Cell Technology Partner k stow R A 1 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 ES Culture of Excellence Email customerservice lifetechindia com Life Technologies SED On Web www lifetechindia com V3 April 2014 12 5 3 2 Create an Instrument Protocol AR Foundation Data Collection Version 3 0 No User is logged in In the left navigation window select Protocol Manager and New a Fill out the Protocol Editor b Name Enter a name for the Run Module c Type Regular d Run Module Select the Run Module created in step 5 3 1 3ZOMY_36 POP4 e Dye Set 5 Color Dye Set f Click OK l BI Ao FOCANDOJIES anam aie Industries Ph 91 11 42208000 Fax 91 11 42208444 Wy of Excellence Email customerservice lifetechindia com 13 Web www lifetechindia com 5 3 3 Setup a Plate for Run In the left navigation window select Plate Manager and New AR Foundation Data Collection Version 3 0 No User is logged in GA instrumen
7. Height is applicable for instruments with a lower detection scale i e ABI 310 ABI 3100 and ABI3130 maximum detection 8000 RFU When using ABI 3500 or ABI 3730 Genetic Analyzers maximum detection at 32000 RFU the Minimum Peak Height needs to be adjusted b Open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the Icon Or In the GeneMapper Manager select the Analysis Methods tab Highlight the desired Analysis Method and then click Open c Select the Peak Detector tab The user specified RFU values of 100 for the blue green yellow and red and 50 for the orange are applicable for the ABI 310 3100 3130 Genetic Analyzers If using an ABI 3500 or 3730 Genetic Analyzer values of 500 for the blue green yellow and red and 200 for the orange are applicable d Click OK and Done after entering the user specified RFU values 6 7 Missing Allele Calls Analysis Method and Bin Set Connection Lost Occasionally the Analysis Method looses its connection with the Bin Set This is the likely explanation when the allele calls and bins cannot be displayed Instead question marks are displayed in the peak labels Follow the instructions below to ensure that the Bin Set is connected to Analysis Method a To connect the Bin Set to the Analysis Method open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking
8. Reaction Valance 25 l 25 l 25ul Cap the tubes and centrifuge briefly to collect the contents 4 4 Thermal Cycling Protocol The 3ZOMY Kit has been optimized for the GeneAmp PCR System 9700 thermal cycler Turn on the thermal cycler at least 30 minutes prior to amplification For ABI GeneAmp System 9700 set ramp speed to 9600 mode For use of alternative thermal cyclers the following ramping rates must be applied heating 0 8 C sec cooling 1 6 C sec Program the thermal cycler for amplification as follows Step Temp Time Cycle No 1 95 C for 15 min 2 94 C for 30 sec 3 58 C for 90 sec 4 72 C for 90 sec For 26 cycles 5 72 C for 30 min Start amplification duration approximately 3 hours Following amplification remove the tubes containing completed PCR amplification reaction from the thermal cycler and place into a suitable holder Centrifuge briefly to collect the contents Remove the caps carefully to avoid aerosol contamination Do not bring amplified material into the pre amplification areas Amplified material should be restricted to the amplification and detection areas BI Pite Technologies FD Your Molecular amp Cell Technology Partner a V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 J Culture of Excellence Email customerservice lifetechindia com 10 Web www lifetechindia com 5 Capillary Electrophoresis Sample Preparation and Instrument
9. Sample File Sample_2 AMELXY X detected B 103 67 00 39583 1_3ZOMY_5_Chrs_17 7 13_ Sample_2 D13S1 1 1 R 143 53 155 63 0 9447 10054 10642 1_3ZOMY_5_Chrs_17 7 13_ Sample_2 D135305 459 01 20 27480 1_3ZOMY_5_Chrs_17 7 13_ Sample_2 D135628 456 97 00 22215 1_3ZOMY_5_Chrs_17 7 13_ 1 2 3 4 2 5 a e e E E E E 6 7 8 8 8 8 8 Sample besazju k bee ae omw Sample prasass 11 alee detanean G fea t9 a25 O OO ojo nzaser same pisss ee oO EE s fme pess R je ka f zem j 10 Sample 2 piesozs t O wo oeo keo O O of nomis isay 1 a pe N N 9 is ml wo wih a Oo o o an Ka a ea la teal e ee l ee e ee M 7 12 pees premju t __ SMe ko 306 32 810 48 819 23 11792 1145713726 11980 13 o e has hes o os 403933 14 me paju faa p e a a O e a S 15 Bes R ms e pa N siza iaa 3ZOMY 5_Chrs 17 7 13 16 ee 2 aoe 7 it B 0 9203 16446 3ZOMY_5_Chrs_17 7 3 17 Sample_2 DXS1187 1 1 G 1 0269 33692 1_3ZOMY_5_Chrs_17 7 13_ 18 Sample_2 DxS2390 1 1 R 0 9631 13754 1_3ZOMY_5_Chrs_17 7 13_ 19 Sample_2 DXY5218 1 1 R 1 073 15017 13995 1_3ZOMY_5_Chrs_17 7 13_ 20 Sample_2 DXY5267 s 1 1 0697 16915 1_3ZOMY_5_Chrs_17 7 13_ 21 Sampe2 GATAL Ooo h 22 me w a l 23 o b ea hsa o o on a Eo o rr rr 2 fez ben Oo R a O o To o ar S Sample 2 pw oo o e es
10. These markers may be used to assess the total number of sex chromosomes when informative d Itis not possible to determine which allele represents the X or Y chromosome 13 The use of the additional PCR mixes for chromosome 21 and sex chromosomes is recommended as back up in case all the markers in the 5 Chromosome Mix are non informative homozygous and can also be used individually for the respective aneuploidy diagnosis BI Pite Technologies n Your Molecular amp Cell Technology Partner am V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 25 J Culture of Excellence Email customerservice lifetechindia com 22 Web www lifetechindia com 14 Analysis of heterozygous markers displaying two allele peaks is performed by calculating peak area ratios Peak1 Peak2 Peak1 is the peak area of the shorter length fragment and Peak2 is the peak area of the longer length fragment Ratio Criteria RC Interpretation Due to the occasional preferential amplification of the smaller allele the ratio between fluorescent peaks may vary within limits shown in the following table Rao 12 inconclusive 1 1 Inconclusive 21 0650 74 O75144 145175 0 65 0 74 075154 155 175 For markers displaying three allele peaks the ratio is always calculated starting with the area of the shortest length fragment Peak1 i e Peak1 Peak2 and Peak1 Peak3 respectively Ratio inconclusive aaa inconcl
11. imported in the Matrices tab when analyzing raw data generated from capillary electrophoresis using the ABI 310 Genetic Analyzer l Pite Technologies SEED On BI Culture of Excellence Email customerservice lifetechindia com Web www lifetechindia com Your Molecular amp Cell Technology Partner V3 Ap ril 2014 z A ISO 13485 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 ES 6 3 Performing Data Analysis a Import the raw data files fsa files from the capillary electrophoresis by selecting File and Add Samples to Project or by clicking on the ue b Navigate to the desired run folder using the folder tree in the left navigation window Double click or use the plus symbol to display all files contained in the folder Highlight the complete folder or selected samples and click on Add to List c The added run folder will now appear in the Samples to Add window Add the samples by clicking on Add d Set the appropriate analysis settings Analysis Method Panel and Size Standard for each sample alternatively fill down the columns by selecting the settings for one sample highlighting the desired number of samples and typing Ctrl D keys to copy the settings Note The matrix has to be set when analyzing fsa files from an ABI 310 Genetic Analyzer icon e Initiate the data analysis by selecting Analysis and Analyze or by clicking on the gt Icon Assign a project name when prompted 6 4 Revie
12. 910 00 for the ABI 31XX 3730 and 500 Genetic Analyzers or the 5 Color Dye Set Single Capillary for the ABI 310 Genetic Analyzer BI Cat 20 911 00 Size Standard 3ZOMY size standard BI Cat 20 920 00 or GeneScan 600 LIZ Size Standard Life Technologies Cat 4366589 2 3 Storage and Handling Requirements a Store all components below 18 C b The activated reaction mixes prepared by adding the 3ZOMY Chromosome Mix to the PCR may be stored at 2 to 8 C for at least 7 days and at below 18 C for at least 90 days c Avoid repeated freeze thawing d Dispose of unused reagents and waste in accordance with country federal state and local regulations e Do not mix reagents from different kit lot numbers i m FED Q BI Life Technologies Fo ania Biological Industries ISO 13485 Ph 91 11 42208000 Fax 91 11 42208444 2003 Culture of Excellence Email customerservice lifetechindia com 6 Web www lifetechindia com 2 4 Symbols used on Labels Lot or batch number y Number of tests Store below temperature shown Biological Industries Culture of Excellence Vite Technologies a de gt Your Molecular amp Cell 109 panne C IN gt 0 Expiry date wal Manufacturer In vitro IVD diagnostic device ISO 13485 Ph 91 11 42208000 Fax 91 11 42208444 2003 Email customerservice lifetechindia com Web www lifetechindia com V3 April 2014 7 3 Befor
13. Cell Technology Partner am V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 2 J Culture of Excellence Email customerservice lifetechindia com PF Web www lifetechindia com Turner Syndrome 45 X Sample 5 Chromosome PCR Mix All informative autosomal STR markers demonstrate a normal 1 1 marker ratio The presence of AMELX and the absence of AMELY and SRY confirm female gender The X chromosome counting markers TS3 and TS7 demonstrate an abnormal female 2 1 marker ratio consistent with the dosage of a single X chromosome All X and XY STR markers are uninformative DXS1187 DXYS267 DXYS218 XHPRT DXS2390 consistent with the dosage of a single X chromosome 4_320MY_8_Chrs_17 7 13_F41_Sample_11_0114 320MY F Mark Sample for Deletion g _3Z0MY_5_Chrs_17 7 13_F11_Sample_11 _0114 320MY BB F Mark Sample for Deletion 400 260 300 380 500 3000 2000 1000 i 0 i i i A i 1 320MY_5_Chrs_17 7 13_F11_Sample_11_0114 i3Z0MY F Mark Sample for Deletion 100 140 180 220 260 300 340 380 420 460 500 F Mark Sample for Deletion 100 140 180 220 260 300 340 380 420 460 300 Triploidy Sample 5 Chromosome PCR Mix All informative STR markers on chromosomes 13 18 21 X and Y demonstrate abnormal 1 2 2 1 or 1 1 1 marker ratios consistent with triploidy 1_3Z0MY_5_Chrs_A12_Sample_3_002 fsa 3Z0M Mark Sample for
14. Deletion 130 170 530 11_32z0MY_5_Chrs_A12_Sample_9_002 fsa 3ZOMY F Mark Sample for Deletion p e 4_3Z0MY_5_Chrs_A12_Sample_9 002fsa szOMy z Mark Sample For Deletion pa Pr aa DASA 170 430 530 3000 2000 1000 ki lI ras A 1 _3Z0MY_5_Chrs_A12_Sample_9_002fsa 3Z0MY IC C E Mark Sample For Deletion 530 BI Life Technologies FD Your Molecular amp Cell Technology Partner am V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 W7 Culture of Excellence Email customerservice lifetechindia com 28 Web www lifetechindia com Down syndrome Trisomy 21 47 XX 21 Sample Chromosome 21 PCR Mix All STR markers demonstrate abnormal 1 2 2 1 or 1 1 1 marker ratios consistent with trisomy 21 M _3Z0MY _Chr24 _ChrX Y_BO2 _ Sample_ 2 004 fsa 3ZOMY 21 v1 Mark Sample For Deletion 1 _3Z0MY _Chr21 _ChrXY_ BO2_ _ Sample Pa 004 fsa 3ZOMY 21 v1 E Mark Sample for Deletion TEA oo 140 180 220 260 300 340 380 420 460 500 ee Turner Syndrome 45 X Sample Sex Chromosome PCR Mix The presence of AMELX and the absence of AMELY and SRY confirm female gender The X chromosome counting markers TS3 and TS7 demonstrate an abnormal female 2 1 marker ratio consistent with the dosage of a single X chromosome All X and XY STR markers are uninformative DXS1187 DXYS267 DX
15. ISO 13485 Ph 91 11 42208000 Fax 91 11 42208444 C Sa Email customerservice lifetechindia com 25 9 Analysis Examples Normal Male 46 XY Sample 5 Chromosome PCR Mix All informative autosomal STR markers demonstrate a normal 1 1 marker ratio The presence of AMELY and SRY is consistent with male gender The X chromosome counting markers TS3 and TS7 demonstrate a 2 1 marker ratio and all the X chromosome STR markers are uninformative in line with the expected dosage of X chromosomes in a normal male The 1 1 marker ratio of the informative pseudo autosomal XY chromosome STR markers DXYS267 and DXYS218 and the non polymorphic XY markers AMELXY and ZFYX confirm a normal male sex chromosomal dosage Mark Sample For Deletion E Mark Sample for Deletion pxsier A a A aasrae ress asa 220 260 300 340 380 420 460 500 E Mark Sample for Deletion Down Syndrome Trisomy 21 47 XX 21 Sample 5 Chromosome PCR Mix All informative autosomal STR markers on chromosome 21 demonstrate abnormal 1 2 2 1 or 1 1 1 marker ratios consistent with trisomy 21 1_320MY_5_Chrs_17 7 13_b_B12_Sample_2 006 3ZOMY F Mark Sample for Deletion 100 140 180 220 260 300 340 380 420 460 500 4 _320MY_5_Chrs_17 7 13_b_B12_Sample_2 00 3ZOMY B B F Mark Sample for Deletion g _3Z0MY 5 Chrs_ ATI 13_ b B12 _Sample ea 004 3z0MY DST mmg ii E Mark Sample for Deletion 180 re ee s
16. OOo Too jen i The 3ZOMY Report Setting is included in the product settings folders Use the 3ZOMY Table Setting with the corresponding 3ZOMY Report Setting Report and Table Settings are imported into GeneMapper through the GeneMapper Manager tool a Select the appropriate Table Setting from the Table Setting drop down menu in the GeneMapper main window b Generate a report by highlighting the Sample file s to be analyzed and then selecting Analysis and Report Manager or by clicking on the Report Manager icon Or In the Report Manager window select the appropriate Report Setting from the Report Setting drop down menu c To review the report data in a Samples Plot Highlight a marker to be reviewed and then click on the ihi icon To edit data in the Samples Plot window right click on the marker and select the appropriate function in the menu box that appears Any changes made will appear immediately in the Report Life Technologies SED On B Your Molecular amp Cell Technology Partner haat V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 ES Culture of Excellence Email customerservice lifetechindia com 19 Web www lifetechindia com 6 6 Modifying the Analysis Method Setting Depending on the ABI Genetic Analyzer used it might be necessary to modify the Minimum Peak Height in the Analysis Method a In the Analysis Method provided for the kit the default Minimum Peak
17. YS218 XHPRT DXS2390 consistent with the dosage of a single X chromosome 4_3Z0MY_Chr24_ChrX _D04_Sample_11_007 fs S20MY XY v1 B B F Mark Sample for Deletion pxs6s03 O pxYs2657 e SS Seso S 420 160 200 240 280 320 360 400 440 xz 2000 i J 4_3Z0MY_Chr24_ChrXY_001_Sample_11_007 fs 3Z0MY XY v1 b B F Mark Sample for Deletion AmE Dxsafe7 o o oooi a S pxscaa O 120 160 200 240 280 320 360 400 440 4000 L E xT Wm msz Zex RHPRT 120 160 200 240 280 320 360 400 440 3000 2000 1000 I a _3Z0MY_Chr21_ChrX Y_D01_Sample_1 _007 f 3ZOM KY vA O m E Mark Sample for Deletion j 200 240 280 320 360 400 440 3000 2000 1000 f i I FIED at BI Life Technologies SEED On Your Molecular amp Cell Technology Partner V3 Ap ril 2014 Biological Industries 2003 y Ph 91 11 42208000 Fax 91 11 42208444 Culture of Excellence Email customerservice lifetechindia com Web www lifetechindia com 10 References Professional guidelines for clinical cytogenetics and clinical molecular genetics QF PCR for the diagnosis of aneuploidy best practice guidelines 2012 v3 01 January 2012 11 Appendixes 11 1 The3ZOMY size standard BI Cat 20 920 00 The 3ZOMY size standard contains 21 DNA fragments of 73 88 123 148 173 198 223 248 273 298 324 349 373 398 423 448 470 495 520 545 and 555 bases in length It is detected as a fourth color in the presen
18. apper Report Format 19 6 6 Modifying the Analysis Method Setting 20 6 7 Missing Allele Calls Analysis Method and Bin Set Connection Lost 20 6 8 Disclaimers 20 7 Interpretation of Results 21 8 Troubleshooting 24 8 1 Unusual conditions 24 8 2 PCR Artifacts 29 8 3 Electrophoretic Artifacts 25 9 Analysis Examples 26 10 References 30 11 Appendixes 30 11 1 The Internal Size Standard 30 11 2 Advantages of Using the Loci in the 3ZOMYKit 30 11 3 Contact Information 31 l BI y Life Technologies a vain N Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 C eS Culture of Excellence Email customerservice lifetechindia com 2 Web www lifetechindia com 1 Product Description and Principles of the Procedure The 3ZOMY Kit is a DNA based multiplex QF PCR Quantitative Fluorescent Polymerase Chain Reaction assay designed to test aneuploidy of 5 chromosomes 13 18 21 X and Y in DNA DNA can be extracted from prenatal samples amniotic fluid AF chorionic villus CVS and fetal tissue samples to determine fetal aneuploidies A parent mother or father sample blood or tissue is used as a control for ensuring the origin of the fetal sample and to prevent sample mixing The product is not intended nor tested to be used for non invasive procedures The 3ZOMY Kit includes a multiplex STR marker set used to perform initial aneuploidy diagnosis of the chromosomes 21 18 13 X and Y and two additional c
19. ce of 3ZOMY amplified DNA in the orange channel 2010 2410 2810 3210 3610 4010 4410 4310 5210 5610 6010 6410 11 2 Advantages of Using the Loci in the 3ZOMY Kit l 3ZOMY QF PCR has the major advantage of providing rapid accurate and cost effective analysis for the diagnosis or exclusion of aneuploidy in chromosomes 13 18 21 X or Y The complete procedure from sample to results takes less than five hours thus enabling your laboratory to provide results in just one day gt Requires small amounts of genomic DNA gt The 3ZOMY Kit s excellent assay performance and large number of highly informative markers significantly reduce the number of re runs In addition a single PCR mix and detection reduces the risk of sample mix up BI Pite Technologies n Your Molecular amp Cell Technology Partner koiaa V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 we Culture of Excellence Email customerservice lifetechindia com 30 Web www lifetechindia com The kit includes 16 different somatic markers for chromosomes 21 18 and 13 and 10 different sex chromosome markers to perform initial aneuploidy diagnosis Two additional chromosome specific marker sets for chromosomes 21 and XY to confirm the initial diagnosis or to be used individually for the trisomy of chromosome 21 and sex chromosome aneuploidy Three unique X chromosome counting markers for reliable detection of Turner s
20. e You Begin 3 1 Precautions a The laboratories should process their own validation studies and quality control QC measures Internal QC samples of Known genotypes should be processed in each assay to assess the validity of the procedure Qe gt oao TT Work under sterile conditions to avoid contamination e g bacterial Do not pool reagents from different lots or from different vials of the same lot Do not use the kit after its expiry date Each test is for single use only Do not use opened or damaged kit reagent vials Work flow in the laboratory should proceed in a unidirectional manner Begin in the reagent preparation area move to the DNA extraction area then to the amplification area and finally to the detection area Pre amplification activities should begin with reagent preparation and proceed to DNA extraction Reagent preparation activities and DNA extraction activities should be performed in separate areas Supplies and equipment should be dedicated to each activity and not used for other activities or moved between areas Gloves should be worn in each area and should be changed before leaving that area Equipment and supplies used for reagent preparation should not be used for DNA extraction activities for pipetting or processing amplified DNA or other sources of target DNA Amplification and detection supplies and equipment should remain in the amplification and detection a
21. errupted repeat can be 1 2 or 3 bases long Microvariants Microvariants are designated by the number of full repeats and the size of the incomplete repeat eg A 9 3 allele indicates 9 full repeats anda 3 base microvariant Maternal cell contamination Amplification of the maternal DNA should be done Partial or complete allele drop out If a In rare cases complete drop out is seen in a normal amplification failure due to sample the profile shows apparent mutation of the primer site homozygosity for an individual marker In the has been reported for the Primer site case of a trisomic sample the profile may AMELY sequence polymorphism show apparent disomy In rare cases ratio Partial drop out is seen as an additional peak skewing due to mutation at a reduced height which can result in of the primer site has skewed inconclusive or apparent 1 2 2 1 been reported for the TS1 allele ratios marker sequences SMM is detected as a novel allele at a single locus and is probably caused by a mitotic Somatic replication error It usually involves 1 2 microsatellite repeats increase or decrease in the repeat mutation unit Can be identified in a profile when present in a subpopulation of cells mosaic SMMs are more frequent in CVS than in AF Have been reported for markers D13S742 D13S634 D13S 628 D13S305 Copy number D18S978 D18S535 variation CNV D18S386 D21 11 D21 1411 D21S1437 XHPRT DXYS218 AMELXY ZFYX and TS7
22. etermined by fragment area ratio calculation BI Piire Technologies n Your Molecular amp Cell Technology Partner am V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 25 J Culture of Excellence Email customerservice lifetechindia com 21 Web www lifetechindia com In a normal female an X chromosome counting marker area ratio of 1 1 is expected Fig 5 In normal males and females with monosomy X a 2 1 ratio is expected Fig 6 5 Fig 5 Area ratio 1 1 Fig 6 Area ratio 2 1 10 Monosomy X pattern is determined by a All X and XY markers showing a homozygous allelic pattern b The AMELY and SRY peaks are not detected c Marker TS3 showing two peaks of differing height area and the peak ratio is classified as 2 1 d Marker TS7 showing two peaks of differing height area and the peak ratio is classified as 2 1 11 Non Polymorphic Markers The AMELXY and SRY markers amplify non polymorphic sequences on the X AMELX and Y AMELY and SRY chromosomes and can be used to determine the presence or absence of a Y chromosome SRY will give a single peak in normal males AMELXY may be used to assess the relative number of X to Y chromosomes 12 Pseudo Autosomal XY Markers a The DXYS267 and DXYS218 markers are polymorphic STR markers present on both the X and Y chromosomes b The ZFY ZFX marker is a non polymorphic non STR marker present on both the X and Y chromosomes c
23. he dye set used is the 5 Color Dye Set 3010 3130 3500 BI Cat 20 910 00 However the procedure is similar for the other instruments as well For further details refer to the User Guide for the instrument used ABI 3500 Before starting the electrophoresis for fragment analysis the following settings need to be set up in the instrument s Data Collection Software Run Module Instrument Protocol and Plate l BI Biological Industries Culture of Excellence Vi Ph 91 11 42208000 Fax 91 11 42208444 Email customerservice lifetechindia com Web www lifetechindia com ife Technologies Your Molecular amp Cell Technology Partner CE wre Q a ISO 13485 2003 V3 April 2014 11 5 3 1 Create a Run Module In the left navigation window select Module Manager and New BB sports ot Viewer Sarum Control Berce Log Cem Lovee Cimen Cevo x Fill out the Run Module Editor according to the Kit s instructions for use a Name Enter a name Type Regular Template FragmentAnalysis36_POP4 default template for the capillary array and polymer used Specific Parameters As specified in section 5 2 Run Module Editor iam Module Description Name 3ZOMY_36_POP4 Type REGULAR v Template FragmerntAnalysis36_POP4 v Description 18 65 Deg Poly_Fill_Vol 6500 38000 steps Current_Stability 0 2000 uAmps PreRun_Voltage 45
24. hromosome specific marker sets for the chromosomes 21 and XY that can be used to confirm the diagnosis The 3ZOMY Kit uses a five dye fluorescence system and is intended for DNA fragment analysis on Applied Bio systems Genetic Analyzers ABI 310 3100 3130 3500 and 3730 The 3ZOMY Kit is for professional and special trained users QF PCR analysis includes amplification detection and analysis of chromosome specific repeated DNA sequences known as short tandem repeat STR Fluorescently labeled primers are used for amplification visualization and quantification of the fluorescently labeled PCR products The resulting PCR products are separated and analyzed by a genetic analyzer DNA amplified from a normal diploid sample will present two alleles of a chromosome specific STR marker as two peaks in a 1 1 ratio when the marker is heterozygous Fig 1 or as one peak when the marker is homozygous Fig 2 The relative amount of the PCR product is quantified by calculating the allele peaks height ratio or areas STRs vary in size between subjects depending on the number of tetra repeats present on each allele Heterozygous STR markers are considered to be informative Figure 1 Heterozygous marker area ratio 1 1 Figure 2 Homozygous monosomic marker The detection of three peaks in a 1 1 1 ratio Fig 3 or two peaks in a 2 1 1 2 ratio Fig 4 may indicate the presence of an additional STR sequence possibly corresponding to an additional
25. i ii 01 1 1 _3Z0MY 5 Chrs_ AP 13_b_B12_Sample_ 21 ooe 3ZOMY IE B F Mark Sample for Deletion 3000 2000 1000 Edwards Syndrome Trisomy 18 47 XY 18 Sample 5 Chromosome PCR Mix BI Life Technologies N Your Molecular amp Cell Technology Partner LN V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 7 Culture of Excellence Email customerservice lifetechindia com 26 Web www lifetechindia com All informative autosomal STR markers on chromosome 18 demonstrate abnormal 1 2 2 1 or 1 1 1 marker ratios consistent with trisomy 18 E Mark Sample For Deletion 1_3Z0MY_5_Chrs_17 7 13_b_C1 1_Sample_6_00 3Z20MY Mark Sample For Deletion 100 140 180 260 300 340 330 420 460 500 F Mark Sample for Deletion Patau Syndrome Trisomy 13 47 XY 13 Sample 5 Chromosome PCR Mix All informative autosomal STR markers on chromosome 13 demonstrate abnormal 1 2 2 1 or 1 1 1 marker ratios consistent with trisomy 13 1_320MY_5_Chrs_17 7 13_b_012_Sample_7_00 3Z0MY Mark Sample For Deletion 1_320MY_S_Chrs_17 7 13_b_D12_Sample_7_ODE 3ZOMY C E Mark Sample for Deletion 100 140 180 220 260 300 340 380 420 460 500 6000 4000 2000 i Li RN 6 ae aw 1_320MY_5_Chrs_17 7 13_b_D12_Sample_ _00 3Z0MY F Mark Sample for Deletion 100 140 180 220 260 300 340 380 420 460 500 BI Life Technologies FD Your Molecular amp
26. mport panels by clicking on the Panel Manager in the left navigation window Panel Manager will now appear highlighted in blue Select File and Import Panels Navigate to and select the appropriate 3ZOMY Kit Panel file txt file on your computer Click Import The imported panel will now be displayed in the left navigation window Add a Bin Set to the imported panel by highlighting the panel in the left navigation window Select File and Import Bin Set Navigate to and select the appropriate 3ZOMY Kit Bin Set file txt file on your computer Click Import The imported Bin Set will appear in the Bin Set drop down list Click Apply and OK to confirm the imported panel and bin settings 6 2 Importing GeneMapper Analysis Settings in the GeneMapper Manager a f Open the GeneMapper Manager by selecting Tools and GeneMapper Manager in the GeneMapper main menu or by clicking on the Icon In the GeneMapper Manager select the Analysis Methods tab and then click Import Navigate to and select the appropriate 3ZOMY Kit Analysis Methods file xml file on your computer Click Import The imported Analysis Method will now be listed in the Analysis Methods tab Repeat the process selecting the appropriate tab and importing the corresponding file for Plot Settings Table Settings Report Settings Size Standards select Done to confirm the imported analysis settings Note A matrix file mtx file needs to be generated or
27. must show all the expected peaks 4 To interpret a result as normal for a particular chromosome at least two informative markers consistent with a normal genotype are required with all other markers being uninformative 5 To interpret a result as abnormal for a particular chromosome at least two informative markers consistent with an abnormal genotype are required with all other markers being uninformative 6 Normal allelic pattern is determined by Marker showing two peaks of similar height area and the peak ratio is classified as 1 1 7 Abnormal allelic pattern is determined by a Marker showing two peaks of differing height area and the peak ratio is classified as 2 1 or 1 2 Trisomic Diallelic pattern b Marker showing three peaks of similar height area and the peak ratio is classified as 1 1 1 Trisomic Triallelic pattern 8 The Kit includes three X chromosome counting markers TS2 TS3 and TS7 to be used to determine the number of X chromosomes when monosomy X is suspected 9 X chromosome counting markers The TS2 TS3 and TS7 markers are non polymorphic X chromosome counting markers that may be used to determine the number of X chromosomes when monosomy X is suspected The X chromosome counting markers define sequences present on the X chromosome and an autosomal chromosome that are amplified using identical primers The amplified marker fragments are separated according to length and the X chromosomal copy number is d
28. n tubes and centrifuge briefly to collect the contents Continue to step 4 3 Unused working solution should be aliquoted into 20ul per tube and frozen 4 2 2 Subsequent use Completely thaw the 20ul working solution tubes to be used Continue to step 4 3 4 3 Amplification Setup Under optimal PCR conditions and using the recommended analysis settings see below acceptable results are consistently obtained at genomic DNA concentrations of 1 10n g PCR reaction The 3ZOMY Kit has been validated using a total PCR reaction volume of 25ul Any change to the reaction volume may impair the Kit s performance We recommend using the following controls in every run Negative control no DNA 46 chromosomes from known origin normal DNA 47 chromosomes from known origin for Chromosome 21 trisomy BI Pite Technologies FD Your Molecular amp Cell Technology Partner hc V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 J 9 Culture of Excellence Email customerservice lifetechindia com Web www lifetechindia com Addition of Sample Samples and controls should be added in a dedicated area separated from the reagent preparation amplification and detection areas Each PCR reaction should be prepared as follows Samples Positive Negative Components Control Control 3ZOMY Working Solution from step 4 2 2 cu 20uI 20ul DNA 1 5ul 1 10ng 1 5ul 1 10ng None H2O Up to 5ul Up to 5ul 5 ul Final PCR
29. nce Excessive amount of DNA on DNA concentration is too high amplification DNA concentration is too low Reduced amount of DNA template can result in preferential amplification of one allele resulting in skewing and or uneven amplification of markers Insufficient template on amplification If sufficient extracted and re suspended in a lower volume DNA is degraded or of poor quality Can occur when peak heights are too high or if a poor or incorrect matrix has been applied to the samples Pull up peaks should be excluded from the analysis Dye blobs appear as broad undefined peaks of a single color and tend to occur relatively early in the data Dye blobs should not be included in the analysis The size standard can be corrected manually in the Size Match Editor or re run the Low sizing quality might be caused by insufficient peak height of the size standard fragments or incorrect identification of the fragments by the software due to sample overloading spectral overlap or pre terminated runs size standard mix Data should not be interpreted The PCR product may be re injected and re analyzed Minor voltage changes or urea crystals passing the laser Re inject samples for confirmation Web www lifetechindia com sample with a new Formamide sample is available it can be re F F j FIED Life Technologies P Your Molecular amp Cell Technology Partner V3 Ap ril 2014
30. nt Protocol 1 Select the instrument protocol that you created in step 5 3 2 e Click Ok R GeneMapper Plate Editor Wet ADI Bo ae pot EE ai haal as D02 E02 F02 G02 H02 A03 B03 E pos H03 a ny l l eee wa He pesoj n M f aassst gpm nem ea senm teanas meneg ea ame wether eee ao ee Caze Cmaj BI Life Technologies FED Your Molecular amp Cell Technology Partner haat V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 7 Culture of Excellence Email customerservice lifetechindia com 15 Web www lifetechindia com l b Select the plate created in Step 5 3 3 Status Pending Link the plate by clicking on the yellow plate position indicator which will turn green when linked Start the run on the C green arrow X Foundation Data Collection Version 3 0 No User is logged in Fie view Service Tools Wizards Help gt ESHI E GA Instruments L Results Group 2 Database Manager a B ga3130x1 EJ Plate Manager T Protocol Manager Sije Module Manager E Run History GA Instruments gt ga3130xl gt 3130XL gt Run Scheduler Find Plates Matching These Criteria Type of Search Scan or Type Plate ID 3Z0MY_5_Chrs BI Biological Industries Culture of Excellence EYEPT viewer EE event Log Append Results Ta Instrument Protocol 0 pe
31. omic DNA extracted from whole blood amniotic fluid and chorionic villus samples The product is not intended nor tested to be used for non invasive procedures The 3ZOMY Kit has been validated using the QlIAamp DNA Blood Mini Kit QiagenCat 51104 for extraction of DNA from human whole blood and amniotic fluid AF and QlAamp DNA Mini Kit QiagenCat 51304 for extraction of DNA from chorionic villus CV biopsies lt is recommended that alternative DNA extraction methods and sample materials using the 3ZOMY Kit is carefully evaluated prior to the results being used for diagnostic use 4 2 Working Solution Preparation 4 2 1 First time use If the complete process is performed in one day the working solution should be prepared before preparing the samples The reverse order is advisable only if the samples are prepared the day before amplification or earlier The working solution is prepared by adding the Chromosome PCR Mix yellow white red caps to the PCR Enzyme orange cap Ensure that the Chromosome PCR Mix is completely thawed before use Centrifuge the Chromosome PCR Mix tube briefly to collect the contents Do not vortex the tubes at this step Add 500ul of Chromosome PCR Mix to one tube of PCR Enzyme Carefully mix by pipetting several times from the bottom of the tube Vortex the working solution and centrifuge briefly to collect the contents Add 20ul of the working solution to separate PCR reaction tubes Cap the reactio
32. on the icon Or In the GeneMapper Manager select the Analysis Methods tab b Highlight the desired Analysis Method and then click Open c In the Analysis Method Editor select the Allele tab If the Bin Set has been lost the Bin Set is set to none Make sure that the correct Bin Set is added in this example 3ZOMY d Click OK and Done If the Analysis Method has been modified the samples need to be reanalyzed 6 8 DISCLAIMER RESULTS OBTAINED WITH ANY IVD KIT SHOULD ONLY BE EMPLOYED AND INTERPRETED WITHIN THE WHOLE CLINICAL PICTURE BIOLOGICAL INDUSTRIES LTD CANNOT BE CONSIDERED RESPONSIBLE FOR ANY CLINICAL DECISIONS TAKEN THIS KIT IS NOT INTENDED TO BE USED FOR THE RISK EVALUATION OF TRISOMY 21 1 i r FED Q BI Life Technologies Po ayan Biological Industries ISO 13485 Ph 91 11 42208000 Fax 91 11 42208444 2003 Culture of Excellence Email customerservice lifetechindia com 20 Web www lifetechindia com 7 Interpretation of Results For interpretation of results see Best Practice Guidelines for QF PCR at http www cmgs org 1 At least one peak should be observed for each marker tested The acceptable range for marker peaks analyzed on the 3130 Genetic Analyzer is between 50 and 6000 RFU and for the 3500 Genetic Analyzer between 175 and 32000 RFU Peak heights falling outside this range must not be analyzed 2 The negative control should not show peaks above 50 RFU 3 The positive control
33. rea at all times h Handling of kit components and samples their use storage and disposal should be in accordance with the procedures defined by national biohazard safety guidelines or regulations i Wear powder free disposable gloves laboratory coats and eye protection when handling specimens and kit reagents Wash hands thoroughly after handling specimens and kit reagents 3 2 Spectral Calibration Proper generation of a matrix file is critical for evaluating fluorescent labeled multiplex systems with ABI Genetic Analyzers A matrix must be generated for each individual instrument The Kit has been calibrated with the following dye sets The 5 Color Dye Set BI Cat 20 910 00 required for spectral calibration on ABI 31XX 3500 and 3730 Genetic Analyzers The 5 Color Dye Set Single Capillary BI Cat 20 911 00 for ABI 310 Genetic Analyzer For protocols and additional information about spectral calibration see the 3ZOMY Spectral Calibration protocol that can be obtained by request from BI s technical support team l Piire Technologies SEED On BI Culture of Excellence Email customerservice lifetechindia com Web www lifetechindia com Your Molecular amp Cell Technology Partner V3 Ap ril 2014 Bj d i s ISO 13485 IOIOgICaAI INGUSTIES Ph 91 11 42208000 Fax 91 11 42208444 ES 4 PCR Sample Preparation and Amplification Setup 4 1 DNA Extraction The 3ZOMYkit is for use with human gen
34. s for Sex Chromosome PCR Mix Pite Technologies STD Oy Your Molecular amp Cell Technology Partner haat V3 Ap ril 2014 DI Industries Ph 91 11 42208000 Fax 91 11 42208444 QT Culture of Excellence Email customerservice lifetechindia com 5 Web www lifetechindia com 2 Product Components and Storage Conditions 2 1 Kit Components The Kit includes reagents for 50 tests 2 x 25 using the 5 Chromosome PCR Mix and 25 1 x 25 additional tests each for Chromosome 21 PCR Mix and Sex Chromosome PCR Mix 5 Chromosome PCR Mix Yellow Cap 20 900 50 part 1 X2 500ul Chromosome 21 PCR Mix White Cap 20 900 50 part 2 X1 500ul Sex Chromosome PCR Mix Red Cap 20 900 50 part 3 X1 500ul PCR Enzyme Orange Cap 20 900 50 part 4 X4 25ul 2 2 Reagents Required But Not Supplied Reagent Preparation Consumables for the thermal cycler Micropipette dispenser with aerosol barrier tips or displacement tips Disposable protective gloves powder free PCR Amplification Thermal cycler ABI GeneAmp PCR System 9700 using 9600 mode Micropipette dispenser with aerosol barrier tips or displacement tips Detection ABI Genetic Analyzer ABI 310 3100 3130 3500 3730 Performance optimized polymers POP 4 or POP 7 Hi Di Formamide Genetic Analysis Grade 1x Genetic Analyzer Buffer Micropipette multipipette dispenser with aerosol barrier tips or displacement tips Spectral calibration dye set 5 Color Dye Set BI Cat 20
35. ts E Resuts Group GA Instruments gt ga3130xl gt Plate Manager E Database Manager Find Plates Matching These Criteria ng 34 20 Type of Search Barcode Vv t Protocol Manager Qh Module Manager 5 Run History EJEPT Viewer E Event Log Ta Instrument Protocol Gi Spatial Calibration Vi EI cepillary Viewer BB capiarray Viewer Gil Spectral Calibration Be Reextraction a 3130x B instrument Status EXEPT Chart E Event Log Hi Spatial Run Schedule Run Scheduler Piate View run view E Capillaries Viewer Bl capiArray Viewer Gi spectral Viewer amp Manual Control service Log Fill out the new Plate Dialog Name Enter a name for the plate a b Application GeneMapper Generic 9 Plate Type 96 Well d Owner Name Enter the name of the owner e Operator Name Enter the name of the operator f Click OK fi New Plate Dialog Name 3ZOMY_5_Chrs n o Application GeneMapper Generic v Plate Type 96 VVell x Owner Name Operator Name Life Technologies SED On B I Your Molecular amp Cell Technology Partner LN Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 28 7 Culture of Excellence Email customerservice lifetechindia com Web www lifetechindia com V3 April 2014 14 Fill out the GeneMapper Plate Editor a Sample Name Enter the sample names b Comment Optional c Results Group 1 Location where the raw data files fsa files will be saved d Instrume
36. usive 0751 44 0 75 1 54 RC 1 is used when peak distance is lt 24bp RC 2 is used when peak distance is 224bp Homozygous markers are considered uninformative 15 Height Ratio a Height ratio may be calculated when an area ratio is classified as inconclusive The same RC is applied as for area ratio calculation b Allele ratios that fall between the normal and abnormal ranges are classed as inconclusive and must be re analyzed c If both normal and abnormal allelic patterns are obtained for a particular chromosome it is recommended that follow up studies be performed to identify the reason BI Pite Technologies n Your Molecular amp Cell Technology Partner haat V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 25 J Culture of Excellence Email customerservice lifetechindia com 23 Web www lifetechindia com 8 Troubleshooting If heterozygous markers results fall in the inconclusive range or are not detected it may be due to a number of factors such as 8 1 Unusual conditions Profiles demonstrate the presence of two genotypes a second genotype is seen at all the markers Skewed allele ratios and or minor third allele Chromosome peak are detected on a chromosome specific Skewed and or extra peaks mosaicism group of markers 15 25 of mosaicism should be investigated further could be detected by QF PCR STRs occasionally demonstrate incomplete repeat sequences The int
37. wing QF PCR Data a Highlight the sample row for the sample s to be selected for analysis in the GeneMapper main window b Display the sample plot by selecting Analysis and Display Plots or by clicking on the Hm icon In the Samples Plot window select the 3ZOMY Plot Setting from the drop down menu To add or remove information shown in the peak labels open the GeneMapper Manager select the Plot Settings tab Highlight the 3ZOMY Plot Setting and then click Open e Select the Labels tab In the Show Labels box select the information to be shown on the labels by using the drop down lists in the Label 1 to Label 4 options To confirm the modified label format click OK and Done Changes in the Plot Settings will be directly applied in the Samples Plot without re analyzing the samples BI Piire Technologies n Your Molecular amp Cell Technology Partner am V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 25 J Culture of Excellence Email customerservice lifetechindia com 18 Web www lifetechindia com 6 5 Using the GeneMapper Report Format The Report format can be used to facilitate the calculation of peak area ratios for the markers included in the analysis a Report Manager Untitled lt _ e File Edit View Tools Help Report Setting BZOMY Aneuploidy ISE DE EAOa E Sample Name Ma Ratio Comment Dye Size1 Size 2 Size 3 A1JA3 A1 A2 PeakAreal PeakArea2 PeakArea3
38. yndrome gt 3ZOMY can be used for maternal cell contamination detection v 3ZOMY is a CE IVD certified kit BI provides a complete technical support service 11 3 Contact Information Life Technologies India Pvt Ltd 306 Aggarwal City Mall Opposite M2K Pitampura Delhi 110034 INDIA Ph 91 11 42208000 8111 8222 Mobile 91 9810521400 Email customerservice lifetechindia com Web www lifetechindia com B P Life Technologies Sry Your Molecular amp Cell Technology Partner 7 V3 Ap ril 2014 Biological Industries Ph 91 11 42208000 Fax 91 11 42208444 2 J Culture of Excellence Email customerservice lifetechindia com 31 Web www lifetechindia com
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