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Quantifiler Kits User`s Manual (PN 4344790F)

1. 123455 7 8 8 101112181415161718192021 2223242526 27 28 29 UH 323334353637 J8 3940 4142434445 Cycle Number DetS RA 1234535 1 8 9101112131415151718 19 2021 2223 242526 21 20 28 1031 32333435 3637 18 3940 414243 4445 Cyclo Number Baseline spikes with certain reactions and normal amplification with other reactions Mechanical or optical misalignment 1 Localize the wells that contain baseline spikes 2 Run the TaqMan RNase P Instrument Verification Plate PN 4310982 3 Perform the instrument function tests Ifa function test fails contact your Applied Biosystems Service Representative If all functional tests pass the reaction plate or the door of the instrument may not have been aligned properly during the run Note See your instrument user guide for instructions on how to perform instrument function tests S O q voresyndwy BunooysajgqnoL Table 5 4 Troubleshooting amplification plots continued A jenueyy sA sN Suy 1 jypueno Recommended Action Observation Possible Cause vata Uncalibrated pure dyes If the pure dyes are not calibrated run the pure we ELLE HELLLEEEELLLEEELL LE damage to the lens
2. Standards Table 3 1 shows an example of one standards dilution series with the Dilution Series concentrations ranging from 50 ng uL Std 1 to 0 023 ng uL or Example 23 pg uL Std 8 A sample at the lowest concentration 2 uL per reaction contains on average 14 to 16 copies of a diploid single copy locus and 7 to 8 copies of a haploid single copy locus Table 3 1 Standards dilution series example Concentration s Dilution Standard ng uL Example Amounts Minimum Amounts Factor Std 1 50 000 50 uL 200 ng uL stock 10 uL 200 ng uL stock 4x 150 uL T 9E glycogen buffer 30 uL T oEo buffer Std 2 16 700 50 uL Std 1 10 uL Std 1 3x 100 uL T 9E glycogen buffer 20 uL T E 9 buffer Std 3 5 560 50 uL Std 2 10 uL Std 2 3x 100 uL Ty 9E9 glycogen buffer 20 uL T E buffer Std 4 1 850 50 pl Std 3 10 uL Std 3 3x 100 uL Ty 9E9 glycogen buffer 20 uL T E buffer Std 5 0 620 50 uL Std 4 10 uL Std 4 3x 100 uL T 9E glycogen buffer 20 uL T E 9 buffer Std 6 0 210 50 uL Std 5 10 uL Std 5 3x 100 uL T 9E glycogen buffer 20 uL T E 9 buffer Std 7 0 068 50 uL Std 6 10 uL Std 6 3x 100 uL T 9E glycogen buffer 20 uL TjoEo buffer Std 8 0 023 50 uL Std 7 10 uL Std 7 3x 100 uL T 9E glycogen buffer 20 uL T E 9 buffer Quantifiler Kits User s Manual 3 3 Chapter 3 PCR Amplification Preparation Guideli
3. Quantifiler Human Kit Quantifiler Y Kit A260 DI Sample Result Result Di o Di j ng ul ng ul Result R Di o Di Result Di o pitt ng uL from DI ng uL from DI A260 A260 007 A 2 74 2 502 2 094 23 6 16 3 3 547 29 4 41 8 007 B 0 685 0 756 1 007 47 0 33 2 0 950 38 6 25 7 007 C 0 137 0 176 0 272 98 8 54 6 0 220 60 3 24 6 9948 A 1 9 2 286 2 215 16 6 3 1 2 562 34 8 12 1 9948 B 0 475 0 496 0 677 42 5 36 4 0 634 33 5 27 8 9948 C 0 095 0 103 0 144 51 1 39 3 0 115 21 1 11 6 Human 2 2 2 270 2 887 31 2 27 2 1 792 18 6 21 1 genomic A Quantifiler Kits User s Manual 6 43 Chapter 6 Experiments and Results Table 6 17 Comparison with A go and dye intercalation 7900HT SDS Quantifiler Human Kit Quantifiler Y Kit Asso DIA Sample Result Result Di o Dj ng ul ng ul Result R Di o i Result DMT o pitt ng uL A from DI ng uL A from DI 260 260 Human 0 55 0 584 0 805 46 3 37 9 0 379 31 0 35 0 genomic B Human 0 11 0 134 0 184 67 4 36 9 0 105 4 3 21 7 genomic C K 562 A 2 76 1 317 1 631 40 9 23 9 0 000 n d n d K 562 B 0 69 0 365 0 474 31 4 29 9 0 000 n d n d K 562 C 0 138 0 104 0 060 56 2 42 1 0 000 n d n d Raji 1 A 2 1 271 1 702 14 9 33 9 2 101 5 0 65 2 Raji 1 B 0 5 0 351 0 483 3 4 37 7 0 547 9 5 56 1 Raji 1 C 0 1 0 085 0 094 6 4 10 6 0 109 8 5 28 3 Raji 2 A 1 98 1 262 1 555 21 5 23 2 2 134 7 8 69 1 Raji 2 B 0 495 0 357 0 446 9
4. Q Q oO 2 g 7 n e n ke IOO n m ogl o w gt Figure 2 2 Example plate setup of reactions using repeat pipettors Quantifiler Kits User s Manual 2 9 Chapter 2 Software Setup Setting Up a Plate Document Overview Setting up a plate document to run Quantifiler kit assays involves 1 Creating a Blank Plate Document page 2 10 Creating Detectors the first time only page 2 11 Adding Detectors to the Plate Document page 2 14 Applying Detectors for Standards page 2 15 Applying Detectors for Unknown Samples page 2 17 Adding Sample Names for Unknown Samples page 2 18 Setting Thermal Cycler Conditions page 2 19 a Aa Au eu Saving the Plate Document page 2 21 2 5 3 Q 2 ku S O 2 ja 7 r N Creating a Blank To create a blank plate document Plate Document 1 Ifthe SDS software is not already started select Start gt Programs gt ABI Prism 7000 gt ABI Prism 7000 SDS Software 2 In the SDS software select File gt New to open the New Document dialog box x Assay Absolute Quantitation Default settings Container 9 Well Clear Template Blank Document 2 10 Quantifiler Kits User s Manual Setting Up a Plate Document To create a blank plate document continued 3 Click OK to use the de
5. N I Omit Well Passive sdd Detector Remove ROX Note Samples with identical sample names are treated as replicates by the SDS software Results for replicate reactions are grouped together automatically for data analysis 2 18 Quantifiler Kits User s Manual Setting Up a Plate Document Setting Thermal Before running a Quantifiler kit assay you need to make two changes Cycler Conditions to the default thermal cycler conditions Thermal profile e Sample volume To set thermal cycler conditions 1 In the plate document select the Instrument tab 2 Press the Shift key and click within the Stage 1 hold step Stage 1 hold step y 50 C for 2 minutes to select it 2 Thermal Profile Auto Increment 2 Stage 1 Stage 2 Stage 3 iw 77 Reps ff Reps ff Reps fio N Q F Press the Shift E key while you a T click within the o Add Cycle Add Hold Add Step Sample Volume 50 uL I Dissociation Protocol 60 0 Cc M 9600 Emulation 3 After the hold step is selected press the Delete key Thermal Profile Auto Increment Stage 1 Stage 2 stage 3 Reps i Reps fi Reps 0 5 0 5 0 10 00 15 0 0 Stage 1 hold step is selected Add Step Sample Volume 50 ul I Dissociation Protocol fon C 9600 Emulation
6. 2 2 2222 5 10 Troubleshooting Amplification Plots 5 12 ASS Hr QUE anreisen 5 16 Quantifiler Kits User s Manual 5 1 Chapter 5 Interpretation of Results Checking Analysis Settings The validity of the results requires correct analysis settings Checking To check analysis settings on the 7000 SDS Analysis Settings on the 7000 SDS 1 Ifthe SDS software is not already started select Start gt Programs gt ABI Prism 7000 gt ABI Prism 7000 SDS Software 2 Select File gt Open 3 Locate the plate document for the assay run of interest select it then click Open 4 Select Analysis gt Analysis Settings 5 For all detectors confirm that the settings are as shown below Analysis Settings x Detector All v r Settings for Threshold 0 200000 Baseline Start cycle 6 Baseline End cycle 15 F Use System Calibration OK amp Reanalyze Cancel Apply 6 Ifthe analysis settings differ from those shown in step 5 a Change the settings to match those in step 5 b Click Apply c Click OK amp Reanalyze to close the dialog box and reanalyze the plate document d View the results using Chapter 4 Data Analysis and Results 5 2 Quantifiler Kits User s Manual Checking Analysis Settings Checking To check analysis settings on the 7900HT SDS Analysis Settings on the 7900HT SDS 1 Ifthe SDS soft
7. ng uL Standard Standard ee Cr Mean Deviation Cr Mean Deviation 0 068 31 90 0 28 33 38 0 44 0 023 33 45 0 48 35 19 0 73 Figure 6 1 and Figure 6 2 show the Cy pay results for all 8 quantification standard dilutions reactions using the Quantifiler Human kit and the Quantifiler Y kit Day 2 Mean 40 35 30 Day 1 123123 Instruments 50 16 7 5 56 1 85 0 62 0 21 0 068 0 023 DNA quantity ng uL Figure 6 1 Precision using the Quantifiler Human kit Quantifiler Kits User s Manual 6 5 Chapter 6 Experiments and Results Day1 Day 2 Mean 40 ee od ooo 3 T2323 Ze Instruments oy oo MMII 15 50 16 7 5 56 1 85 0 62 0 21 0 068 0 023 DNA quantity ng uL Figure 6 2 Precision using the Quantifiler Y kit The data show that at lower DNA concentrations the Cy values increased and the standard deviation increased most likely because of stochastic effects For each sample the C values obtained using the Quantifiler Human kit are lower than those obtained using the Quantifiler Y kit because there are two copies of the autosomal human target locus and only one copy of the Y chromosome target locus The C values do not vary significantly from run to run or from instrument to instrument The C value from one sample run on three different 7000 instruments varies with an average standard deviation of 0 3 Systematic differences between instrument
8. DNA C values with Quantifiler Human DNA Calculated Quantitation with Quantifiler Human ee e Auto Baseline Manual 32 10 OL et 29 waren 3 De 5 A 3 Oo 60 zes u a Ne wre 07 5777705 Pence 2 eooo DR 777 7077 12 KL CET SL 77777 23 0 40 80 0 40 80 Sample Sample Figure 6 27 Comparison of C values and the corresponding calculated quantities Auto Baseline and Manual analysis modes Quantifiler Human Kit 6 4 5 Discussion 6 4 5 1 Precision and Accuracy 7500 System Comparison No statistically significant differences were observed in Cp slope and R values between replicate samples run on the 7500 System SDS Software v1 2 3 using both Quantifiler kits 7500 to 7000 System Comparison Statistically significant differences in Cp slope and R values were observed in samples run on the 7500 System SDS Software v1 2 3 versus the 7000 System SDS Software v1 0 using both Quantifiler kits However the data obtained from both instrument types are within the previously established parameter ranges published in the Quantifiler User s Manual Chapter 5 Table 5 1 6 4 5 2 Reproducibility and Sensitivity Sensitivity Similar Cy values and calculated DNA quantities were obtained at each of the standard curve concentrations demonstrating similar sensitivity results between the 7000 System SDS Software v1 0 and 7500 System SDS Software v1 2 3 6 64 Quantifiler Kits User s Manual Section
9. Figure 6 22 Average R values Replicate standard curves 6 4 4 2 Reproducibility and Sensitivity 6 58 Two sample DNAs were quantitated for this experiment Eight 3 fold serial dilutions for each sample were run five replicates per dilution 40 wells per sample The Cy values were generated in Manual analysis mode then the quantities were calculated using the standard curve on each plate Figure 6 23 shows average Cy values each point n 90 replicates across a set of four serial dilutions 2 ng uL to 0 5 ng uL with the Quantifiler Human Kit and the corresponding quantitated concentrations for one DNA sample Similar results were obtained for the second DNA sample and the Quantifiler Y Human Male Kit data not shown As the data show differences in Cy values do not affect calculated quantities calculated quantities were normalized resulting in comparable concentrations on both instrument types Quantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Cr 31 28 25 DNA Cr values with Quantifiler Human kit 7000 Instruments e 7500 Systems O7 nd a nate EEE ome pas u Poste Era il 0 40 Sample Calculated Quantities ng ul DNA Calculated Quantitation with Quantifiler Human kit e 7000 Instruments e 7500 Systems e ee oo Po EEK 9 oe ee o ee o 40 8
10. Quantifiler Kits User s Manual 2 41 Q So I 2 g 7 n io n ke a 2 g 3 fe 2 2 DO 7 Pr I Q f N Chapter 2 Software Setup Creating a Plate After you create a template you can use it to create a plate document Document from a Template To create a plate document from a template l If the SDS software is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 Select File gt New and in the New Document dialog box and make the following selections For Assay select Absolute Quantitation For Container select 96 Well Clear Plate For Template select an appropriate template from the list Note Ifthe template is not available in the list click Browse to locate and select an appropriate template Complete the plate document setup e Copy detectors page 2 34 e Apply detectors for standards page 2 35 e Apply detectors for unknown samples page 2 36 e Set thermal cycler conditions page 2 38 Note The tasks that you perform vary according to which settings were defined in the template Save the plate document page 2 39 Note For Files of Type select ABI PRISM SDS Single Plate sds 2 42 Quantifiler Kits User s Manual Setting Up a Plate Document Template Q oO I 2 g 7 n ie n
11. a zw L o m li Eli J ilill u 1 Bg 6D owo_Run_demo_3100_2003 05 07_519_0min_D05_07 fsa Omin owo _Run_demo_3100_2003 05 07_519_Omin_D0S_07 fs we 40min BY owo Run deme 1100_2003 05 07 810 Drin 00807 toa Oran owe Run der a 100 2003 05 072618Jpmin D087 ten 7 ran en 4 ve a N meee dadha 1 l i TB 108 owo_ Run demo 2100_2002 05 07_510_Imin_ED65_00 fsa Imin E owo _Run_demo _2003 05 07_510_imin re SE EB 10Y cwe_Run_demo_3100_2003 05 07_619_tmin_ 05_09 tsa 1min ewe _Run_ ee en 19_1min_ EDS 1400 1 ER ini 2 700 Bati midis ed A aL een zn nen Ey 3 a lu Mi A ch ems Ue r EAR S FE re 3109 309 9 10 Si 70 85 fa omm oes 3003 05 07 510 rnin J06 D2 tzu Smin a Sees ers nen 3100_2003 05 07_619_9min_A00_02 ME SR owo_Run_der Te p100 200s aS 07 519_3min_A00_02 tsa 3min 2100 Fo HER 4 11 U iii oer eee N i ah 9B ewe_Run_demo_3100_2003 05 07_519_4min_DDO_DB 1sa 4min DE 86 cwe_Run_demo_3100_2003 08 07 508 18 oa 9Y cwe_Run_demo_3100_2003 05 07_819_4min_DD6_08 5a Amin BGR owo_Run_ dem 100 2003 05 0726 Sion Anin Doe D0800 tsa aim EK m 150 Ba 210 20 70 300 330 300 i y a A 2100 100 5 di wi a eNO meres Cay re oe AE 138 sewc_Run_demo_3100_2003 05 07_519 Amin_FOB 12 152 min EIER 120 cee Run_demo 3100 2002 05 07 510 bmin_FOb_12 fsa Smin O 13Ycuc Run demo 3100_ 2003 0507 619 8min _F06_12 tsa Smin ER 1R owo Run demo 3100_2902 05 07 610 8min FO6_12 lt sa Smin 2100 10
12. oO n ke Barcode eal Cancel Quantifiler Kits User s Manual 2 31 Chapter 2 Software Setup Creating Before you set up the plate document you need to create detectors in Detectors the SDS software for running Quantifiler kit assays After the detectors are created you do not need to create detectors for subsequent runs of Quantifiler kit assays and you can skip to Copying Detectors to the Plate Document on page 2 34 To create detectors 1 With a new plate document open select Tools gt Detector Manager 2 Create a detector for the Quantifiler Human kit a In the lower left part of the Detector Manager click New then complete the dialog box Add Detector x Name Quantifiler Human Group Default z Description Reporter FAM Color m Notes Created Jul 17 2003 3 07 08 PM Last Modified Jul 17 2003 3 07 08 PM Cancel b Click OK to return to the Detector Manager a 2 o 3 fe 2 2 DO 7 Pr I Q f N 2 32 Quantifiler Kits User s Manual Setting Up a Plate Document To create detectors continued 3 Create a detector for the Quantifiler Y Human Male kit a In the Detector Manager click New and complete the dialog box Ada petector S Name Quantifiler Group Default x Description Reporter FAM Quencher Non Fluorescent Color No
13. Green R Roinestad I Boland C Hennessy L Developmental Validation of the Quantifiler Real Time PCR Kits for the Quantification of Human Nuclear DNA Samples Journal of Forensic Science July 2005 Vol 50 No 4 Kutyavin I V Lukhtanov E A Gamper H B and Meyer R B 1997 Oligonucleotides with conjugated dihydropyrroloindole tripeptides base composition and backbone effects on hybridization Nucleic Acids Res 25 3718 3723 Lakowicz J R 1983 Energy Transfer In Principles of Fluorescence Spectroscopy New York Plenum Press 303 339 Martens H and Naes T 1989 Multivariate Calibration Chichester John Wiley amp Sons Scientific Working Group on DNA Analysis Methods Revised validation guidelines approved July 2003 Available at http www fbi gov hq lab fsc backissu july2004 standards 2004_03_standards02 htm Quantifiler Kits User s Manual Bibliography 1 Bibliography 2 Quantifiler Kits User s Manual Index Numerics 5 nuclease assay 1 3 1 4 7000 SDS analysis settings checking 5 2 baseline settings 4 3 detectors creating 2 11 detectors settings 5 2 fluorescence detection on 1 7 PCR reactions running on 3 7 plate document analyzing 4 3 plate document setting up 2 10 results viewing 4 4 starting 2 5 supported configuration 2 4 template setting up 2 22 threshold settings 4 3 validation SDS v1 0 software 6 3 validation SDS v1 2 3 software 6 66 7500 Real Time PCR System vali
14. a 7 r I Q oS O N 2 28 Quantifiler Kits User s Manual About Plate Documents Example Plate You can arrange the reactions in any well of the reaction plate but Document Setup you need to set up the plate document so that it corresponds exactly to the arrangement of the standards and unknown samples in the wells of the reaction plate Figure 2 3 shows one example of arranging reactions when running two Quantifiler kit assays on one 96 well plate e Wells Al through D12 gray correspond to reactions using the Quantifiler Human kit e Wells El through H12 white correspond to reactions using the Quantifiler Y kit Note For each Quantifiler kit assay there are eight DNA quantification standards and two reactions for each standard See Preparing the DNA Quantification Standard on page 3 2 for more information about the DNA quantification standards 1 2 3 4 5 6 7 8 9 10 11 12 Std 1 Std1 Std 2 Std 2 Std3 Std 3 Std 4 Std 4 Std5 Std 5 Std 6 Std 6 Std 7 Std7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std5 Std 6 Std 6 Std 7 Std 7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNK
15. 4 Click Print to print the report Exporting the You can export the results in tab delimited txt format and later Results open the exported files using spreadsheet software To export the results 1 Select File gt Export 2 Select the results to export e Setup Table e Results Table e Multicomponent e Clipped Q N D c lt s a N Q N a I So N 3 Select whether you want to export data from all wells or selected wells 4 Select the SDS format of data to export 5 Select Group by replicates if you want the replicates to be grouped together in the exported results 4 10 Quantifiler Kits User s Manual Viewing Results To export the results continued 6 Locate then select the folder where you want to save the exported results file 7 Enter the File Name then click Export Quantifiler Kits User s Manual I 2 g 7 oO D D gt 5 D lt o 7 Chapter 4 Data Analysis and Results Q N D c lt Ss fa N Q N a I So 4 12 Quantifiler Kits User s Manual 03 2012 Part Number 4344790 Rev F Interpretation of Results This chapter covers Checking Analysis Settee occ sce ui 5 2 Examining the Standard Curve san kiera 5 4 Troubleshooting the Standard Curve 222222220 5 6 Using the Internal PCR Control System
16. Exporting the Results page 4 10 For information about interpreting and troubleshooting the standard curve see Examining the Standard Curve on page 5 4 and Troubleshooting the Standard Curve on page 5 6 To view the standard curve 1 In the Results tab select the Standard Curve tab 2 In the Detector drop down list select the detector that corresponds to the kit that you are using e Quantifiler Human or e Quantifiler Y 3 View the Cy values for the quantification standard reactions and the calculated regression line slope intercept and R values The amplification plot can display one of the following e Plot of normalized reporter signal R versus cycle number for each reaction e C versus well position on the assay plate For more information about the amplification plot see Real Time Data Analysis on page 1 10 Quantifiler Kits User s Manual Viewing Results Viewing the For troubleshooting information see Troubleshooting Amplification Amplification Plot Plots on page 5 12 To view the amplification plot 1 After the run is finished select the Results tab then select the Amplification Plot tab 2 In the Detector drop down list select the detector e Quantifiler Human or Quantifiler Y e IPC 3 Select the appropriate samples in the 96 well grid or the sample table to the left of the amplification plot 4 Make sure that the
17. Quantifiler Kits User s Manual 2 5 3 Q 2 ku S O 2 a 7 N Chapter 2 Software Setup 2 20 To set thermal cycler conditions continued 4 Make sure that the thermal profile appears as follows Stage 1 Stage 2 5 Change the Sample Volume to 25 uL and make sure that the 9600 Emulation box is selected Note Selecting the 9600 Emulation box reduces the ramp rate Thermal Profile Auto Increment Stage 1 Stage 2 Reps fi Reps Set the volume to Add Step Sample Volume 25 05 25 uL F Dissociation Protocol 60 0 C 600 Emulation Make sure that this box is selected Quantifiler Kits User s Manual Setting Up a Plate Document Saving the Plate Before running the reaction plate save the plate document as an SDS Document Document sds file Note To save the plate document as a template see Setting Up a Plate Document Template on page 2 22 To save the plate document 1 Select File gt Save 2 Select the location for the plate document 3 Enter a file name 4 For Save as type select SDS Documents sds 5 Click Save N Q Q oO n g n n e x n gt Quantifiler Kits User s Manual 2 21 Chapter 2 Software Setup Setting Up a Plate Document Template Purpose A plate document template re
18. Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Examples of the user attention words appear below Note The size of the column affects the run time Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password IMPORTANT You must create a separate Sample Entry Spreadsheet for each 96 well plate Safety alert words also appear in user documentation For more information see Safety Alert Words on page ix Quantifiler Kits User s Manual Safety Safety Alert Words Chemical Hazard Warning Safety Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N 7Co Indicates a potentially hazardous situa
19. and 22 were casework DNA extracts from fabric clothing or surface swabs All DNA samples were prepared by organic extraction The DNA samples were quantified using the QuantiBlot Human DNA Quantitation Kit Applied Biosystems and the Quantifiler Human kit performed on both the 7000 SDS and 7900HT SDS The QuantiBlot kit was used in the chemiluminescent autoradiography mode Tests with the Quantifiler kits for the 7000 SDS and 7900HT SDS were performed according to the standard procedure Using the results from the Quantifiler Human kit and the 7000 SDS between 0 8 and 1 4 ng human genomic DNA was added to each Identifiler kit reaction with many of the samples added at approximately 1 0 ng per reaction Identifiler kit reactions were processed on the ABI PRISM 3100 Genetic Analyzer and analyzed using GeneScan Software v3 7 1 and Genotyper Software v3 7 for use with the Windows NT operating system The STR profiles obtained from using the Identifiler kit were analyzed Successful STR profiles produced complete profiles with peak heights between 200 and 4000 relative fluorescence units RFU Quantifiler Kits User s Manual Results Section 6 3 Casework Sample Analysis According to the results from the Quantifiler Human kit reactions run on the 7000 SDS the range of DNA concentrations was 0 06 ng uL to 2 61 ng uL Table 6 19 Successful STR profiles were obtained for the 28 samples that were analyzed Figure 6 18 Th
20. fluorescence detection of 1 7 G geometric phase amplification plot 1 11 glycogen adding to T E buffer 3 2 guidelines chemical safety x chemical waste safety xi waste disposal xii H hazards biological xii chemical waste xi Quantifiler Kits User s Manual IMPORTANT description ix Information Development department contacting xiv instrument powering on for the 7000 SDS 2 6 for the 7900HT SDS 2 27 Internal PCR Control system See IPC system IPC system about assay 1 3 components 5 10 detectors for creating on the 7000 SDS 2 13 detectors for creating on the 7900HT SDS 2 34 interpreting results of 5 10 invalid results from 5 11 italic text when to use vii L linear phase amplification plot 1 11 M manuals See documentation related materials not included with Quantifiler kits 1 17 menu commands conventions for describing vii minor groove binder description 1 4 mixture studies on the 7000 SDS 6 21 on the 7900HT SDS 6 40 MSDSs description x obtaining x xiv referring to x xi multicomponent data exporting 7900HT SDS 4 10 printing 7900HT SDS 4 10 processing of 1 9 Quantifiler Kits User s Manual N negative results 5 11 New Document dialog box 7000 SDS 2 10 7900HT SDS 2 31 nonfluorescent quencher description 1 4 O Optical Adhesive Cover sealing plate with 3 6 P passive reference multicomponent analysis usein 1 9 normalization using 1 10 selecting i
21. 0 1 2 3 4 5 15 60 High molecular weight DNA melee oa S 3 Figure 6 10 DNase degradation of human genomic DNA The results from the Quantifiler kits Figures 6 11 and 6 12 showed higher Cy values with longer DNase exposure times corresponding to lower amounts of amplifiable DNA in the samples According to results from the Quantifiler Human kit the amount of amplifiable DNA decreased from 12 0 ng uL to 1 2 ng uL at the 5 minute time point and to 0 11 ng uL at the 15 minute time point At the 60 minute time point no amplifiable DNA was detected Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Delta Rn 4 1234567 8 91011121314 1516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 6 11 Degraded DNA Quantifiler Human kit amplification plot Minutes 0 1 2 3 4 5 Delta Rn 15 60 1 123 4 5 6 7 8 910111213141516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 6 12 Degraded DNA Quantifiler Y kit amplification plot Using the DNA quantification results from the Quant
22. 2 12 creating 7900HT SDS 2 32 Quantifiler Y detector creating 7000 SDS 2 12 creating 7900HT SDS 2 33 quantity assessing 5 16 entering for detector 7000 SDS 2 16 exporting 7000 SDS 4 6 printing 7000 SDS 4 6 viewing 7000 SDS 4 5 viewing 7900HT SDS 4 9 Index 4 R R value interpreting 5 4 viewing 7000 SDS 4 4 viewing 7900HT SDS 4 8 radioactive waste handling xii raw data about 1 8 printing 7000 SDS 4 6 printing 7900HT SDS 4 10 reactions examples of arranging 2 8 2 29 real time data analysis 1 10 regression line formula 5 4 replicates grouping in exported results 7900HT SDS 4 10 results how grouped 2 18 2 37 report exporting and printing on the 7000 SDS 4 6 exporting and printing on the 7900HT SDS 4 10 viewing on the 7000 SDs 4 5 reporter signal normalized about 1 10 viewing in amplification plot 7000 SDS 4 4 viewing in amplification plot 7900HT SDS 4 8 reproducibility 6 7 results exporting 7900HT SDS 4 10 viewing 7000 SDS 4 4 viewing 7900HT SDS 4 8 R See reporter signal normalized ROX selecting in Well Inspector 7000 SDS 2 15 2 17 selecting in Well Inspector 7900HT SDS 2 36 2 37 Quantifiler Kits User s Manual S safety biological hazards chemical waste xi safety alert words CAUTIONS ix DANGERS ix description ix IMPORTANTS ix WARNINGS ix sample name adding for unknown samples 7000 SDS 2 18 adding for unknown samples 7900HT SDS 2 37 entering for
23. 6 1 12 Comparison with Azs and Dye Intercalation The concentration of DNA was measured for 13 human genomic DNA samples using the A method a dye intercalation method and the Quantifiler kits DNA Samples Six human genomic DNA samples were obtained from different Tested commercial sources Table 6 12 Human DNA samples tested with A and dye intercalation DNA Sex Extraction Source 007 Male Blood 9948 Male Cell line Quantifiler Kits User s Manual 6 31 Chapter 6 Experiments and Results 6 32 Experiment Table 6 12 Human DNA samples tested with A and dye intercalation DNA Sex Extraction Source Human genomic Male Blood Raji 1 Male Cell line Raji 2 Male Cell line K 562 Female Cell line Using the concentrations provided by the supplier the DNA samples were diluted to 2 0 ng uL A 0 5 ng uL B and 0 1 ng uL C Note All dilutions were made in T E Buffer with 20 ug mL glycogen added as a carrier and stabilizer All sample dilutions were quantified using the following methods As Because the concentrations of the dilutions extended below the detection limit of the spectrophotometer ultraviolet absorbance at 260 nm was measured for only the highest dilution 2 0 ng uL DNA concentration was calculated from the formula Concentration ug mL 50 x Aygo The results calculated for the 2 0 ng uL dilutions were then extrapolated for the
24. Exponential Signal is detected and increases in direct proportion to the increase of PCR product As PCR product continues to increase the ratio of AmpliTaq Gold polymerase to PCR product decreases During the geometric phase amplification is characterized by a high and constant efficiency It occurs between the first detectable rise in fluorescence and before the beginning of the linear phase During the geometric phase a plot of DNA concentration versus cycle number on a log scale should approximate a straight line with a slope Typically the SDS system is sufficiently sensitive to detect at least 3 cycles in the geometric phase assuming reasonably optimized PCR conditions When the template concentration reaches 1078 M PCR product stops accumulating exponentially Phase 2 Linear During the linear phase the slope of the amplification plot decreases steadily At this point one or more components of the PCR has decreased below a critical concentration and the amplification efficiency begins to decrease This phase is termed linear because amplification approximates an arithmetic progression rather than a geometric increase Because the amplification efficiency is continually decreasing during the linear phase it exhibits low precision Phase 3 Plateau The amplification plot achieves the plateau phase when the PCR stops the R signal remains relatively constant and the template concentration reaches a plateau at about 107
25. Oo Quantifiler Kits User s Manual 2 43 Chapter 2 Software Setup a o 3 fe 2 2 m 7 Fr I Q oS O N 2 44 Quantifiler Kits User s Manual 03 2012 Part Number 4344790 Rev F Chapter 3 PCR Amplification Quantifiler Kits User s Manual PCR Amplification This chapter covers Preparing the DNA Quantification Standard 3 2 Preparing the Reactions ans 000060 0bepedseetans 3 5 Running the Reactions on nah ann 3 7 Quantifiler Kits User s Manual 3 1 Chapter 3 PCR Amplification Preparing the DNA Quantification Standard Required e Pipettors Materials e Pipette tips e Quantifiler Human DNA Standard Note The same standard can be used for both Quantifiler kits Ty Eo buffer 10 mM Tris HCl pH 8 0 0 1 mM Na EDTA 20 pg mL glycogen optional Note If you use T E buffer with glycogen you can store the DNA quantification standards for up to 2 weeks at 2 to 8 C Guidelines for The standard dilution series example shown in Table 3 1 on page 3 3 Calculating the is suitable for general use Standards IMPORTANT Applied Biosystems recommends Dilution Series Three fold dilution series with eight concentration points in the standard series for each assay e Minimum input volume of 10 uL DNA for dilutions to ensure accuracy of pipetting 3 2 Quantifiler Kits User s Manual Preparing the DNA Quantification Standard
26. Select File gt New and in the New Document dialog box and make the following selections For Assay select Absolute Quantitation For Container select 96 Well Clear For Template select an appropriate template from the list Note Ifthe template is not available in the list click Browse to locate and select an appropriate template 2 5 3 Q 2 ku S O 2 a 7 N 3 Complete the plate document setup e Add detectors to the plate document page 2 14 e Apply detectors for standards and for unknown samples page 2 15 and page 2 17 Set thermal cycler conditions page 2 19 Note The tasks that you perform vary according to which settings were defined in the template 4 Save the plate document page 2 21 Note For Save as type select SDS Documents sds 2 24 Quantifiler Kits User s Manual Section 2 2 7900HT SDS Software Setup Section 2 2 7900HT SDS Software Setup This section covers AEE ie Oils Heke re ee 2 26 Starting the 7900HT Real Time PCR System 2 27 About Plate Documents seris irern ep arnis a ARE Os 2 28 Seting Up a Plite Document aaa 2 31 Setting Up a Plate Document Template 2 40 So on g n n e D x n O i lt j xo Quantifiler Kits User s Manual 2 25 Chapter 2 Software Setup Overview Purpose During software setup you start up
27. 106 3A 20 _Non_Casework_sample fsa 3A 20x UM 10Y 34_20x_Non_Casework_sample fsa 3A 20x BM 10R 3A20x_Non_Casework_sample tsa 3A 20x O sh a a th EB 128 16_4x_Non_Casework_sample fsa 16_4 DE 126 16 _4 _Non_Casework_sample tsa 16_4x I 127 16_4x_Non_Casework_sample fsa 16 4x BIB 12h 16 4x_Non_Casework_sample tsa 7 16_4 2400 0 16B 4_25x_Non_Casework_sample fsa 4 25x DE 186 4 25x_Non_Casework sample fsa 74_25x 16Y 4_25x_Non_Casework_sample fsa 4_25x BE 16R 4_25 _Non_Casework_sample fsa 4_26x 17B Positive Control094 a fsa positive _Control9947a EB 176 Positive Control9947a tsa positive Control0Qd7a 17Y Positive_Control994 a fsa positive _Control9947a WM 17R Positive Control9947a fsa positive_Control99d7a 24B Negative _Control fsa Negative_Control WE 245 Negative_Control tsa Negative_Control 24Y Negative_Control fsa Negative_Control BEE 24R Negative_Control fsa Negative_Control 3B 8A_30x_Cutting_from_shirt fsa 8A_30x BM 36 8 30x_Cutting_from shirt fsa 8A 30x 3 8 30x_Cutting_from_shirt fsa 8A_ 30x HIM 3 8 30x_Cutting from _shit tsa 8A 30x 6B 17A_10x_Cutting_from_t shirt tsa 17A_10x BM 58 17A_10x_Cutting_from_t shirt fsa 4 17A_10x 6Y gt 17A_10x_Cutting_from_t shirt fsa 17A 10x IR 17A 10x_Cutting_from_t shirt fsa 17A 10x 15B 26A_10x_Cutting_from_fabric fsa 26A_ 10x HIM 155 268 10x_Cutting_from_fabric fsa 268 10x
28. 15 m E adili da Oa 2B ewo_Run_demo_3100_2003 05 07_620_16min_407_01 fsa 4 15min Oa 26 ewe_Run_demo_3100_2003 05 07_620_14min_A07_01 fsa 16min COR 2Y ewo_Run demo 3100_2003 05 07_ 20_t8min_ 07_01 fsa 7 15min HIM 2R owe Run_demo_3100 2003 05 07 520_18min A07 01 fsa 15min 200 2100 von 60 m H zewo _Run_demo _3100_2003 05 07 619_thr_405 01 fsa thr BIB 10 0 Run demo _3100_2003 05 07519_1hr_A05_01 fsa thr F R 1 ome Run dama AAN MALMENT AIA the AR MI fes ihe WA irn Rin dama A NAT AAD the AR NI few the Figure 6 13 STR analysis using degraded DNA Quantifiler Kits User s Manual Section 6 1 ABI Prism 7000 Sequence Detection System Validation SDS Software v1 0 6 1 10 Comparisons with Other Methods Purified DNA samples were quantified using the Quantifiler Human kit and the Quantifiler Y kit The results were compared to results obtained from measuring absorbance at 260 nm A260 using a dye intercalation method and using the Quantiblot Human DNA Quantitation Kit Applied Biosystems The methods tested show different sensitivity ranges and different specificities Table 6 9 Comparison sensitivity and specificity of methods Method Sensitivity Specificity A260 Cannot detect DNA in the picogram Not specific for human genomic DNA range Detects single stranded DNA double stranded DNA and RNA Dye intercalation 25 pg mL Not specific for human genomic DNA Quantiblot kit
29. 16 0 60 7 Raji 2 C 0 099 0 110 0 113 14 1 2 5 0 091 7 7 17 1 K 562 A 2 76 1 317 1 360 50 7 3 3 neg n d n d K 562 B 0 69 0 365 0 379 45 1 3 9 neg n d n d K 562 C 0 138 0 104 0 096 30 4 7 9 neg n d n d a Dye intercalation method 6 34 Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 The different methods produced similar quantification results Table 6 14 Average differences from Asso and dye intercalation Average Difference Method Quantifiler Human Kit Quantifiler Y Kit A260 23 3 34 9 Dye intercalation 19 0 48 0 6 1 13 Assay Background An experiment was performed to check the assay system for false positive results that would indicate the presence of human DNA in a sample that contained none Experiment For each Quantifiler kit 48 negative control reactions were set up PCR Mixes were prepared and dispensed into wells of the reaction plate according to the standard procedure For each negative control reaction 2 uL of Ti E Buffer was added All standard assay parameters were used except that the number of thermal cycles was extended from 40 to 50 for increased stringency Results Figures 6 14 and 6 15 show that all 48 reactions with each assay were negative for their respective human DNA targets The IPC reactions amplified for all reactions in both assays indicating that the assa
30. 2 3 Comparisons with Other Methods 7900HT SDS 6 42 Experiment Six human genomic DNA samples were obtained from different commercial sources Table 6 16 DNA samples tested with A and dye intercalation 7900HT SDS DNA Sex Extraction Source 007 Male Blood 9948 Male Cell line Human genomic Male Blood Raji 1 Male Cell line Raji 2 Male Cell line K 562 Female Cell line Using the concentrations provided by the supplier the DNA samples were diluted to 2 0 ng uL A 0 5 ng uL B and 0 1 ng uL C Note All dilutions were made in T E Buffer with 20 ug mL glycogen added as a carrier and stabilizer All sample dilutions were quantified using the following methods A469 Because the concentrations of the dilutions extended below the detection limit of the spectrophotometer absorbance at 260 nm was measured only for the highest dilution 2 0 ng L DNA concentration was calculated from the formula Concentration ug mL 50 x Ayg The results calculated for the 2 0 ng uL dilutions were then extrapolated for the higher dilutions 0 5 ng uL and 0 1 ng uL using the known dilution factors Dye intercalation The microplate assay mode was used and the plate was read on a 7700 SDS All of the sample dilutions were within the detection range of the assay The assay was run using the A bacteriophage DNA quantification standard supplied with the kit and a quantification standard ba
31. 2 4 Quantifiler Kits User s Manual Starting the 7000 SDS Starting the 7000 SDS Overview Starting the 7000 SDS involves 1 Starting the Computer 2 Powering On the Instrument page 2 6 3 Starting SDS Software page 2 6 Starting the Computer 1 Ifyou are using the laptop computer open it by pushing in the front center button holding it and lifting up the lid 2 Press the power button on the computer y je Q oO 72 g 7 n e n xe Laptop power button 3 Inthe Enter User name field of the login window type your name or the user name associated with the computer 4 If required type your password in the Password field Quantifiler Kits User s Manual 2 5 Chapter 2 Software Setup Powering On the Note Wait for the computer to finish starting up before powering on Instrument the 7000 instrument EN WARNING PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating Disconnect power before servicing the instrument Press the power button on the lower left front of the instrument 2 5 3 Q 2 ku S O 2 a 7 N Power button Starting SDS Select Start gt ABI Prism 7000 gt ABI Prism 7000 SDS Software Software The software attempts to initialize the instrument and displays a message in the status bar for a few seconds Then the c
32. 40x_Cutting_trom_cloth fsa 14 40x 32Y 14 40x_Cutting_from_cloth f 3 14A_40x 320 144 40x_Cuming_trom_cloth tsa f 144 40x 32R 14 40x_Cutting_from_cloth tza 144 40x hu Lal Ida a hid as Ma a Ada at 33B 29A_10x_Cutting_from_tabrio fsa 20A_10 x 336 29A_10x_Cutting_from_fabrio fsa 20A_10 x 33Y 29A_10x_Cutting_from_tabric tsa 29A_10 x 33R 294_10x_Cuming_trom_fabric 1sa 29A_10 x 2000 1500 o ki 4i Ah ak ikh 4 AA A ala a AA sa Aa A A 30B 22A_100x_Cutting_trom_carpet tsa 22A_100x 366 22A_100x_Custing_from_carpet fza 22A_100x 36Y 22A_100x_Cutting_from_oarpet fsa 22A_100x JGR 22A_100x_Cuting_from_carpet fsa 7224100x 2000 z ail ai oj A AM AA A A AAA a a Aa da Aa a als 37B 7A_10x_Outting_from_cap tsa 7A 10x 370 7A_10x_Cuning_trom_cap fsa TA_10x 37Y 7A_10x_Cuning _from_oap tsa 7A_10x PR 7A_10x_Cuming_from_cap tsa 7A_10x 0000 0000o T alu u A n salia dadd A Ahh 43B 8A_10x_Cutting_from_shirt fea 8A_10x 436 8A_10x_Cutting_from_shirt fsa 8A_10x 48 8A_10x_Cutting_from_shirt fsa 3A 10 FOR 8A_10x_Cutting_from_shirt tsa SA_10x Figure 6 18 STR profiles of casework samples 6 50 Quantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Section 6 4 Applied Biosystems Overview Validation Experiments Performed 7500 Real Time PCR System Validation
33. 5 T T oEo buffer contents of 3 2 TaqMan MGB probe 1 4 targets about 1 3 task selecting for detector 7000 SDS 2 16 selecting for detector 7900HT SDS 2 35 Technical Support contacting xiv Index 5 template creating plate document from 7000 SDS 2 24 creating plate document from 7900HT SDS 2 42 saving 7000 SDS 2 23 saving 7900HT SDS 2 41 setting up for plate documents 7000 SDS 2 22 setting up for plate documents 7900HT SDS 2 40 template plate document See sdt file text conventions vii thermal cycler conditions printing 7900HT SDS 4 10 setting 7000 SDS 2 19 setting 7900HT SDS 2 38 threshold settings for the 7000 SDS 4 3 settings for the 7900HT SDS 4 7 threshold cycle calculation of 1 13 exporting 7000 SDS 4 6 in standard curve 5 4 normal range for IPC system 5 11 printing 7000 SDS 4 6 relationship to initial template amount 1 14 viewing in amplification plot 7000 SDS 4 4 viewing in amplification plot 7900HT SDS 4 8 viewing in standard curve 7000 SDS 4 4 viewing in standard curve 7900HT SDS 4 8 training information on xiv U unknown samples detectors applying for 7000 SDS 2 17 detectors applying for 7900HT SDS 2 36 names adding 7000 SDS 2 18 names adding 7900HT SDS 2 37 user attention words described viii Index 6 V validation 7000 SDS SDS v1 0 software 6 3 7000 SDS SDS v1 2 3 software 6 66 7500 Real Time PCR System SDS v1 2 3 softwar
34. 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Calculated Quantities Data obtained using the Quantifiler Human Kit showed no statistically significant difference when the calculated quantities obtained from the 7000 and 7500 systems were compared p 0 22 with 95 confidence However minimally significant differences were observed between the two instrument types for calculated quantities using the Quantifiler Y Human Male Kit To further explore the extent of the difference between the two instrument types the percent differences between the calculated quantities within the concentration range of 2 ng uL to 0 5 ng uL were determined This range was selected because it represents the optimal input range for most STR kits In this range there was at most an 18 concentration difference between calculated quantities using the 7000 and the 7500 systems The impact of the slight differences in calculated quantities should have minimal effect on results of STR analysis However laboratories should perform the appropriate studies to verify optimal input amounts for amplification 6 4 5 3 Auto Baseline Analysis Versus Manual Analysis 6 4 6 Conclusion No statistically significant difference was observed for C values and calculated quantities derived using the Auto Baseline and Manual analysis modes on the 7500 System SDS Software v1 2 3 This validation study demonstrates that the Applied Biosystems 75
35. 6 5 Specificity with human DNA panel Result Sex Quantifiler Human Kit Quantifiler Y Kit Male 240 Female 260 _ 6 1 4 Specificity with a Non Human Panel Samples were obtained either as purified DNA or as whole blood from individual animals For some of the purified DNA samples the sex of the donor animals was unknown for remaining samples the sex and identity of the animals was known For some species multiple individuals were tested Experiment For many of the reactions approximately 0 25 to 1 0 ng of DNA was used in each reaction For a few reactions up to 40 ng of DNA was used in one reaction SDS software was used to analyze the data and calculate the Cy fam value Cy FAM Value Result Cr fam lt 40 No amplification after 40 cycles Quantifiler Kits User s Manual 6 11 Chapter 6 Experiments and Results 6 12 Results The two human control samples that were tested show expected results as shown in Table 6 5 on page 6 11 Quantifiler Human Kit Results The Quantifiler Human kit detected DNA from humans and apes with some less efficient detection of one other primate The Quantifiler Human kit Detected DNA from all of the higher ape DNA samples chimpanzee gorilla and orangutan at an efficiency similar to that of humans Detected DNA from macaque monkeys at a significantly reduced efficiency possibly because of partial homology betwe
36. 9850 0 9800 Average R Per Run o Software v1 0 m Software v1 2 3 f f Run 01 Run 02 Run 03 Run 01 Run 02 Run 03 Quantifiler Human Quantifiler Y Figure 6 34 Average R values Quantifiler Human Kit and Quantifiler Y Human Male Kit SDS Software v1 0 and v1 2 3 6 5 4 2 Reproducibility and Sensitivity Two sample DNAs were quantitated for this experiment Eight 2 fold serial dilutions for each sample were run four replicates per dilution 32 wells per sample The Cy values were generated in Manual analysis mode then the quantities were calculated using the standard curve on each plate Figure 6 35 shows the C values for 007 and 9948B across a set of eight serial dilutions 30 ng uL to 0 1 ng uL with the Quantifiler Human Kit and the corresponding quantitated concentrations As the data show differences in C values do not affect calculated quantities calculated quantities were normalized resulting in comparable concentrations from results generated with both software versions Quantifiler Kits User s Manual 6 73 Chapter 6 Experiments and Results Software v1 0 and Software v1 2 3 C values Quantity Calculations between software v1 0 and software 34 ae v1 2 3 analysis u i ens EA i i 3 e Quantifiler Human 007
37. M Martens and Naes 1989 Quantifiler Kits User s Manual 1 11 Chapter1 Overview Relationship of Amplified PCR Product to Initial Template Concentration 1 12 Because of the progressive cleavage of TaqMan fluorescent probes during the PCR as the concentration of amplified product increases in a sample so does the R value The following equation describes the relationship of amplified PCR product to initial template during the geometric phase N NC E where N is the concentration of amplified product at any cycle N is the initial concentration of target template E is the efficiency of the system and c is the cycle number For example with the dilutions of RNase P target in the TaqMan RNase P Instrument Verification Plate the ratio of template concentration to detectable signal is preserved in the geometric phase for all dilutions Figure 1 9 As the rate of amplification approaches a plateau the amount of product is no longer proportional to the initial number of template copies Copy Number 20000 10000 5000 2500 1250 Cycle Number Figure 1 9 Amplification plot from a real time run of an RNase P Instrument Verification Plate Quantifiler Kits User s Manual About the Threshold About the Threshold Cycle How C Values Are Determined Real Time Data Analysis
38. Proteus vulgaris Pseudomonas aeruginosa Hafnia alvei Yersinia enterocolitica Campylobacter coli Providencia stuartii Vibrio parahaemolyticus Alcaligenes faecalis Quantifiler Kits User s Manual 6 15 Chapter 6 Experiments and Results 6 1 6 Sensitivity 6 16 DNA Samples Tested Experiment Results Human genomic DNA samples were obtained from different commercial sources For each DNA sample a dilution series was made and each dilution was tested with the Quantifiler Human kit and the Quantifiler Y kit Five different human DNA samples were tested Table 6 8 Human DNA samples tested for sensitivity Sample Extraction Source 007 Human male blood 9948 Human male cell line Human genomic Human male blood Raji Human male cell line K 562 Human female cell line Using the concentrations provided by the suppliers five fold serial dilutions ofthe DNA samples were made Concentrations ranged from 10 ng uL to 0 016 ng uL 16 pg uL Note All dilutions were made in T E Buffer with 20 ug mL glycogen added as a carrier and stabilizer For each 25 uL reaction 2 0 uL of DNA sample was used A plot of the C values versus the known DNA quantities showed the expected log linear relationship between the two quantities All dilutions including samples at the lowest concentration 16 pg uL gave positive results for the Quantifiler Human kit and the Quant
39. Quantifiler Human kit C results 7900HT SDS Quantifiler Kits User s Manual 6 39 Chapter 6 Experiments and Results 50 16 7 556 1 85 0 62 0 21 0 068 0 023 DNA quantity ng uL Figure 6 17 Precision Quantifiler Y kit C results 7900HT SDS 6 2 2 Mixture Studies 7900HT SDS 6 40 Experiment An experiment was performed to demonstrate the specificity of the Quantifiler Human kit and the Quantifiler Y kit in analyzing mixtures of human genomic DNA from male and female sources The mixture studies were designed to simulate circumstances in which a small component of male DNA must be discerned from a high background of female DNA Purified genomic DNA from the Raji male and K 562 female cell lines were combined in ratios of 1 1 1 4 1 16 1 64 1 256 and 1 1024 Raji K 562 The male DNA was added at a constant level of 0 05 ng uL in all samples and the female DNA was present at amounts ranging from 0 05 ng uL in the 1 1 sample to 50 ng uL in the 1 1024 sample The DNA amounts were based on the DNA concentrations provided by the suppliers and were not calibrated with the Quantifiler kits The mixtures were tested with the Quantifiler Human kit assay and the Quantifiler Y kit assay to determine the concentrations of total human genomic DNA Quantifiler Human kit and male DNA only Quantifiler Y kit For each sample three replicate reactions were performed for each assay Each as
40. Quantifiler kits page 2 32 Template To create a plate document template 1 Ifthe SDS software is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 2 40 Quantifiler Kits User s Manual Setting Up a Plate Document Template To create a plate document template continued 2 Select File gt New then complete the New Document dialog box x Assay Absolute Quantification Standard Curve 7 Container 96 Wells Clear Plate 7 Template Blank Template z Browse Barcode ual Cancel Apply the desired template settings to the plate document e Copy detectors page 2 34 Apply detectors for standards page 2 35 e Apply detectors for unknown samples page 2 36 e Set thermal cycler conditions page 2 38 Select File gt Save As and complete the Save As dialog box a For Files of Type select ABI PRISM SDS Template Document sdt b Locate and select the Templates folder within the software folder X Program Files gt Applied Biosystems gt 7900HTSDS gt Templates where X is the hard drive on which the SDS software is installed Note Saving the template file in the Templates folder makes it available in the Template drop down list of the New Document dialog box see step 2 in Creating a Plate Document from a Template on page 2 42 c Enter a name for the template For example enter Quantifiler Template d Click Save
41. SDS Software v1 2 3 The Quantifiler Human DNA Quantification Kit and the Quantifiler Y Human Male DNA Quantification Kit were tested see the experiments listed below using the Applied Biosystems 7500 Real Time PCR System with SDS Software v1 2 3 running on the Windows XP operating system The results were then compared to the previously validated ABI PRISM 7000 Sequence Detection System with SDS Software v1 0 The experimental data generated demonstrate that the 7500 System SDS Software v1 2 3 e Provides accurate results when used with the Quantifiler kits for the analysis of genomic DNA samples Produced results that are statistically similar to the results produced on the previously validated 7000 System SDS Software v1 0 e Precision and Accuracy e Reproducibility and Sensitivity Background e Auto Baseline versus Manual analysis 6 4 1 Materials and Methods 6 4 1 1 Reagents To minimize variables from hand pipetting and lot to lot reagent differences the following set up procedures were used throughout the study Eight serial dilutions were made with one lot of standard DNA provided with the Quantifiler kits first dilution prepared with 500 uL DNA and 1 000 uL 10 mM Tris HCl pH 8 0 and 0 1 mM Na EDTA Tj Ep buffer Quantifiler Kits User s Manual 6 51 Chapter 6 Experiments and Results e One manufactured lot of each kit was used for all validation studies Kit Part Numbe
42. The SDS software uses a threshold setting to define the level of detectable fluorescence Based on the number of cycles required to reach the threshold the SDS software can compare test samples quantitatively A sample with a higher starting template copy number reaches the threshold earlier than a sample with a lower starting template copy number The threshold cycle C for a specified amplification plot occurs when the fluorescent signal increases beyond the value of the threshold setting The C value depends on Starting template copy number Efficiency of DNA amplification by the PCR system To determine the Cy value the SDS software uses the R values collected from a predefined range of PCR cycles called the baseline the default baseline occurs between cycles 6 and 15 on the 7000 SDS and between cycles 3 and 15 on the 7900HT SDS 1 The software generates a baseline subtracted amplification plot of AR versus cycle number 2 An algorithm defines the cycle where the AR value crosses the threshold setting the default threshold setting is 0 2 as the threshold cycle Cy Quantifiler Kits User s Manual 1 13 Chapter 1 Overview Relationship of Threshold Cycles to Initial Template Amount 1 14 The following equation describes the exponential amplification of the PCR Xn Xm 1 Ey T where X number of target molecules at cycle n so that n gt m X n number of target molecules at cycle m Ey eff
43. applied to the correct detector and reanalyze 5 6 Quantifiler Kits User s Manual Troubleshooting the Standard Curve The examples shown in the following sections can be caused by errors made in applying the detectors for standards when setting up the plate document For instructions on how to apply the detectors for standards see e Page 2 15 7000 SDS e Page 2 35 7900HT SDS Note The standard curves shown in these examples are not optimal and should not be used Example 1 Observation Almost all of the Cy values for the DNA quantification standard reactions lie outside of the standard curve and form a straight horizontal line Quantifiler Kits User s Manual 5 7 Chapter 5 Interpretation of Results Possible Cause When applying detectors for the standards the Task and Quantity were applied to the IPC detector instead of to the Quantifiler Human detector x Well s Al Sample Name Use _ Detector _ Reporter Quenchq Task Quantity Color V Quantifiler Human FAM none Quantifiler Y FAM none M PC vIC none Standard 50 Unknown m ask and Quantity J Omit Well applied Passive to wrong sdd Detector Remove ROX detector Example 2 Observation One point lies outside of the standard curve By Ti Wen Tool Instrument Anahats Window Hab aisis DOSU Abra Ta A AEA ee ADA A Outlier Possible Cause When applying detect
44. as an ABI Document PRISM SDS Single Plate sds file Note To save the document as a template see Setting Up a Plate Document Template on page 2 40 To save the plate document 1 Select File gt Save As 2 For Files of Type select ABI PRISM SDS Single Plate sds Quantifiler Kits User s Manual 2 39 Q So I 2 g 7 n io D n O ke a 2 g 3 fe 2 2 DO 7 Pr I Q f N Chapter 2 Software Setup To save the plate document continued 3 Navigate to where you want to save the plate document file 4 In the File Name field enter a name for the plate document 5 Click Save Setting Up a Plate Document Template Purpose A plate document template reduces the time required to set up a plate document This section describes how to create an SDS Template Document sdt set up for running Quantifiler kit assays Template Settings In addition to plate document settings assay and container templates can contain e Assay specific detectors e Well assignments for quantification standards with detectors tasks and quantity e Well assignments for unknown samples with detectors and tasks Instrument settings thermal cycler conditions and reaction volume settings Creating a Plate This procedure assumes that you have created the detectors for Document running reactions using the
45. corresponds exactly to the arrangement of the standards and unknown samples in the wells of the reaction plate Figure 2 1 shows one example of arranging reactions when running two Quantifiler kits on one 96 well reaction plate e Wells Al through D12 gray correspond to reactions using the Quantifiler Human kit e Wells El through H12 white correspond to reactions using the Quantifiler Y kit Note For each Quantifiler kit assay there are eight DNA quantification standards and two reactions for each standard See Preparing the DNA Quantification Standard on page 3 2 for more information about the DNA quantification standards 1 2 3 4 5 6 7 8 9 10 11 12 Std 1 Std1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std5 Std 6 Std 6 Std 7 Std7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std5 Std 6 Std 6 Std 7 Std 7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN 2 5 3 Q 2 ku S O 2 DO 7 r N IO nm
46. detector 7000 SDS 2 16 entering for detector 7900HT SDS 2 36 SDS document See sds file sds file description 7000 SDS 2 7 description 7900HT SDS 2 28 saving 7000 SDS 2 21 saving 7900HT SDS 2 39 See also plate document 7000 SDS and plate document 7900HT SDS SDS software starting for 7000 SDS 2 6 starting for 7900HT SDS 2 27 SDS template See sdt file sdt file description 7000 SDS 2 7 description 7900HT SDS 2 28 saving 7000 SDS 2 23 See also template sensitivity ofassay 5 16 tests 6 16 Services and Support obtaining xiv setup table exporting 7900HT SDS 4 10 single plate document See sds file 7900HT SDS slope of standard curve about 5 4 differs significantly from 3 33 5 6 interpreting 5 5 Quantifiler Kits User s Manual viewing 7000 SDS 4 4 viewing 7900HT SDS 4 8 specificity with bacterial pools panel 6 14 with human DNA panel 6 10 with non human panel 6 11 stability 6 17 standard curve about results 5 4 differences in Cy values of replicates 5 9 interpreting 5 4 outlier in example of 5 8 print setup 7000 SDS 4 6 replicates example of four 5 9 straight horizontal line 5 7 troubleshooting 5 6 viewing 7000 SDS 4 4 viewing 7900HT SDS 4 8 standards applying detectors for 7000 SDS 2 15 applying detectors for 7900HT SDS 2 35 See also DNA quantification standards stochastic effects 5 16 storage recommendations for kits 1 16 strand displacement in 5 nuclease assay 1
47. expected because the amounts of DNA added to the mixtures were based only on the DNA concentrations provided by the suppliers and were not calibrated with the Quantifiler kits In all samples the male DNA was detected and quantified accurately regardless of the amount of female DNA present Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 ca Quantifiler Y kit o Quantifiler Human kit 100 000 10 000 1 000 DNA Quantity ng uL 0 100 0 010 1 1 1 5 1 16 1 64 1 256 1 1024 Male Female Mixture Ratio Figure 6 9 DNA quantities determined in mixture studies 6 1 9 Degraded DNA Studies Experiment Forensic samples may be exposed to environmental conditions that degrade DNA molecules and reduce their amplification efficiency in PCR reactions Exposure to environmental conditions can reduce the overall DNA concentration and may cause fragmentation of full length DNA molecules into smaller fragments DNA fragmentation makes it difficult to amplify longer segments such as the larger STR loci Because of such potential occurrences the validation of forensic DNA methods often involves studies of the effects of degradation on the amplification and detection of DNA The Quantifiler kits were tested with DNA degraded with the DNA nuclease DNase I The degraded DNA samples were tested with the Quantifiler Human kit and the Quantifiler Y kit to determine the
48. of Components IPC template DNA a synthetic sequence not found in nature Two primers for amplifying the IPC template DNA One TaqMan MGB probe labeled with VIC dye for detecting the amplified IPC DNA Quantifiler Kits User s Manual 1 3 Chapter1 Overview About the Probes The TaqMan MGB probes contain A reporter dye FAM dye or VIC dye linked to the 5 end of the probe A minor groove binder MGB at the 3 end of the probe This modification increases the melting temperature T m without increasing probe length Afonina et al 1997 Kutyavin et al 1997 which allows the design of shorter probes A nonfluorescent quencher NFQ at the 3 end of the probe Because the quencher does not fluoresce Applied Biosystems sequence detection systems can measure reporter dye contributions more accurately 5 Nuclease The 5 nuclease assay process Figures 1 1 through 1 5 takes place Assay Process during PCR amplification This process occurs in every cycle and does not interfere with the exponential accumulation of product Nonfluorescent quencher GB Minor groove binder Reporter P AmpliTaq Gold DNA Polymerase Figure 1 1 Legend for 5 nuclease assay process figures During PCR the TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites Figure 1 2 When the probe is intact Figures 1 2 and 1 3
49. or dust on dyes and recalibrate w the fiters andlor mirror Note See your instrument user guide for 5 I instructions on how to run pure dyes and 2 FH Tr Seins recalibrate an PEHEE EEA TH aa u 060 1224 5 6 7 01011 1213141516 1718102021 2229 24 26 26 3720293031 22333435 3537 38 394041 42424445 Abnormal amplification plots in one column of reactions Delia Rn 122345678 910111213141516 1718 10 2021 22 29 24 25 2627 20 20 30 31 32 33 34 35 36 a7 30 an 40 No defined amplification plots Incorrect detector selected on the amplification plot or incorrect detector applied to the reactions when setting up the plate document 1 Make sure that the correct detector is selected on the amplification plot 2 If the amplification plots are still not defined a From the plate document double click a well to view the Well Inspector b Verify that the detector settings are correct and reanalyze Dalia Rn vs Cycle Deta Rn 040 fi ie 122456879 A101 121314 15161718 19 20 21 2223 24 25 1627 26 20 3031 32 3334 353
50. quantity of amplifiable DNA in each time point Results obtained using the Quantifiler kits were used to calculate DNA input for an STR assay using an ABI PRISM 3100 Genetic Analyzer A time course of exposure to DNase I was performed on a sample of high molecular weight human genomic DNA to generate a series of samples with varying levels of degradation The time points in the DNase I treatment were 0 minutes untreated 1 minute 2 minutes 3 minutes 4 minutes 5 minutes 15 minutes and 60 minutes Samples from all time points were run on a 2 agarose gel for 25 minutes and visualized by staining with ethidium bromide The Quantifiler Kits User s Manual 6 23 Chapter 6 Experiments and Results 6 24 Results treated DNA samples were examined by agarose gel electrophoresis to determine the average size of the DNA fragments at each time point The degraded DNA samples were tested with the Quantifiler Human kit and the Quantifiler Y kit to determine the quantity of amplifiable DNA in each time point Using the results from the Quantifiler kits the volumes of DNA required for AmpF STR Identifiler kit reactions were calculated so that 1 0 ng uL was added for each reaction The PCR products were run on an ABI PRISM 3100 Genetic Analyzer Agarose gel electrophoresis showed that the DNase I treatment reduced the average size of DNA fragments to 100 basepairs bp or less within the first 5 minutes Figure 6 10 Minutes degraded
51. s Manual 6 61 Chapter 6 Experiments and Results 6 4 4 3 Background Figure 6 24 shows background amplification plots for 95 NTCs and one positive control for both kits one plate each run on the 7000 System SDS Software v1 0 Figure 6 25 shows background amplification plots for the 7500 System SDS Software v1 2 3 On all instruments the 95 NTC samples yielded negative results all C values gt 40 with both Quantifiler kits 20 ai 22 a3 at a5 25 a7 a0 28 30 N 2 33 U 3S I3 S7 30 38 W0 129 A7 38 39 20 21 22 23 21 25 26 27 2B 29 30 3 32 33 34 35 36 3T 38 3 AD Quantifiler Human Kit Quantifiler Y Human Male Kit Figure 6 24 Background amplification plots 7000 System SDS Software v1 0 sossoor os f 1106 006 18 19 20 21 22 29 24 25 20 27 28 29 30 31 32 33 34 35 30 37383 40 4810 20 21 22 23 24 25 20 27 28 29 30 31 32 33 34 35 3 373 3 4 Quantifiler Human Kit Quantifiler Y Human Male Kit Figure 6 25 Background amplification plots 7500 System SDS Software v1 2 3 6 62 Quantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 6 4 4 4 Auto Baseline Analysis Versus Manual Analysis C Pre
52. the procedure keep hands away until the heated cover and sample block reach room temperature 5 Inthe SDS software open the plate document that you set up for the run 6 Select the Instrument tab then click Start 3 8 Quantifiler Kits User s Manual Running the Reactions Running the Plate To run the plate on the 7900HT SDS on the 7900HT SDS 1 In the SDS software select the Instrument tab for the plate document In the Real Time tab click Open Close to rotate the instrument tray to the OUT position Place the plate in the instrument tray so that e Well Al is in the upper left corner The notched corner is in the upper right corner Click Start to rotate the instrument tray to the IN position and to start the run Note The instrument may pause to allow the heated cover to heat to the appropriate temperature before beginning the run The SDS software collects and saves the run data and the Real Time tab displays the instrument status and run progress After the run is complete remove the plate from the instrument a Click Open Close in the Instrument tab of the plate document that is open and connected to the 7900HT instrument The instrument tray rotates to the OUT position b Remove the plate from the instrument c Click Open Close in the Instrument tab to rotate the instrument tray to the IN position Quantifiler Kits User s Manual Chapte
53. v1 2 3 3 ee 3 3 e Quantifiler Human 9948 v1 2 3 30 pir 10 oe HEE an 5 28 5 o e eto 3 i i i i en ee e Quantifiler Human 007 v1 0 26 3 a Genie Human 9948 v1 0 1 i e e Quantifiler Human 007 v1 2 3 3388 Guenter Human 9948 v1 2 3 m e 244 i 3 i i e 3 Se 22 T T T T T T 1 0 1 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Well location Well location Figure 6 35 Average C values and quantitated DNA concentrations 007 and 9948B Quantifiler Human Kit Figure 6 36 shows Cy results for the Quantifiler Y Human Male Kit that differ slightly between the v1 0 analysis and the v1 2 3 analysis However differences in C values do not affect calculated quantities calculated quantities were normalized resulting in comparable concentrations from results generated with both software versions Software v1 0 and Software v1 2 3 Cr values Quantity Calculations between software v1 0 and software 34 40 s v1 2 3 analysis ES x gt Quantifiler Y 007 v1 0 siges 2333 S Guanthler 007 v 23 30 i f 8 4 se e Quantifiler Y 9948 v1 2 3 iiig sess o pij iit 3 sess 83 7 Hi ee ses ATT a m M e e 6 x 22 0 1 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Well location Well location Figure 6 36 Average C values and quantitated DNA concentrations 007 and 9948B Quantifiler Y Human Male Kit Quantifiler Kits User s Manual 6 74 Section 6 5 ABI PRISM 7000 Sequence Detection System Validatio
54. 0 Sample Figure 6 23 C values and quantitated concentrations Quantifiler Human Kit comparable data were obtained for the Quantifiler Y Human Male Kit Quantifiler Kits User s Manual 6 59 Chapter 6 Experiments and Results Table 6 21 shows the average calculated quantities for each DNA sample obtained with the Quantifiler Human Kit For sample concentrations between 2 ng uL and 0 5 ng uL the percent difference between the quantitated values between instrument types did not exceed 16 No statistically significant difference was observed for calculated quantities obtained using the Quantifiler Human Kit on the two instrument types Table 6 21 Average Calculated DNA Quantities Quantifiler Human Kit ane ona 7000Avg 7000 Calculated 7500 Between 7000 amp of 7000 aty Sample Qty ng L Dev Qty Dev 7500 Calculated Value from ng uL Qty ng L 7500 Qty Value Raji 9 33 0 51 9 14 0 33 0 19 2 04 4 58 0 15 4 24 0 12 0 34 7 72 2 30 0 11 2 09 0 04 0 21 9 63 1 16 0 05 1 07 0 03 0 09 8 01 0 59 0 03 0 55 0 01 0 04 6 91 0 27 0 01 0 26 0 01 0 01 3 43 0 15 0 01 0 15 0 01 0 00 3 24 0 08 0 00 0 07 0 00 0 01 8 04 9948 4 65 0 15 5 02 0 20 0 37 7 58 2 33 0 02 2 34 0 05 0 01 0 36 1 16 0 05 1 09 0 03 0 07 5 98 0 59 0 02 0 50 0 03 0 08 15 52 0 31 0 02 0 27 0 01 0 04 12 31 0 17 0 01 0 15 0 01 0 02 10 80 0 08 0 01 0 06 0 00 0 03 38 59 0 05 0 01 0 04 0
55. 00 0 01 18 14 6 60 Quantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Table 6 22 shows the average calculated quantities for each DNA sample obtained with the Quantifiler Y Human Male Kit For sample concentrations of 2 ng uL to 0 5 ng uL the percent difference between the quantitated values between instrument types did not exceed 18 A minimal statistical difference was observed for calculated quantities obtained using the Quantifiler Y Human Male Kit on the two instrument types p 0 0027 Table 6 22 Average Calculated DNA Quantities Quantifiler Y Human Male Kit Difference Difference SPNA Calculated Std Galculated 7300Std 700087300 Value from Qty ng L Dev Qty ng uL Calculated 7500 Qty Qty ng L Value Raji 9 12 0 40 9 09 0 07 0 03 0 34 4 60 0 20 4 66 0 04 0 06 1 29 2 53 0 07 2 36 0 05 0 17 7 04 1 29 0 09 1 19 0 03 0 10 8 12 0 66 0 05 0 62 0 03 0 05 7 36 0 33 0 02 0 30 0 02 0 02 7 89 0 15 0 02 0 14 0 01 0 02 11 55 0 070 0 02 0 057 0 01 0 01 19 85 9948 4 71 0 12 4 56 0 06 0 15 3 19 2 43 0 14 2 30 0 06 0 12 5 14 1 34 0 09 1 13 0 05 0 21 17 34 0 68 0 03 0 62 0 03 0 06 9 93 0 33 0 03 0 28 0 03 0 05 15 60 0 18 0 01 0 14 0 01 0 04 24 87 0 08 0 00 0 05 0 00 0 02 34 65 0 04 0 00 0 03 0 00 0 01 38 29 Quantifiler Kits User
56. 00 Real Time PCR System with SDS Software v1 2 3 is a robust reliable and reproducible system for performing DNA quantification using the Quantifiler kits When statistically comparing 7500 System SDS Software v1 2 3 results Cp slope and R values to results obtained using previously validated ABI PRISM 7000 Sequence Detection System with SDS Software v1 0 Differences in calculated quantities are minimal Quantifiler Y Human Male Kit or insignificant Quantifiler Human Kit for unknown samples using the 7500 and 7000 systems The differences observed should have little effect on resulting STR amplification based on calculated DNA quantities e No significant difference is observed between C values and calculated quantities derived by using Auto Baseline and Manual analysis modes Quantifiler Kits User s Manual 6 65 Chapter 6 Experiments and Results Section 6 5 ABI Prism 7000 Sequence Overview Validation Experiments Performed Detection System Validation SDS Software v1 2 3 The Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit were tested see the experiments listed below using the ABI PRISM 7000 Sequence Detection System with SDS Software v1 2 3 running on the Windows 2000 operating system then compared to the previously validated ABI PRISM 7000 Sequence Detection System with SDS Software v1 0 The experimental data generated demonstrate that th
57. 04 0 11 0 15 1 1 43 Cutting from 1 2 2 61 3 75 1 4 sweatshirt 48 Cutting from cotton 0 4 0 52 0 87 1 1 Cutting from 0 4 0 94 0 97 1 0 sweatshirt Cutting from cloth 0 4 0 31 0 56 1 1 a9 Cutting from fabric 0 04 0 23 0 34 1 1 G Cutting from leather 0 08 0 10 0 18 1 2 B Cutting from carpet 0 4 0 76 0 95 1 3 amp Cutting from cloth 1 6 1 89 2 95 1 1 amp Cutting from shirt 1 2 2 29 3 10 1 2 amp Swab from hammer 0 6 0 47 0 58 1 1 E Cutting from cloth 0 4 0 45 0 58 1 1 amp Cutting from fabric 0 08 0 16 0 18 1 3 Cutting from carpet 0 4 1 45 1 62 1 3 amp Cutting from cap 0 4 0 45 0 46 1 0 amp Cutting from shirt 1 2 2 29 3 10 1 3 a See Figure 6 18 for STR profiles 6 48 Quantifiler Kits User s Manual Section 6 3 Casework Sample Analysis 1B 1_100x_Non_Casework_sample fsa 1_100x UM 1Y 1_100x_Non_Casework_sample fsa 1_100x 1_100x_Non_Casework_sample fsa 1_100x 1R 1_100x_Non_Casework_sample fsa 1_100x 2400 0 EB 28 25_25x_Non_Casework_sample fsa 25 25x 2G 25 25x_Non_Casework_sample fsa 25_25x UM 2Y 25_25x_Non_Casework_sample fsa 25_25x 2R 25_25x_Non_Casework_sample fsa 25_26x 2400 LE OEM 58 18_25x_Non_Casework_sample tsa 15_25x IM 5Y7 15_25x_Non_Casework_sample fsa 15 25x A asa A 15_25x_Non_Casework_sample fsa 15_25x 5R 15_25x_Non_Casework_sample fsa 15_25x ao ln 3 E 108 34 20x_Non_Casework_sample fsa 3A 20x BE
58. 0x_Cutting_from sweatshirt fsa 4A 30x 21B 12A_10x_Cutting_from_eloth fsa 12A_10x 21Y 12A_10x_Cutting_from_oloth fsa 12A_10x 112A 10x_Cutting_from_oloth tsa 7 12A_10x 12A 10x_Cutting_from_cloth fsa 12A_10x 228 27A10x_Cutting_trom_fabric tsa 27A 10x DE 225 27 10x_Cutting_trom_fabric fsa 27A_10 oa OM 227 27A 10x_Cutting_trom_fabric tsa 27A 10x 22R ZTA 10x_Cutting_from_fabrio fsa 27A_10x 20B 6A_10x_Cutting_from_leather fsa 5A_10x 5A_10x_Cutting_from_leather fsa 5A_10x 26Y 5A_10x_Cutting_from_leather fsa 5A 10x 15A 10x_Cutting_from leather fsa 5A 10x Mau ma da 4 pho AMA 27B Cutting _from_carpet tsa Cutting_from_carpet DE 276 Cutting_trom_carpet fsa Cutting from_carpet 27V Cutting_from_carpet tsa Cutting_from_carpet BEE 270R Cutting _trom_carpet fsa Cutting from_carpet mE 298 Cutting _from_oloth fsa Cutting_from_cloth EB 295 Cutting _trom_oloth fsa Cutting_from_oloth OE 207 Cutting _from_cloth fsa Cutting_from_cloth ME 29R Cutting_trom_oloth fsa Cutting_from_oloth a id A 10x_Cutting_from_shirt fsa 18_10x 3 10x_Cutting_from_shit fsa 18_10x 10x_Cutting_from_shirt fsa 18_10x 10x_Cutting_from_shirt fsa 18_10x 31B Swab_from_hammer fsa Swab_from_hammer 31Y Swab_from_hammer fsa Swab_from_hammer 316 Swab_from_hammer fsa Swab_from_hammer SIR Swab_from_hammer fsa Swab_from_hammer 32B 144
59. 16 26A_10x_Cutting_from_fabrio fsa 26A_ 10x BE NR 264 10x_Cutting_from_fabrio fsa 26A_10x cP E 8B 10A_10x_Cutting_from_poly_cotton fsa 7 10A_10x 8G 10A _10x_Cutting_from_poly_cotton fsa 10A_10x 8Y gt 10A_10x_Cutting_from_poly_cotton fsa 410A 10x BE R 10A10x_Cuting_from_poly_cotton fsa 10A 10x 2000 0 9B 25A_10x_Cutting_from_denim fsa 258 10x 9G 25A 10x_Cutting_from_denim tsa 254 10x GY 25A_10x_Cutting_from_denim fsa 26A_10x OR 26 10x_Cutting_from_denim fsa 25A_10x 2000 0 EB 118 Cutting_from_sock fsa Cutting_from_sock 116 Cutting_from_sock fsa Cutting_from_sock O 11Y Cutting_from_sock fsa Cutting_from_sock 11R Cutting_from_sock fsa Cutting_from_sock 2000 0 EHE 128 Cutting from_sweatshirt tsa 4 Cutting_from_sweatshirt COM 139 Cutting_from_sweatshirt fsa Cutting_from_swaatshirt 136 Cutting_from_sweatshirt fsa Cutting_from_sweatshirt 13R Outting_from_sweatshirt fsa Cutting_from_sweatshirt Quantifiler Kits User s Manual 6 49 Chapter 6 Experiments and Results DE 148 114_10x_Cuting_from_cotton fsa 11A_10x m 146 11A_1Dx_Custing_from_ostton fsa 11A_10x I tar 11a 10x Cutting_from_ootton isa 114 10x 14R 11A 10x_Cutting_from_cotton tsa 114 10x AE 208 44 30x_Cutting_from sweatshirt fsa 4A 30x DE 206 44 30x_Cutting_from sweatshirt fsa 4A 30x 20Y 4A 30x_Cutting_from sweatshirt fsa 4A 30x BE 20R 4A 3
60. 2 5 For 7900HT SDS setup procedures see page 2 27 Set up a plate document for the run For 7000 SDS software procedures see page 2 7 For 7900HT SDS software procedures see page 2 31 To run the plate on the 7000 SDS l Lift the handle at the bottom of the door on the front of the instrument until the door is raised completely Gently push the carriage back until it stops and locks into place Do not push here to open Lift handle to open Position the plate in the instrument thermal block so that e Well Al is in the upper left corner The notched corner of the plate is in the upper right corner Quantifiler Kits User s Manual 3 7 Chapter 3 PCR Amplification To run the plate on the 7000 SDS continued 3 Gently push then release the carriage to unlatch it The carriage automatically slides forward into position over the sample plate Do not pull the door forward by the handle Gently push carriage back and release 4 After the door moves to the front pull the handle down into place to close the cover N oy Up el Do not pull the door handle to move the carriage forward This may cause serious damage to the door or the door mechanism AN VUOI PHYSICAL INJURY HAZARD During instrument operation the temperature of the heated cover can be as high as 108 C and the temperature of the sample block can be as high as 100 C Before performing
61. 2 ng uL to 0 03125 ng uL Specific for human genomic DNA a Value obtained from the manufacturer s documentation 6 1 11 Comparison with Azs and Quantiblot Kit Resolution Panel The concentration of DNA was measured for 50 human genomic DNA samples using a A569 method the Quantiblot kit and the Quantifiler kits The DNA quantification results were compared The resolution panel a set of 50 human genomic DNA samples purified from blood was tested The samples were database type samples because they were extracted from blood specimens and had uniform high concentrations of DNA between approximately 10 and 20 ng ul All samples were within the range of sensitivity for the Aygo method Quantifiler Kits User s Manual 6 27 Chapter 6 Experiments and Results Experiment Results Each DNA sample was quantified using A6 method Absorbance at 260 nm was measured DNA concentration was calculated using the formula Concentration ug mL 50 x Aygo e Quantiblot kit DNA was quantified using a protocol for chemiluminescence detection with film autoradiography e Quantifiler kits DNA was quantified using the standard procedure For each sample the percent differences between Quantifiler kit results and results from the other two methods were calculated The differences were expressed as a percentage of the reference method For each method the average percent differences from Quantifiler kit results were calculat
62. 6 a7 38 39 40 Dyele Number Abnormal AR values and some negative Ra values Incorrect passive reference was selected when setting up the plate document Confirm the diagnosis 1 From the plate document double click a well to view the Well Inspector 2 Observe which Passive Reference is selected Note ROX should be selected as the Passive Reference G 4ajdeyD synsey Jo uoneJs4dheju jenueyy sasn SHY Jejynuend GL G Table 5 4 Troubleshooting amplification plots continued Observation Possible Cause Recommended Action Deta Rn Data Re 4 15 16 17 16 19 20 Cycle Number Reactions in rows B C and D show poor amplification and reactions in the rows E F and G show good amplification Instrument door was not aligned properly on the reaction plate 1 Localize the wells that show poor amplification 2 Run the TaqMan RNase P Instrument Verification Plate PN 4310982 3 Check the calibration of the regions of interest ROI 4 Perform the instrument function tests Ifa function test fails contact your Applied Biosystems Service Representative If all functional tests pass the reaction plate or the door of the instrument may not have been aligned properly during the run Note See your in
63. 60 QB 8 M 11 2 20 13 51 21 2 32 5 11 77 5 6 41 2 9 M 9 8 20 15 09 54 0 24 6 13 06 33 3 34 7 10 M 9 7 20 13 98 44 1 30 1 12 29 26 7 38 6 11 M 13 0 20 11 27 13 3 43 7 12 85 1 2 35 8 12 M 13 3 30 9 92 25 1 66 9 11 59 12 5 61 4 13 M nd 14 13 90 n d 0 7 11 31 n d 19 2 14 F 12 8 16 13 90 9 0 13 1 neg n d n d 15 M 15 7 16 12 62 19 4 21 1 13 89 11 2 13 2 16 M 12 1 24 13 09 8 2 45 5 10 78 10 9 55 1 17 M nd 20 12 81 n d 36 0 14 36 n d 28 2 18 M 13 5 24 8 18 39 4 65 9 10 25 24 1 57 3 19 M 13 2 20 10 37 21 4 48 2 13 12 0 6 34 4 20 M 12 9 16 12 69 1 2 20 7 12 36 3 8 22 8 21 M 11 0 14 13 48 22 9 3 7 13 00 18 5 7 1 22 M 11 5 24 12 23 6 6 49 0 12 85 12 0 46 5 23 M 10 9 14 10 91 0 6 22 1 11 73 8 1 16 2 24 M 12 4 20 15 19 22 8 24 1 14 38 16 2 28 1 25 M 10 8 20 15 21 41 5 24 0 18 07 68 1 9 7 26 F 13 9 20 14 00 1 1 30 0 n d n d 27 F 11 5 32 13 16 14 4 58 9 n d n d 28 F 11 5 40 10 51 8 6 73 7 n d n d Quantifiler Kits User s Manual 6 29 Chapter 6 Experiments and Results Table 6 10 Comparison with A and Quantiblot kit continued Quantifiler Human Kit Quantifiler Y Kit Sample Sex neat eat Diff Diff Diff Diff ng L ng uL gris from rom Ba dom l fom l A260 QB Acco QB 29 F 11 2 20 10 45 6 3 47 8 n d n d 30 F 16 0 20 12 56 21 5 37 2 n d n d 31 F 10 9 20 12 12 11 7 39 4 n d n d 32 F 10 9 40 9 42 13 6 76 5 n d n d 33 F 11 5 20 13 95 21 3 30 3 n
64. 8 1 1 Developmental validation that is conducted shall be appropriately documented DAB 1998 DAB Guideline 8 1 2 Novel forensic DNA methodologies shall undergo developmental validation to ensure the accuracy precision and reproducibility of the procedure DAB 1998 BEP 6 55 65 dictate eis iiasddeeteed ehe 6 4 2 Reprod Di ars ihrer 6 7 DAB Guideline 8 1 2 2 Species specificity sensitivity stability and mixture studies are conducted DAB 1998 6 1 3 Specificity with a Human DNA Panel 6 10 6 1 4 Specificity with a Non Human Panel 6 11 6 1 5 Specificity with a Bacterial Pools Panel 6 14 CLS Ban user ong geiko taxa guenceenurdae ad 6 16 Ml TOMY atiatc cay sebecbuiesicausebes dEi RESER 6 17 6 1 3 Mile SUES ee 6 21 6 1 9 Degraded DNA Studies ns ana nass 6 23 6 1 10 Comparisons with Other Methods 6 27 6 1 11 Comparison with Aj and Quantiblot Kit 6 27 6 1 12 Comparison with Az and Dye Intercalation 6 31 6 1 13 Assay Background us 3 8200 Een 6 35 Quantifiler Kits User s Manual 6 3 Chapter 6 Experiments and Results 6 1 1 Precision 6 4 Experiment Results The precision of the Quantifiler Human kit and the Quantifiler Y kit was tested by performing runs on different instruments and on different days One set of eight serial dilutions of the Quantifiler Human DNA Standard was prepared The dilutions
65. 9 24 9 0 606 22 4 69 7 Raji 2 C 0 099 0 110 0 081 17 7 26 1 0 126 27 0 14 0 a Dye intercalation method 6 44 Quantifiler Kits User s Manual Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 The different methods produced similar quantification results Table 6 18 Average differences from A and dye intercalation 7900HT Average Difference Method Quantifiler Human Kit Quantifiler Y Kit A260 34 8 23 5 Dye intercalation 29 8 34 9 Quantifiler Kits User s Manual 6 45 Chapter 6 Experiments and Results Section 6 3 Casework Sample Analysis 6 46 Case Type Studies Experiment There is a recommended optimal DNA concentration range for using AmpF STR PCR Amplification kits The recommended amount of DNA input for the AmpF STR Identifiler PCR Amplification Kit is 0 5 to 1 25 ng human DNA total per reaction and for four dye assays such as the AmpF STR Profiler Plus PCR Amplification Kit 1 0 to 2 5 ng DNA quantification is specified as a requirement by the Scientific Working Group on DNA Analysis Methods SWGDAM as a preliminary step to STR genotyping Scientific Working Group on DNA Analysis Methods 2000 A set of samples consisting of both non casework and casework samples was tested Of the sample set 6 samples were non casework consisting primarily of blood sample extracts from single sources
66. 96 Well Optical Reaction Plates Applied Biosystems PN 4306737 Optical Adhesive Covers Starter Kit 20 covers 1 compression pad 1 applicator Applied Biosystems PN 4313663 Optical Adhesive Covers 100 covers Applied Biosystems PN 4311971 MicroAmp Splash Free Support Base Applied Biosystems PN 4312063 Quantifiler Kits User s Manual Chapter 1 Overview Table 1 4 User supplied materials continued Material Source Mid to Low Throughput Setup MicroAmp Optical Tubes 8 tubes strip 125 Applied Biosystems strips PN 4316567 MicroAmp 96 Well Tray Retainer Set Applied Biosystems PN 403081 Optical Caps 8 caps strip 300 strips Applied Biosystems PN 4323032 Compression pad from Optical Adhesive Applied Biosystems Covers Starter Kit PN 4313663 Note Not necessary if using Optical Caps Table 1 5 Documents Document Applied Biosystems Part Number ABI Prism 7000 Sequence Detection System 4330228 User Guide ABI PRISM 7900HT Sequence Detection 4317596 System User Guide 1 18 Quantifiler Kits User s Manual Chapter 2 Software Setup Quantifiler Kits User s Manual Software Setup This chapter covers Section 2 1 7000 SDS Software Setup 2000 2 3 Der sus 2 4 Starts the 7000 SDS ivy sande dn ded EEVA eee ade 2 5 About Plate Doeuments u 3 220 Reed 2 7 Setting Up a Plate Document ne
67. I 2 g 7 n e n ke 4 Select Start gt Programs gt Applied Biosystems gt SDS 2 0 At startup the software attempts to establish communication with the 7900HT instrument If the connection is successful the software displays in the status bar Quantifiler Kits User s Manual 2 27 Chapter 2 Software Setup About Plate Documents How Plate Running a reaction plate on the 7900HT Real Time PCR System Documents Are requires creating and setting up a plate document using the SDS Used software A plate document is a representation of the arrangement of samples standards and unknowns and reagents on the reaction plate The SDS software uses the plate document to e Coordinate the instrument operation such as thermal cycling and data collection e Organize and store the data gathered during the run e Analyze the data from the run Plate Document You can use SDS software to create two types of plate document Types files Plate Document Type File Extension Description Single plate document sds Primary file to use when performing a run Required for all experiments Template plate sdt File that already contains run parameters that are document commonly used in plate documents such as detectors thermal cycler conditions and so on Streamlines the creation of the SDS document sds file a p n o amp fe 2 2
68. N UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN IO nm oo WD gt Figure 2 3 Example arrangement of reactions with two kits y Q oO I 2 g 7 n e D n O ke Quantifiler Kits User s Manual 2 29 Chapter 2 Software Setup Figure 2 4 shows another example of arranging reactions when running two Quantifiler kits on one 96 well reaction plate if you are using repeat pipettors e Wells Al through D6 gray correspond to reactions using the Quantifiler Human kit e Wells A7 through H12 white correspond to reactions using the Quantifiler Y kit Note For each Quantifiler kit assay there are eight DNA quantification standards and two reactions for each standard See Preparing the DNA Quantification Standard on page 3 2 for more information about the DNA quantification standards 1 2 3 4 5 6 7 8 9 10 11 12 Std 1 Std 1 UNKN UNKN UNKN UNKN Std 1 Std 1 UNKN UNKN UNKN UNKN Std 2 Std 2 UNKN UNKN UNKN UNKN Std 2 Std 2 UNKN UNKN UNKN UNKN Std 3 Std 3 UNKN UNKN UNKN UNKN Std 3 Std 3 UNKN UNKN UNKN UNKN Std 4 Std 4 UNKN UNKN UNKN UNKN Std 4 Std 4 UNKN UNKN UNKN UNKN Std 5 Std 5 UNKN UNKN UNKN UNKN Std 5 Std 5 UNKN UNKN UNKN UNKN Std 6 St
69. NA it is thought that it is extracted and purified with the DNA The presence of hematin in DNA samples may interfere with PCR by inhibiting polymerase activity Bovine serum albumin BSA is used in enzymatic reactions because it appears to increase the efficiency ofthe PCR reaction most likely acting as a chelating agent with many inhibitors BSA is added to the Quantifiler kit and AmpF STR kit reaction mixes specifically to counteract the presence of PCR inhibitors Human genomic DNA was mixed with varying concentrations of hematin 0 uM 10 uM 12 uM 14 uM 16 uM 18 uM 20 uM and 40 uM 2 0 uL of each DNA hematin mix containing 1 0 ng total of human DNA was quantified using the Quantifiler Human kit and Quantifiler Y kit the same amounts of samples were added to reactions using the AmpF STR Identifiler PCR Amplification Kit Identifiler kit reactions were analyzed on a 3100 instrument Data were analyzed with GeneScan Software v3 7 1 and Genotyper Software v3 7 for use with the Windows NT operating system Amplification plots Figures 6 5 and 6 6 showed lower AR values and higher Cy values as the concentration of hematin increased Cy results and corresponding quantification results were relatively stable up to 14 uM hematin with results more affected at higher concentrations As the concentration of hematin increased the PCR efficiency in the Quantifiler kit reactions and the Identifiler kit reactions decreased For
70. Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit User s Manual Applied Biosystems Applied Biosystems Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit User Manual Applied KS Bipsystems 2012 Life Technologies Corporation All rights reserved Information in this document is subject to change without notice Life Technologies Corporation assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF TRADEMARKS Life Technologies Corporation AB Design ABI PRISM AmpF STR Genotyper GeneScan Identifiler MicroAmp Profiler Plus QuantiBlot Quantifiler and VIC are registered trademarks and FAM MicroAmp and ROX are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular System
71. R value Measure of the closeness of fit between the standard curve regression line and the individual Cy data points of quantification standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points e Regression coefficients Slope Indicates the PCR amplification efficiency for the assay A slope of 3 3 indicates 100 amplification efficiency Y intercept Indicates the expected Cy value for a sample with Qty 1 for example 1 ng uL An R value gt 0 99 indicates a close fit between the standard curve regression line and the individual C data points of quantification standard reactions If the R value is lt 0 98 check the following e Quantity values entered for quantification standards in the Well Inspector during plate document setup e Making of serial dilutions of quantification standards e Loading of reactions for quantification standards e Failure of reactions containing quantification standards e C value for Std 8 of the DNA quantification standard 23 pg uL if using the Quantifiler Y kit Quantifiler Kits User s Manual Examining the Standard Curve R Value lt 0 98 Ifthe R value is lt 0 98 for the Quantifiler Y kit only you may choose for Quantifiler Y to omit Std 8 of the DNA quantification standard 23 pg L from Kit Only analysis At the lowest concentration point there are only 7 to 8 copies per 2 uL reaction of the haploid target loc
72. RTANT Although you do not enter units for Quantity you must use a consistent unit for example ng uL for all standard quantities The units used for standard quantities defines the quantification units for analysis results 2 5 3 Q 2 ku S O 2 a 7 N Note Leave the IPC detector Task for standard reactions set to Unknown Quantity values are not needed for IPC detectors d Enter the Sample Name for example Std 1 Std 2 and so on For example Well Inspector q x Well s A1 A2 Sample Name Sta 1 Use Detector__ Reporter Quenchd Task Quantity Cotor Vv IPC VIC none Unknown Task for IPC set M Quantifiler Human FAM none Standard 50 Quantifiler Y FAM none Unknown to Unknown default F Omit Well Passive dd Detector Remove ROX v 2 16 Quantifiler Kits User s Manual Setting Up a Plate Document Applying You need to apply detectors to the plate document for the wells on the Detectors for reaction plate that contain unknown samples Unknown IMPORTANT If you run reactions for the Quantifiler Human kit and Samples the Quantifiler Y kit on the same plate apply detectors for unknown samples for each kit separately To apply detectors for unknown samples 1 On the Plate tab select the wells that correspond to all unknown samples for one Quantifiler kit 2 With the well s selected select V
73. SDS Software v1 0 Quantifiler Kits User s Manual 6 75 Chapter 6 Experiments and Results Figure 6 39 shows the background results for 16 NTCs and one positive control for both kits reanalyzed on the 7000 System SDS Software v1 2 3 One out of 16 NTCs for the Quantifiler Human Kit resulted in a lt 40 CT value 38 26 C Overall the NTC results do not change when analyzed with version 1 2 3 PEYSS conga tnplteabor Plo Siar ube fean par C ye amter Quantifiler Human Kit Quantifiler Y Human Male Kit Figure 6 39 Background amplification plots 7000 System SDS Software v1 2 3 6 5 4 4 Auto Baseline Analysis Versus Manual Analysis C Precision and For Manual to Auto Baseline analysis comparisons Accuracy e Data from initial runs were collected with SDS Software v 1 2 3 and analyzed in Manual analysis mode then reanalyzed in Auto Baseline analysis mode default threshold 0 2 e The Cy values were compared to each other Figures 6 40 and 6 41 show the average Cy values between Auto Baseline analysis and Manual analysis There is a lt 2 difference between the two analysis methods for both kits 6 76 Quantifiler Kits User s Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Cr 32 30 28 26 24 22 Average Cr values from AutoBaseline and Manual Analysis e Quantifiler Human Aut
74. TCs which served as background samples Figure 6 29 shows the experimental plate layout Quantifiler Kits User s Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Standard Quantifiler Y Human Kit Human Male Kit 01 020304 05 06 07 08 09 10 11 12 01 02 03 04 05 06 07 08 09 10 11 12 o o 8 A GOOOOOO OCC lt gt L JOOK XII 00 0 00 00000 ees Ionrmooar ro nmo Standard DNA 1 DNA 2 NTC Standard DNA1 DNA2 NTC curve curve Figure 6 29 Plate Layout Reproducibility and sensitivity experiments 7000 Systems One plate was run with each type of Quantifiler kit To demonstrate reproducibility and sensitivity the replicate DNA samples were quantitated and the results were compared between each software version 6 5 3 Data Collection The standard thermal cycling protocol 9600 Emulation mode described in the Chapter 3 PCR Amplification was used for both studies 6 5 4 Data Analysis Initial Data Compiling and Analysis All runs were analyzed initially using Manual analysis mode with the baseline set to 3 to 15 and the threshold set at 0 2 Average values and standard deviations for C slope and R were calculated for all replicate samples in a run The instrument was then upgraded to SDS Software v1 2 3 then the same run files were reanalyzed and exported with the same analysis settings Quantifiler Kits User s Manu
75. Threshold is set to 0 20 the default setting Results Table The results table displays e Well position of samples e Sample names e Detector assignments Task assignments e Cy values Quantity e Mean and standard deviation for C values and Quantity if replicate groups were defined in assay setup Viewing the View the Qty column to determine the quantity of DNA present in Results Table each sample Note Units for calculated quantities are not displayed but are the same as those specified for the quantification standards when you set up the plate document y I 2 g 7 oO D D gt 5 D lt 9 D Note Quantities are calculated only if quantification standards were run and set up correctly in the software Otherwise only C values are shown For more information about the quantities reported see Assessing Quantity on page 5 16 Quantifiler Kits User s Manual 4 9 Chapter 4 Data Analysis and Results Printing the To print the results Results 1 Select File gt Print Report 2 Select the data to include in the report by selecting the corresponding boxes for e Document Information Thermal Cycler Conditions e Detector Information e Well Status Summary e Raw Data Plot e Multicomponent Data Plot e Amplification Plot 3 Click Page Setup then select e Header footer information and placement e Layout orientation and size
76. ain performance parameters for the Quantifiler kits were also tested separately using the ABI PRISM 7900HT Sequence Detection System 7900HT SDS The experiments performed for the 7900HT SDS were less exhaustive than those for the 7000 instrument see previous section and were performed to test and compare the most sensitive parameters of assay performance between the two instrument platforms 8 2 1 Precision 7900HT SOS san en 6 37 6 2 2 Mixture Studies 7900HT SDS casccaevciacvevsens 6 40 6 2 3 Comparisons with Other Methods 7900HT SDS 6 42 6 2 1 Precision 7900HT SDS Experiment One set of eight serial dilutions of the Quantifiler Human DNA Standard was prepared The dilutions ranged from 50 ng uL to 23 pg uL in three fold increments Three identical runs containing both Quantifiler Human and Quantifiler Y kits were performed each containing duplicate reactions of the dilutions for each assay The three runs were performed on different days on the same 7900HT SDS instrument all using standard thermal cycler conditions for the Quantifiler kits The C ram Values were recorded and the means and standard deviations of the Cy pay values were calculated for each of the eight dilutions using the Quantifiler Human kit and the Quantifiler Y kit Quantifiler Kits User s Manual 6 37 Chapter 6 Experiments and Results 6 38 Results Table 6 15 shows the means and standard deviations of the Cy fam values calculated f
77. al 6 69 Chapter 6 Experiments and Results For Manual to Auto Baseline analysis comparisons the run files from the 7000 System SDS Software v1 2 3 were reanalyzed using the Auto Baseline mode and a threshold of 0 2 6 5 4 1 Precision and Accuracy For the precision and accuracy tests between the two software versions the average C average slope and average R values were determined C Results Figures 6 30 to 6 32 show Cy values obtained using the SDS Software v1 0 and v1 2 3 The data consistently show that SDS Software v1 2 3 yields lower Cy values 2 difference Average Cr values fromSoftware v1 0 and v1 2 3 Cr e Quantifiler Human v1 0 28 4 e Quantifiler Human v1 2 3 26 24 22 2 1 5 1 0 5 0 0 5 1 1 5 2 Log Concentration Figure 6 30 Average C values Quantifiler Human Kit SDS Software v1 0 and v1 2 3 error bars indicate standard deviations 6 70 Quantifiler Kits User s Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Average C values from Software v1 0 and v1 2 3 e Quantifiler Y v1 0 e Quantifiler Y v1 2 3 2 1 5 1 0 5 0 0 5 1 1 5 Log Concentration N Figure 6 31 Average C values Quantifiler Y Human Male Kit SDS Software v1 0 and v1 2 3 error bars indicate standard deviations Software v1 0 and v1 2 3 analysis C values from 3 plates of Qua
78. antity values are not needed for IPC detectors Quantifiler Kits User s Manual 2 35 j Q So I 2 g 7 n io n ke a 2 o 3 fe 2 2 DO 7 Pr I Q f N Chapter 2 Software Setup To apply detectors for quantification standards continued Step 2 continued c Enter the Sample Name for example Std 1 Std 2 and so on d Make sure that ROX is selected for the Passive Reference For example Setup Instrument well A1 A2 Bil Sample Name sta 1 Detector Reporter Color x IPC VIC unknown O _ x Quantifiler Human FAM Standard 5E1 m u Quantifiler Y FAM O m Task for IPC set to Unknown default Applying You need to apply detectors to the plate document for the wells on the Detectors for reaction plate that contain unknown samples Unknown IMPORTANT If you run reactions for the Quantifiler Human kit and Samples the Quantifiler Y kit on the same plate apply detectors for unknown samples for each kit separately To apply detectors for unknown samples 1 In the plate grid press the Ctrl key and select the wells that contain unknown samples for one kit 2 In the Well Inspector select the Use boxes for the detectors in the selected wells e IPC e Quantifiler Human or Quantifiler Y For example Setup Instrument Well
79. at the compression pad is used Delta Rn EEE Ee fit jj jj Il 12245678 0101121314 151617 18 19 90 21 723 24 25 9827 28 2A 30 31 22 33 34 35 3 37 38 30 4N Cycle Number AR and C values inconsistent with replicates Incorrect volume of Quantifiler PCR Reaction Mix added to some reactions Confirm the cause 1 Select the Component tab Affected wells should generate significantly different amounts of fluorescence compared to unaffected replicates 2 Select the Spectra tab Wells with the incorrect volume of Quantifiler PCR Reaction Mix should generate significantly different amounts of fluorescence compared to unaffected wells G 1 deyo synsey Jo uoneJs4dheju enue sasn SHY Jejynuend EL G Table 5 4 Troubleshooting amplification plots continued o PTT TT jessie Tt HH 12345678 910111213141516 17 10 19 20 21 2223 24 25 26 77 20 29 I0 31 32 33 34 95 95 37 39 39 40 Cycle Number Jagged amplification plots Observation Possible Cause Recommended Action i aan Weak lamp or improper Replace the lamp or make sure that the lamp Ler replacement was replaced properly Bae Delta Rn AS
80. ata Analysis on page 1 10 4 4 Quantifiler Kits User s Manual Viewing Results Viewing the For troubleshooting information see Troubleshooting Amplification Amplification Plot Plots on page 5 12 To view the amplification plot 1 In the Results tab select the Amplification Plot tab 2 In the Detector drop down list select a detector e Quantifiler Human or Quantifiler Y e IPC 3 Select the appropriate samples in the table below the amplification plot 4 Make sure that the Threshold is set to 0 20 the default setting Note If you move the threshold bar it changes from green to red to indicate reanalysis is needed After reanalysis it changes from red to green j 2 g 7 g D rr 3 gt 5 D lt o 7 Viewing the The report summarizes the quantity of DNA present in the samples Report For information about the quantities reported see Assessing Quantity on page 5 16 To view the report 1 In the analyzed plate document select the Results tab then select the Report tab 2 Select the reactions in the 96 well plate representation below the report to display the results in the report 3 View the Qty column to determine the quantity of DNA in each sample Note Quantities are calculated only if quantification standards were run and set up correctly in the software Otherwise only Cy values are shown Qua
81. biological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov Quantifiler Kits User s Manual Safety e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Additional information about biohazard guidelines is available at http www cdc gov Quantifiler Kits User s Manual xiij Preface How to Obtain More Information Related Documentation Send Us Your Comments e ABI Pr sm 7000 Sequence Detection System User Guide Describes the 7000 SDS hardware and software and provides information on preparing maintaining and troubleshooting the system e ABI PRISM 7900HT Sequence Detection System User Guide Describes the 7900HT SDS hardware and software and provides information on preparing maintaining and troubleshooting the system Note For additional documentation see How to Obtain Support below Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com How to Obtain Support xiv For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Obtain worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Sear
82. calculated as the mean of the DNA quantity two standard deviation units for each sample and expressed as a percentage of the mean quantification result Quantifiler Kits User s Manual 6 7 Chapter 6 Experiments and Results Results The following tables show the DNA quantity calculated for all samples and dilutions tested for all three runs using the Quantifiler Human kit Table 6 3 and the Quantifiler Y kit Table 6 4 Table 6 3 Reproducibility using the Quantifiler Human kit DNA ntity ng uL o Sample Eai on Be Run 1 Run 2 Run 3 Mean percent 007 A 2 580 2 830 2 900 2 770 0 168 12 15 007 B 0 894 0 779 0 892 0 855 0 066 15 40 007 C 0 216 0 160 0 192 0 189 0 028 29 68 9948 A 2 300 2 240 2 210 2 250 0 046 4 07 9948 B 0 504 0 481 0 573 0 519 0 048 18 44 9948 C 0 123 0 132 0 132 0 129 0 005 8 06 Human genomic A 1 810 1 790 2 240 1 947 0 254 26 12 Human genomic B 0 495 0 468 0 504 0 489 0 019 7 66 Human genomic C 0 128 0 106 0 106 0 113 0 013 22 41 K 562 A 1 360 1 350 1 360 1 357 0 006 0 85 K 562 B 0 379 0 425 0 460 0 421 0 041 19 28 K 562 C 0 096 0 126 0 096 0 106 0 017 32 42 Raji 1 A 1 920 1 800 1 770 1 830 0 079 8 67 Raji 1 B 0 484 0 402 0 466 0 451 0 043 19 13 Raji 1 C 0 149 0 120 0 104 0 124 0 023 36 69 Raji 2 A 1 720 1 860 1 700 1 760 0 087 9 91 Raji 2 B 0 419 0 407 0 408 0 411 0 007 3 24 Raji 2 C 0 113 0 088 0 061 0 087 0 026 59 50 6 8 Quan
83. calculations For paired t Tests analysis MicroSoft Excel Analysis ToolPak software was used 6 4 4 1 Precision and Accuracy For the precision and accuracy tests between the two instrument types the following values were determined e Average Cy e Average Slope e Average R 95 confidence intervals CI by ANOVA analysis Quantifiler Kits User s Manual 6 55 Chapter 6 Experiments and Results C Results Table 6 20 shows the average Cy values 95 CI for the 7500 System SDS Software v1 2 3 and the 7000 System SDS Software v1 0 at each standard curve dilution Table 6 20 C Values 95 Cl 7500 System 7000 System Standard Curve Dilution Average Average ng uL C Value C Value Range 95 CI C Value C Value Range 95 Cl 95 CI 95 CI 50 23 29 23 21 to 23 37 23 05 22 97 to 23 13 16 7 24 98 24 90 to 25 06 24 56 24 48 to 24 64 5 56 26 53 26 44 to 26 61 26 08 26 00 to 26 16 1 85 28 05 27 97 to 28 14 27 53 27 45 to 27 61 0 62 29 44 29 36 to 29 53 29 00 28 92 to 29 09 0 21 30 86 30 78 to 30 94 30 33 30 25 to 30 41 0 068 32 40 32 32 to 32 48 31 61 31 53 to 31 70 0 023 33 98 33 88 to 34 05 33 03 32 95 to 33 11 Statistically the two instrument types resulted in significantly different C values p lt 0 0001 when compared with the ANOVA analysis No significant difference in Cy values was observed when comparing results from instruments of the same type 6 56 Q
84. ch through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches Quantifiler Kits User s Manual Overview This chapter covers Farin een 1 2 Chemistry then ea eT PRR Ee EGS 1 3 Instrument Dein aan 1 7 SDS Sotware DENE ade beck nee dr ArT Ai 1 8 Real Time Data Anabsis na 1 10 Procedural vertan bauer es 1 15 Materials and Equipment ss seed saa rosan 1 16 Quantifiler Kits User s Manual 1 1 Chapter1 Overview Product Overview Purpose Product Description 1 2 The Quantifiler Human DNA Quantification Kit PN 4343895 and the Quantifiler Y Human Male DNA Quantification Kit PN 4343906 are designed to quantify the total amount of amplifiable human and higher primate DNA or human male DNA in a sample The results from using the kits can aid in determining Ifsufficient human DNA or human male DNA is present to proceed with short tandem repeat STR analysis e How much sample to use in STR analysis applications The Quantifiler kits contain all the necessary reagents for the amplification detection and quantification of a human specific DNA target or a human male specific DNA target The reagents are designed and optimized for use with the following Applied Biosystems instr
85. cision and Accuracy Cr Reproducibility and Sensitivity Quantifiler Kits User s Manual For Auto Baseline to Manual analysis comparisons The SDS software v1 2 3 data from the experiments described on the previous pages were reanalyzed in Auto Baseline mode default threshold 0 2 e The Cy values were compared to each other Figure 6 26 shows the Cy values obtained using the Auto Baseline and Manual analysis modes with the Quantifiler Human Kit Similar data were obtained for the Quantifiler Y Human Male Kit No statistically significant differences were observed for C values generated using the Auto Baseline and Manual analysis modes with either Quantifiler kit Raji DNA Cy values with Quantifiler Human e Auto Baseline e Manual oo Ao 32 00 se td ge 29 00 u j oO tag ap 26 00 u a 23 00 Aaa 0 40 80 120 160 Sample Figure 6 26 Comparison of C values between Auto Baseline and Manual analysis modes Figure 6 27 shows the Cy values and calculated quantities obtained using the Auto Baseline and Manual analysis modes with the Quantifiler Human Kit Similar data were obtained for the Quantifiler Y Human Male Kit No statistically significant differences were observed for C values and calculated quantities derived using the Auto Baseline and Manual analysis modes with either Quantifiler kit 6 63 Chapter 6 Experiments and Results
86. d n d 34 F 10 4 20 12 14 16 7 39 3 n d n d 35 F 11 1 40 12 38 11 3 69 1 n d n d 36 F 10 5 20 13 38 28 0 33 1 n d n d 37 F 12 0 24 12 50 4 2 47 9 n d n d 38 F 10 8 20 9 59 11 0 52 1 n d n d 39 F 11 4 16 10 42 8 8 34 9 n d n d 40 F 10 4 40 11 16 7 3 72 1 n d n d 41 F 12 6 20 12 49 0 9 37 6 n d n d 42 F 12 5 28 8 68 30 3 69 0 n d n d 43 F 12 2 20 13 57 11 5 32 2 n d n d 44 F 9 8 16 9 42 3 9 41 1 n d n d 45 F 12 4 16 10 96 11 6 31 5 n d n d 46 F 12 2 16 11 49 5 4 28 2 n d n d 47 F 10 4 40 12 93 24 1 67 7 n d n d 48 F 12 3 20 12 23 0 6 38 9 n d n d 6 30 Quantifiler Kits User s Manual Section 6 1 ABI Prism 7000 Sequence Detection System Validation SDS Software v1 0 Table 6 10 Comparison with A and Quantiblot kit continued Quantifiler Human Kit Quantifiler Y Kit A260 QB Sample Sex Result Result Diff Diff Diff Diff ng L ng uL ing from from a from from H Acco QB H A260 QB 49 F 10 7 40 15 02 40 4 62 5 n d n d 50 F 12 8 32 13 50 5 5 57 8 n d n d a Quantiblot kit method b Negative result The different methods produced similar quantification results Table 6 11 Average differences from Ag and Quantiblot kit Average Difference Method Quantifiler Human Kit Quantifiler Y Kit A260 16 9 15 1 Quantiblot 42 0 35 5
87. d 6 UNKN UNKN UNKN UNKN Std 6 Std 6 UNKN UNKN UNKN UNKN Std 7 Std 7 UNKN UNKN UNKN UNKN Std 7 Std 7 UNKN UNKN UNKN UNKN Std 8 Std 8 UNKN UNKN UNKN UNKN Std 8 Std 8 UNKN UNKN UNKN UNKN IO n m oo WD gt Figure 2 4 Example arrangement of reactions using repeat pipettors a g 3 fe 2 2 DO 7 Pr I Q f N 2 30 Quantifiler Kits User s Manual Setting Up a Plate Document Setting Up a Plate Document Overview Setting up a plate document involves 1 Creating a Blank Plate Document page 2 31 Creating Detectors page 2 32 Copying Detectors to the Plate Document page 2 34 Applying Detectors for Standards page 2 35 Applying Detectors for Unknown Samples page 2 36 Adding Sample Names for Unknown Samples page 2 37 Setting Thermal Cycler Conditions page 2 38 ma Oy Ne ee Saving the Plate Document page 2 39 Creating a Blank To create a blank plate document Plate Document 1 Ifthe SDS software is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 2 Select File gt New complete the New Document dialog box then click OK x Assay Absolute Quantification Standard Curve 7 Container 96 Wells Clear Plate 7 Template Blank Template x Browse Q So I 2 g 7 n e
88. d step 50 C for 2 minutes a Press the Shift key and click within the Stage 1 hold step Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Data Collection Stage 1 Stage 2 Stage 3 aj Repeats 40 95 0 95 0 Press the Shift key and _ click within the Stage 1 hold step Add Cycle Aa Hold Adel Step Sample Volume GAF eo 4 9600 Emulation Stage 1 Stage 2 Stage 3 Hold step is selected b After the hold step is selected press the Delete key Make sure that the thermal profile appears as follows Stage 1 Stage 2 Repeats ao 95 0 2 38 Quantifiler Kits User s Manual Setting Up a Plate Document To set thermal cycler conditions continued 4 Set the Sample Volume to 25 uL and make sure that the 9600 Emulation box is selected Note Selecting the 9600 Emulation box reduces the ramp rate Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Data Collection Stage 1 Stage 2 Repeats faa 95 0 95 0 Add Cycle AddHold Add Step Sample Volumet _ Set the volume to Delete S A i iati elete Step dd Dissociation Stage O Emulation 25 uL Make sure that this box is selected 5 Make sure that the default settings are kept on the remaining tabs Auto Increment e Ramp Rate Data Collection Saving the Plate Before running the reaction plate save the plate document
89. dation SDS v1 2 3 software 6 51 7900HT SDS analysis settings checking 5 3 baseline settings 4 7 fluorescence detection on 1 7 PCR reactions running on 3 9 plate document analyzing 4 7 plate document setting up 2 31 results viewing 4 8 starting 2 27 supported configuration 2 26 template setting up 2 40 threshold settings 4 7 validation SDS v2 0 software 6 37 9600 Emulation box selection on the 7000 SDS 2 20 selection on the 7900HT SDS 2 39 Quantifiler Kits User s Manual A amplification plot abnormal plots in one column example of 5 14 about 1 10 baseline spikes example of 5 13 example 1 10 inconsistent replicates example of 5 12 jagged plot example of 5 13 phases of 1 11 print setup 4 6 printing on the 7900HT SDS 4 10 troubleshooting 5 12 undefined plots example of 5 14 viewing on the 7000 SDS 4 5 viewing on the 7900HT SDS 4 9 analysis settings checking on the 7000 SDS 5 2 checking on the 7900HT SDS 5 3 verifying on the 7000 SDS 4 3 verifying on the 7900HT SDS 4 7 Applied Biosystems contacting xiv customer feedback on documentation xiv Information Development department xiv Services and Support xiv Technical Support xiv B background assay 6 35 baseline about 1 13 settings for the 7000 SDS 4 3 settings for the 7900HT SDS 4 7 biohazard warning xii biohazardous waste handling xii biological hazard safety See biohazard warning bold text when to use vii Index 1 C casework sample analy
90. duces the time required to set up a plate document This section describes how to create an SDS Template Document sdt for running Quantifiler kit assays Template Settings In addition to plate document settings assay and container templates can contain e Assay specific detectors e Well assignments for quantification standards with detectors tasks and quantity Well assignments for unknown samples with detectors and tasks Instrument settings thermal cycler conditions and reaction volume settings 2 5 3 Q 2 ku S O 2 ja 7 r N Creating a Plate This procedure assumes that you have created the detectors for Document running reactions using the Quantifiler kits page 2 11 Template To create a plate document template 1 Ifthe SDS software is not already started select Start gt Programs gt ABI Prism 7000 gt ABI Prism 7000 SDS Software 2 Select File gt New complete the New Document dialog box then click OK x Assay Absolute Quantitation x Container 96 Well Clear B Template Blank Document I Browse 3 Apply the desired template settings to the plate document e Add detectors to the plate document page 2 14 e Apply detectors for standards and for unknown samples page 2 15 and page 2 17 Set thermal cycler conditions page 2 19 2 22 Quantifiler Kits User s Manual Setting Up a Plate Document T
91. dye components Then an algorithm applies matrix calculations to determine the contributions of each component dye to the composite spectrum The software uses the pure dye spectra generated as part of instrument calibration to solve for coefficients a b and c and to calculate the mean standard error MSE in the following equation Measured spectrum a FAM b VIC c ROX d Background MSE where coefficients a b and c represent the contribution of each dye to the composite spectrum The MSE value indicates how closely the collective multicomponent spectrum conforms to the raw spectra Note The example equation above assumes that pure dye components exist for FAM dye VIC dye and ROX dye and for the instrument background 3 FAM dye component o 12000 o c 8 8000 K VIC dye component 3 4000 r ROX dye component ra 0 Background 93 EBEBBEEES HEBBBBEEEEBE component e 8 MSE 2 73 er E 63 r 53 0 10 0 30 0 50 6978 1 10 1 30 Time Figure 1 7 Typical component contributions in a multiplex reaction Quantifiler Kits User s Manual 1 9 Chapter1 Overview Normalization of Reporter Signals The SDS software displays cycle by cycle changes in normalized reporter signal R The SDS software normalizes each reporter signal by dividing it by the fluorescent signal of the passive reference dye Because the passive reference is one component of the PCR master mix
92. e and probe Quantifiler Human DNA Standard 200 ng L purified DNA 1 tube 15 to 25 C standard 120 uL Quantifiler PCR Reaction Mix AmpliTaq Gold DNA 1 tube 2 to 8 C Polymerase dNTPs with 5 mL dUTP Passive Reference and optimized buffer components Additional Follow the additional guidelines for storing the primer mixes Storage Guidelines For Primer Mixes e Minimize freeze thaw cycles Keep protected from direct exposure to light Excessive exposure to light may affect the fluorescent probes Quantifiler Kits User s Manual Materials and Equipment Equipment and Tables 1 3 through 1 5 list required and optional equipment and Materials Not materials not supplied with the Quantifiler kits Unless otherwise Included noted many of the items are available from major laboratory suppliers MLS Table 1 3 Equipment Equipment Source Applied Biosystems 7900HT Real Time PCR System no automation ABI Prism 7000 Sequence Detection System Contact your local Applied Biosystems sales representative Tabletop centrifuge with 96 well plate MLS adapters optional Table 1 4 User supplied materials Material Source Quantifiler Human DNA Quantification Kit Applied Biosystems PN 4343895 Quantifiler Y Human Male DNA Quantification Kit Applied Biosystems PN 4343906 Glycogen 20 mg 1 mL Roche Applied Science PN 901 393 High Throughput Setup
93. e 6 51 7900HT Real Time PCR System SDS v2 0 software 6 37 background assay 6 35 comparisons with other methods 6 27 comparisons with other methods 7900HT SDS 6 42 degraded DNA studies 6 23 importance of 6 2 mixture studies 6 21 mixture studies 7900HT SDS 6 40 precision 6 4 precision 7900HT SDS 6 37 reproducibility 6 7 sensitivity 6 16 specificity with bacterial pools panel 6 14 specificity with human DNA panel 6 10 specificity with non human panel 6 11 stability 6 17 W WARNING description ix waste disposal guidelines xii Y y intercept of standard curve interpreting 5 4 viewing 7000 SDS 4 4 viewing 7900HT SDS 4 8 Quantifiler Kits User s Manual Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com Applied Biosystems Applied Biosystems is committed to providing the world s leading technology and information for life scientists Printed in the USA 03 2012 Part Number 4344790 Rev F
94. e 7000 System SDS Software v1 2 3 Provides accurate results when used with the Quantifiler kits for the analysis of genomic DNA samples Produced results that are similar to the results produced on the previously validated 7000 System SDS Software v1 0 e Precision and Accuracy e Reproducibility and Sensitivity Background e Auto Baseline versus Manual analysis 6 5 1 Materials and Methods 6 5 1 1 Reagents 6 66 To minimize variables from hand pipetting and lot to lot reagent differences the following set up procedures were used throughout the study Eight serial dilutions were made with one lot of standard DNA provided with the Quantifiler kits first dilution prepared with 500 uL DNA and 1 000 uL 10mM Tris HCl pH 8 0 and 0 1 mM Na EDTA T oEo buffer Quantifiler Kits User s Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 6 5 1 2 Instruments One manufactured lot of each kit was used for all validation studies Kit Part Number Lot Number Quantifiler Human Kit 4343895 0501022 Quantifiler Y Human Male Kit 4343906 0501020 One ABI PRISM 7000 Sequence Detection System was used for this study under the following conditions All experiments were run initially using SDS Software v1 0 The 7000 system computer was upgraded to SDS Software v1 2 3 The 7000 System SDS Software v1 2 3 was calibrated by an Applied Biosyste
95. e analysis settings differ from those shown here change them to match the settings before clicking OK 3 Select Analysis gt Analyze Quantifiler Kits User s Manual 4 3 2 v D c lt fe m 2 a N o je N Chapter 4 Data Analysis and Results Viewing Results Overview Viewing the results of data analysis can involve one or more of the following e Viewing the Standard Curve page 4 4 e Viewing the Amplification Plot page 4 5 Viewing the Report page 4 5 Printing or Exporting the Report page 4 6 Viewing the For information about interpreting and troubleshooting the standard Standard Curve curve see Examining the Standard Curve on page 5 4 and Troubleshooting the Standard Curve on page 5 6 To view the standard curve 1 In the Results tab select the Standard Curve tab 2 In the Detector drop down list select the detector that corresponds to the kit that you are using e Quantifiler Human or e Quantifiler Y 3 View the Cy values for the quantification standard reactions and the calculated regression line slope y intercept and R values Amplification Plot The amplification plot can display one of the following Results e Plot of normalized reporter signal R versus cycle number for each reaction e Cry versus well position on the assay plate For more information about the amplification plot see Real Time D
96. ed For comparisons with the Quantifiler Y kit only results from male samples were used Results Table 6 13 shows the DNA concentrations calculated for all samples using the A method the dye intercalation method Quantifiler Human kit and Quantifiler Y kit It also shows the percent differences calculated for the comparisons between the Quantifiler Human kit or the Quantifiler Y kit and the A method and the dye intercalation method Quantifiler Kits User s Manual 6 33 Chapter 6 Experiments and Results Table 6 13 Comparison with Aj and dye intercalation Quantifiler Human Kit Quantifiler Y Kit Rem Reue age ar N I na E rail Reet from from a Benai from from 260 260 007 A 2 74 2 502 2 580 5 8 3 1 3 760 37 2 50 3 007 B 0 685 0 756 0 894 30 5 18 3 1 180 72 3 56 2 007 C 0 137 0 176 0 216 57 7 22 6 0 238 73 7 35 1 9948 A 1 9 2 286 2 300 21 1 0 6 2 590 36 3 13 3 9948 B 0 475 0 496 0 504 6 1 1 5 0 810 70 5 63 2 9948 C 0 095 0 103 0 123 29 5 19 4 0 146 53 7 41 7 Human 2 2 2 270 1 810 17 7 20 3 2 010 8 6 11 5 genomic A Human 0 55 0 584 0 495 10 0 15 2 0 577 4 9 1 1 genomic B Human 0 11 0 134 0 128 16 4 4 8 0 081 26 2 39 6 genomic C Raji 1 A 2 1 271 1 920 4 0 51 0 2 500 25 0 96 7 Raji 1 B 0 5 0 351 0 484 3 2 38 1 0 679 35 8 93 7 Raji 1 C 0 1 0 085 0 149 49 0 76 1 0 123 23 0 45 4 Raji 2 A 1 98 1 262 1 720 13 1 36 3 2 630 32 8 108 4 Raji 2 B 0 495 0 357 0 419 15 4 17 3 0 574
97. ed For comparisons with the Quantifiler Y kit only results from male samples were used Table 6 10 shows the DNA quantification results for all 50 samples in the resolution panel and for the three methods The table also shows the percent differences between the results from the Quantifiler kits and the other two methods There is no A data for two samples 13 and 17 and all female samples were excluded from the comparisons to the Quantifiler Y kit results Table 6 10 Comparison with Ag and Quantiblot kit Quantifiler Human Kit Quantifiler Y Kit Sample Sex aoe a Diff Diff Diff Diff ng uL ng uL eat dam am Ban dam nn 7 A260 QB Acco QB 1 M 17 5 20 6 69 61 7 66 6 10 13 41 9 49 4 2 M 15 4 20 14 3 7 1 28 5 16 78 9 0 16 1 3 M 13 9 30 15 48 11 4 48 4 14 30 2 9 52 3 4 M 11 4 20 12 44 9 6 37 8 12 45 9 7 37 8 5 M 10 3 20 12 69 23 2 36 6 11 00 6 8 45 0 6 M 13 9 20 12 54 9 8 37 3 13 56 2 4 32 2 7 M 11 5 40 13 78 20 1 65 6 12 28 7 1 69 3 6 28 Quantifiler Kits User s Manual Table 6 10 Comparison with Azg and Quantiblot kit continued Section 6 1 ABI Prism 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Human Kit Quantifiler Y Kit Sample Sex cout RSI Diff Diff Diff Diff ng L ng uL Ei from from on trom from A260 QB A2
98. egative sample results and reactions affected by e The presence of PCR inhibitors e Assay setup e A chemistry or instrument failure The following components of the IPC system are present in the Quantifiler PCR mix e Synthetic DNA template Primers that hybridize specifically to the synthetic DNA template e Probe labeled with VIC dye In the amplification plot window of the SDS software observe amplification of the FAM dye Quantifiler Human detector or Quantifiler Y detector and the VIC dye IPC detector then use Table 5 3 to interpret the IPC results Table 5 3 Interpreting IPC amplification results Quantifiler Human or Quantifiler Y FAM Dye IPC VIC Dye Interpretation No amplification Amplification True negative No amplification No amplification Invalid result Amplification low Cy and high AR No amplification Disregard IPC result Amplification high C and low AR No amplification Partial PCR inhibition 5 10 Note Positive amplification is when the C value for the detector is lt 40 Because samples contain unknown amounts of DNA a large range of C values is possible Because the IPC system template DNA is added to the reaction at a fixed concentration the Cr vic should range from 20 to 30 Quantifiler Kits User s Manual True Negative Results Invalid IPC Results Disregarding IPC Results Partial PCR Inhibition Det
99. emplate To create a plate document template continued 4 Select File gt Save As and complete the Save As dialog box a For Save as type select SDS Templates sdt b Locate and select the Templates folder within the software folder X Program Files gt ABI Prism 7000 gt Templates where X is the hard drive on which the SDS software is installed Note Saving the template file in the Templates folder makes the template available in the Template drop down list of the New Document dialog box see step 2 in Creating a Plate Document from a Template on page 2 24 c For File name enter a name for the template For example enter Quantifiler Template So oO oO 2 g 7 n e n xe Save the template file in the Templates folder Po l Save in G Temes gt x e Oct iv AQ RNase P Install sdt My Documents Va My Computer ge JE My Network Pla Filename QuerifierTempite D x Save as type SDS Templates sdt BE Cancel 4 Enter a name for the template d Click Save Quantifiler Kits User s Manual 2 23 Chapter 2 Software Setup Creating a Plate After you create a template you can use it to create a plate document Document from a Template To create a plate document from a template 1 Ifthe SDS software is not already started select Start gt Programs gt ABI Prism 7000 gt ABI Prism 7000 SDS Software 2
100. en the primers and probe and the macaque DNA Did not detect DNA from the remaining species Quantifiler Y Kit Results The Quantifiler Y kit detected DNA from male humans and chimpanzees but from no other species tested Of the DNA samples that were detected using the Quantifiler Human Kit gorilla chimpanzee orangutan and macaque the Quantifiler Y kit e Detected DNA from the chimpanzees Did not detect DNA from the male gorilla Did not detect DNA from the female orangutans or macaques Table 6 6 Specificity with non human panel Result Organism Sex Quantifiler Quantifiler Y Human Kit Kit Gorillla 2 Female Chimpanzee 2 Unknown Orangutan 2 Female Macaque 2 Female b Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Table 6 6 Specificity with non human panel continued Result Organism Sex Quantifiler Quantifiler Y Human Kit Kit Cat Unknown Dog Unknown E u Pig Unknown in u Cow Unknown u Mouse Unknown u Rabbit Unknown u Hamster Unknown u u Rat Unknown Chicken Unknown z u Fish Unknown B Gorilla Male gt 7 Cat Male u Dog 2 Male Z u Mouse Male u Rabbit Male Rat Male Horse 2 Male Bovine Male E Sheep Male E E Pig Male u Deer Male u Quantifiler Kits Us
101. ent Applying You need to apply the detectors to the plate document for the wells on Detectors for the reaction plate that contain DNA quantification standards Repeat Standards _ the procedure until you complete applying detector tasks quantities and sample names for all quantification standards IMPORTANT Set up detectors for each quantity and for each kit separately For example set up detectors for Std 1 for the Quantifiler Human kit first and then for Std 2 for the Quantifiler Human kit and so on until you finish setting up the detectors for all wells containing quantification standards To apply detectors for quantification standards l In the plate grid press the Ctrl key while you select the wells that correspond to a specific quantification standard for one kit Complete the Well Inspector a Select the Use boxes for the applicable detectors e IPC e Quantifiler Human or Quantifiler Y b For the Quantifiler Human or Quantifiler Y detector e Click Unknown in the Task column then select Standard from the drop down list e Select the Quantity field and enter the quantity of DNA in the well IMPORTANT Although you do not enter units for Quantity you must use a consistent unit for example ng uL for all standard quantities The units used for standard quantities defines the quantification units for analysis results Note Leave the IPC detector Task for standard reactions set to Unknown Qu
102. er s Manual 6 13 Chapter 6 Experiments and Results Table 6 6 Specificity with non human panel continued Result Organism Sex al as Quantifiler Quantifiler Y Human Kit Kit Chicken Male Human Female Human Male a Sex confirmed by STR analysis b Weak but positive amplification with higher C values and lower R values than normal for the input amount of DNA in the reaction 6 1 5 Specificity with a Bacterial Pools Panel The bacterial pools panel contained purified genomic DNA from 53 bacterial species and one yeast species The panel included Common gram negative and gram positive species e Species associated with the human gut for example Proteus Providencia Alcaligenes e Species associated with food Lactobacillus spp e Species associated with spoilage and decomposition for example Pseudomonas Flavobacterium Clostridium Candida e Species associated with human enteric disease for example Salmonella Escherichia coli Yersinia Several species of Bacillus a common and pervasive bacterial genus Experiment There were approximately 1 x 10 genome copies of each species in 6 14 each reaction SDS software was used to analyze the data and calculate the Cy fam value Cr FAM Value Result Cr ram lt 40 No amplification after 40 cycles Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Seq
103. ermining the Normal Range for IPC Evaluating PCR Inhibition Quantifiler Kits User s Manual Using the Internal PCR Control System With a true negative result e FAM dye signal indicates that the human specific target failed to amplify e VIC dye signal Cy y c between 20 and 30 indicates that the IPC target was amplified so the PCR was not inhibited If the human specific target and the IPC target failed to amplify it is not possible to distinguish between the absence of DNA and PCR inhibition With extremely high concentrations of human genomic DNA gt 10 ng uL competition between the human specific and IPC PCR reactions appears to suppress IPC amplification for that sample If the target amplifies with low Cy and high AR results it is unlikely that PCR inhibitors are present In these cases appearance of suppression or failure of IPC amplification can be disregarded Weak amplification high C value and low AR value of the human target and no amplification of the IPC may indicate partial PCR inhibition in the sample To determine the normal range of Cy values for the IPC view the VIC dye signal in the amplification plots for the quantification standards If the assays were set up properly and the buffer used to dilute the quantification standards was free of PCR inhibitors the reactions should show normal IPC amplification across a broad range of input DNA If the IPC amplification for certain samples appear
104. ese samples contained the minimum amount of DNA recommended for optimal Identifiler kit results 50 pg uL in a 10 uL reaction For some samples in the original set the volume of DNA sample remaining after DNA quantification was insufficient to perform STR assays these samples were not included in the data presented Table 6 19 Input for STR analysis of casework samples Quantity ng L Input Amount STR Sample Quantifiler Quantifiler for Identifiler QuantiBlot Kit Human Kit Human Kit and Kit ng and 7000 SDS 7900HT SDS Non casework 0 4 0 42 0 4 1 0 Non casework 0 4 0 50 0 50 1 2 Non casework 0 4 0 23 0 38 1 3 Non casework 0 4 0 54 0 56 1 3 Non casework 0 16 0 17 0 23 1 0 Non casework 0 4 0 67 0 65 1 4 Positive control n d n d n d 1 0 Negative control n d n d n d 0 0 Cutting from shirt 0 4 0 78 0 88 1 1 Cutting from shirt 0 4 0 66 0 99 1 1 Cutting from fabric 0 06 0 093 0 11 1 2 Cutting from fabric 0 06 0 060 0 087 0 8 Quantifiler Kits User s Manual 6 47 Chapter 6 Experiments and Results Table 6 19 Input for STR analysis of casework samples continued Quantity ng L Input Amount STR Sample Quantifiler Quantifiler for Identifiler QuantiBlot Kit Human Kit Human Kit and Kit ng and 7000 SDS 7900HT SDS Cutting from denim 0 16 0 10 0 13 1 3 Cutting from sock 0
105. fault settings and to view a blank plate document o x rism 7000 SDS Software Plate1 BB Fie view Tools Instrument Analysis window Help DeesazEr ml h x oO Q oO 2 g 7 n e n ke Creating Before you set up the plate document you need to create detectors in Detectors the SDS software for running Quantifiler kit assays After the detectors are created you do not need to create detectors for subsequent runs of Quantifiler kit assays and you can skip to Adding Detectors to the Plate Document on page 2 14 To create detectors 1 Select Tools gt Detector Manager 2 In the lower left part of the Detector Manager dialog box select File gt New to open the New Detector dialog box Quantifiler Kits User s Manual 2 11 Chapter 2 Software Setup To create detectors continued 3 Create a detector for the Quantifiler Human kit New Detector x Name fQuantifiler Human Description Po Reporter Dye FAM Enter Quantifiler Human Select FAM Quencher Dye none ake sure none is Q color J selected Click to select a color 7 Notes o 3 e 7 A N Create Another OK Cancel o ee Pal je oO N 4 Click Create Another to add the Quantifiler Human detector and to reset the New Detector dialog box 5 Create a de
106. glo w gt Figure 2 1 Example plate setup of reactions with two kits 2 8 Quantifiler Kits User s Manual About Plate Documents Figure 2 2 shows another example of arranging reactions when running two Quantifiler kits on one 96 well reaction plate if you are using repeat pipettors e Wells Al through D6 gray correspond to reactions using the Quantifiler Human kit e Wells A7 through H12 white correspond to reactions using the Quantifiler Y kit Note For each Quantifiler kit assay there are eight DNA quantification standards and two reactions for each standard See Preparing the DNA Quantification Standard on page 3 2 for more information about the DNA quantification standards 1 2 3 4 5 6 7 8 9 10 11 12 Std 1 Std 1 UNKN UNKN UNKN UNKN Std 1 Std 1 UNKN UNKN UNKN UNKN Std 2 Std 2 UNKN UNKN UNKN UNKN Std 2 Std 2 UNKN UNKN UNKN UNKN Std 3 Std 3 UNKN UNKN UNKN UNKN Std 3 Std 3 UNKN UNKN UNKN UNKN Std 4 Std 4 UNKN UNKN UNKN UNKN Std 4 Std 4 UNKN UNKN UNKN UNKN Std 5 Std 5 UNKN UNKN UNKN UNKN Std 5 Std 5 UNKN UNKN UNKN UNKN Std 6 Std 6 UNKN UNKN UNKN UNKN Std 6 Std 6 UNKN UNKN UNKN UNKN Std 7 Std 7 UNKN UNKN UNKN UNKN Std 7 Std 7 UNKN UNKN UNKN UNKN Std 8 Std 8 UNKN UNKN UNKN UNKN Std 8 Std 8 UNKN UNKN UNKN UNKN
107. h Results page a Select Fax or Email below the document title b Click RETRIEVE DOCUMENTS at the end of the document list c Enter the required information d Click View Deliver Selected Documents Now Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical Waste Neem CHEMICAL WASTE HAZARD Some wastes Hazard produced by the operation of the instrument or system are potentially hazardous and can cause injury illness or death Chemical Waste To minimize the hazards of chemical waste Safety Guidelines Quantifiler Kits User s Manual e Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS xi Preface Waste Disposal Biological Hazard xii Safety e Minimize the inhalation of chemicals Do not
108. he detectors for all wells containing quantification standards 3 To apply detectors for quantification standards N D 1 Select View gt Well Inspector to open the dialog box A fo 4 Well s Sample Name o Use Detector _ Reporter Quenchd Task Quantity Color o C PC VIC none Unknown F Quantifiler Human FAM none Unknown 5 MT Quantifiler Y FAM none Unknown I Omit Well Passive dd Detector Remove Make sure that ROX is selected Note The Well Inspector displays the detectors that were added to the plate document 2 On the Plate tab select wells that correspond to a specific quantification standard for one kit J ABI Prism 7000 SDS Software Plate2 B File View Tools Instrument Analysis Window Help DsueRgErm Wells selected Quantifiler Kits User s Manual 2 15 Chapter 2 Software Setup To apply detectors for quantification standards continued 3 With the wells selected go to the Well Inspector and a Select the Use boxes for the applicable detectors e IPC e Quantifiler Human or Quantifiler Y b For the Quantifiler Human or Quantifiler Y detector click Unknown in the Task column then select Standard from the drop down list c For the Quantifiler Human or Quantifiler Y detector select the Quantity field for the appropriate detector and enter the quantity of DNA in the well IMPO
109. iciency of target amplification between 0 and 1 n m number of cycles elapsed between cycle m and cycle n Amplicons designed and optimized according to Applied Biosystems guidelines amplicon size lt 150 bp have amplification efficiencies that approach 100 Therefore Ey so that X n EA Mayne To define the significance in amplified product of one thermal cycle set n m 1 so that i Xm 2 2X m x n Therefore each cycle in the PCR reaction corresponds to a two fold increase in product Likewise a difference in Cy values of 1 equates to a two fold difference in initial template amount Quantifiler Kits User s Manual Procedural Overview Procedural Overview Use of the Quantifiler kits involves the following workflow Software Setup Y PCR Amplification Data Analysis Interpretation of Results Quantifiler Kits User s Manual 1 15 Chapter 1 Overview Materials and Equipment Kit Contents and Each Quantifiler kit contains materials sufficient to perform 400 Storage reactions at a 25 uL reaction volume Table 1 2 Quantifiler kit contents Reagent Contents Quantity Storage Quantifiler Human Primer Mix or e Forward and reverse 3 tubes 15 to 25 C te f primers to amplify 1 4 mL Quantifiler Y Human Male Primer Mix human DNA or human s ch male DNA target e Probe to detect human DNA or human male DNA target e IPC system primers templat
110. iew gt Well Inspector and check the Use boxes for the applicable detectors e Quantifiler Human or Quantifiler Y e IPC For example Well Inspector x Well s B5 D12 Sample Name Detector _ Reporterf uenchd Task Quantity Color Quantifiler Human FAM none Unknown Quantifiler Y FAM none Unknown IPC VIC none Unknown I Omit Well je Q oO 72 g 7 n e n xe Passive sdd Detector Remove ROX Make sure that ROX is selected 3 Ifyou are running both kits on the reaction plate repeat steps 1 and 2 for the unknown samples for the other kit 4 Select View gt Well Inspector to close the Well Inspector Quantifiler Kits User s Manual 2 17 Chapter 2 Software Setup Adding Sample Repeat the procedure to add sample names for all unknown samples Names for Unknown Samples To add sample names for unknown samples 1 On the Plate tab select one well containing an unknown sample 2 With the well selected select View gt Well Inspector and enter the Sample Name For example Well Inspector x Well s B5 Sample Name ibe Daactr_ Repu Oven Quantifiler Human FAM none Unknown T Quantifiler Y FAM none Unknown M PC vic none Unknown Quantity 2 5 3 Q 2 ku S ja 2 a 7
111. ifiler kit before performing STR analysis Quantifiler Kits User s Manual Experiments and Results This chapter covers Overview Section 6 1 Section 6 2 Section 6 3 Section 6 4 Section 6 5 OEO N N E T E E ning 6 2 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 6 3 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 6 37 Casework Sample Analysis 04 6 46 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 6 50 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 6 65 Note The information in Sections 6 4 and 6 5 is also contained in the Quantifiler Kits User Bulletin Validation Using SDS Software Version 1 2 3 on the Applied Biosystems 7500 Real Time PCR System and the ABI PRISM 7000 Sequence Detection System PN 4374659 Rev A 4 2006 Quantifiler Kits User s Manual 6 1 Chapter 6 Experiments and Results Overview About This Chapter Importance of Validation Experiments This chapter provides results of the validation experiments performed by Applied Biosystems using the Quantifiler Human DNA Quantification Kit and the Quantifiler Y Human Male DNA Quantification Kit Although the Quantifiler kits are not DNA genotyping assays they are intended for use before performing genotyping assays such as the AmpF STR PCR A
112. ifiler Human kit 1 0 ng of each DNA sample was added to Identifiler kit reactions As the concentration of amplifiable DNA decreased because of degradation the sample volume required in the reaction increased Identifiler kit results at 1 0 ng uL produced complete STR profiles up to the 5 minute time point although the amount of amplifiable DNA according to the Quantifiler kit was reduced by 90 relative to the untreated control Figure 6 13 The peak heights were reduced for the more degraded samples but profiles were still detected The 15 minute time point contained only 1 of the original amount of Quantifiler Kits User s Manual 6 25 Chapter 6 Experiments and Results 6 26 amplifiable DNA and produced only a partial STR profile of mostly smaller molecular weight loci At 60 minutes no DNA was detected by the Quantifiler kits Figures 6 11 and 6 12 or the Identifiler kit Figure 6 13 The Quantifiler kits can be used to report the amount of amplifiable DNA in a sample but not the amount of DNA degradation Using the quantification data from the kits to determine the amount of sample input for STR analysis may help to correct for the loss of amplifiable DNA because of degradation but if the level of DNA degradation is so high that the remaining DNA fragments are too small the sample will not amplify by using the Quantifiler kits or the STR kits
113. ifiler Y kit For each dilution series the data points formed an acceptable standard curve The small differences in C values among the dilutions of different DNA samples likely reflect differences in the quantification measurements made by each supplier Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 007 m 9948 Human genomic x Raji x K 562 007 m 9948 Human genomic x Raji 6 1 7 Stability x Cr FAM 0 1 1 10 100 DNA Quantity ng uL Figure 6 3 Sensitivity using the Quantifiler Human kit 40 00 38 00 1 36 00 34 00 32 00 30 00 28 00 26 00 24 00 22 00 20 00 0 01 0 1 1 10 100 DNA Quantity ng uL Cy FAM Figure 6 4 Sensitivity using the Quantifiler Y kit DNA samples from various origins are commonly contaminated with organic and inorganic compounds that inhibit the amplification of nucleic acids by PCR These PCR inhibitors can interfere with the reaction and cause varying levels of reduced PCR efficiency including complete inhibition of PCR A wide variety of PCR inhibitors has been reported including in DNA samples extracted Quantifiler Kits User s Manual 6 17 Chapter 6 Experiments and Results 6 18 Experiment Results from blood stains One example is hematin which has been found in DNA samples extracted from blood stains Because the solubility of hematin is similar to that of D
114. ion data from the CCD camera and applies data analysis algorithms 1 7 Chapter1 Overview SDS Software Overview 0 110 0 083 0 055 0 027 0 000 1 8 Composite Spectrum FAM dye This section describes how the SDS software analyzes raw run data from real time runs Raw data consists of the spectral data between 500 nm to 660 nm collected by the SDS software during a sequence detection run Figure 1 6 shows a composite fluorescence spectrum from a single well containing the passive reference one probe labeled with FAM dye and a nonfluorescent quencher and one probe labeled with VIC dye and a nonfluorescent quencher The example shows how the overlapping component dye spectra contribute to the composite spectrum Composite spectrum all dyes ROX dye Background MSE VIC dye Pure Dye components Background from background calibration run Wavelength Figure 1 6 Example of a composite spectrum Quantifiler Kits User s Manual SDS Software Overview Processing During the multicomponent transformation the SDS software uses Multicomponent algorithms to determine the contribution of each dye Data An algorithm removes the background component stored in the background calibration file to eliminate the contribution of background fluorescence in the raw data The software uses the extracted pure dye standards to express the composite spectrum in terms of the pure
115. it is present at the same concentration in all wells of the reaction plate By normalizing the reporter signal using the passive reference the software can account for minor variations in signal caused by pipetting inaccuracies and make better well to well comparisons of reporter signal Real Time Data Analysis Amplification Plot Example 1 10 The 7000 SDS and the 7900HT SDS can be used to determine the relative quantity of a target nucleic acid sequence in a sample by analyzing the cycle to cycle change in fluorescent signal as a result of amplification during a PCR Figure 1 8 When using TaqMan probes with the 7000 SDS or 7900HT SDS the fluorescent signal or normalized reporter R increases as the amount of specific amplified product increases Figure 1 8 shows the amplification of PCR product in a plot of R vs cycle number during PCR This amplification plot contains three distinct phases that characterize the progression of the PCR Phase 3 plateau l Phase 2 linear Phase 1 geometric Cycle Number Figure 1 8 Phases of PCR amplification Quantifiler Kits User s Manual Real Time Data Analysis Phases of Initially R appears as a flat line because the fluorescent signal is Amplification below the detection limit of the sequence detector Phase 1 Geometric
116. lated DNA quantities This validation study demonstrates that the ABI PRISM 7000 Real Time PCR system with SDS Software v1 2 3 is a robust reliable and reproducible system for performing DNA quantification using the Quantifiler kits When comparing 7000 System SDS Software v1 2 3 results Cp slope and R values to results obtained using the previously validated 7000 System SDS Software v1 0 e Small percentage differences are observed in Cp slope and R values e Differences in calculated quantities are minimal for unknown samples using the 7000 System SDS Software v1 2 3 and 7000 System SDS Software v1 0 The differences observed should have little effect on resulting STR amplification based on calculated DNA quantities e No significant difference is observed between C values and calculated quantities derived using Auto Baseline and Manual analysis modes Quantifiler Kits User s Manual Bibliography Afonina I Zivarts M Kutyavin I et al 1997 Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder Nucleic Acids Res 25 2657 2660 DNA Advisory Board Quality assurance standards for forensic DNA testing laboratories approved October1998 Forensic Science Communications 2000 2 3 Available at http www fbi gov programs lab fsc backissu july2000 codispre htm F rster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Ann of Phys Leipzig 2 55 75
117. leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood e After emptying the waste container seal it with the cap provided e Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations If potentially hazardous waste is generated when you operate the instrument you must e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply ANGIE BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective eyewear clothing and gloves Read and follow the guidelines in these publications U S Department of Health and Human Services guidelines published in Biosafety in Micro
118. ls Panel 22 2255 6 14 Quantifiler Kits User s Manual SONSIEVILYE stesso nee eal 2 Bon ae ne Site E a eee 6 16 Stability 4 2 ee la 6 17 Mixture Studies 2 2 32 8a seule a ara gale ese ge esa eles 6 21 Degraded DNA Studies 0 e ee eee 6 23 Comparisons with Other Methods 22222 eee eee 6 27 Comparison with Apso and Quantiblot Kit 0 0 00 eee 6 27 Comparison with Aago and Dye Intercalation 00 6 31 Assay Background eeri ea aa aE tae 6 35 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 6 37 Precision 7900HT SDS 2 000 ee 6 37 Mixture Studies 7900HT SDS 0 000 eee es 6 40 Comparisons with Other Methods 7900HT SDS 6 41 Section 6 3 Casework Sample Analysis 2 0000 e eee eee 6 45 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 6 50 Materials and Methods 0 0 ccc eee ees 6 50 Experimental Setup 0000 eee ee 6 51 Data Collections lt lt b 2 tata eae aia MY LG Pea Cet Be eh ath goed 6 54 Data Analysis rs ea ek ns Ae 6 54 Discussion 2 re Re Pie a 6 63 GONEIUSION u eee use ROME hen ae ae lie ah wee 6 64 Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 1 2 0 0 eee ee 6 66 Materials and Methods 0 0 0 e eee eee teens 6 66 Ex
119. lyze a run after it is complete and reanalyze the run Each time that you open a plate document to convert the saved raw data into analyzed data e After you make changes to the plate document such as sample names To analyze the plate document 1 Open the plate document to analyze 2 Verify the analysis settings a Select Analysis gt Analysis Settings to open the Analysis Settings dialog box b Verify that the settings are as shown below then click OK Automatic Ct Manual Ct Threshold 0 20 C Automatic Baseline Manual Baseline sanje 4 stop 15 4 IMPORTANT If the analysis settings differ from those shown here change them to match the settings before clicking OK 3 Select Analysis gt Analyze for the software to convert the raw data to analyzed data 4 Select the Results tab to view the results Quantifiler Kits User s Manual 4 7 y I 2 g 7 oO D D gt 5 D lt 0 D 2 D D lt s m a m 72 r I N Chapter 4 Data Analysis and Results Viewing Results Overview Viewing the Standard Curve Amplification Plot Results Viewing the results of data analysis can involve one or more ofthe following e Viewing the Standard Curve page 4 8 e Viewing the Amplification Plot page 4 9 e Viewing the Results Table page 4 9 Printing the Results page 4 10
120. mplification kits By testing the procedure with samples commonly encountered in forensic and parentage laboratories the validation process clarifies attributes and limitations that are critical for sound data interpretation in casework Experiments to evaluate the performance of the Quantifiler kits were performed at Applied Biosystems according to the DNA Advisory Board DAB Quality Assurance Standards For Forensic DNA Testing Laboratories DAB 1998 These DAB standards describe the quality assurance requirements that a laboratory should follow to ensure the quality and integrity of the data and competency of the laboratory The DAB defines a laboratory as a facility in which forensic DNA testing is performed Additional validation was performed according to guidelines from the Scientific Working Group on DNA Analysis Methods SWGDAM The experiments focused on kit performance parameters relevant to the intended use of the kits as human specific DNA quantification assays and as a part of a forensic DNA genotyping procedure Each laboratory using the Quantifiler Human DNA Quantification Kit or the Quantifiler Y Human Male DNA Quantification Kit should perform appropriate validation studies Quantifiler Kits User s Manual Section 6 1 ABI Prism 7000 Sequence Detection System Validation SDS Software v1 0 Section 6 1 ABI PRism 7000 Sequence Detection System Validation SDS Software v1 0 DAB DNA Advisory Board Guideline
121. ms service engineer background calibration pure dye calibration RNase P run For the following experiments v1 0 data was reanalyzed using SDS Software v1 2 3 Precision and Accuracy Reproducibility and Sensitivity Background For Auto Baseline versus Manual analysis experiments new data were collected using SDS Software v1 2 3 analyzed in Auto Baseline mode then reanalyzed in Manual mode 6 5 2 Experimental Setup Precision and Accuracy Testing On each 96 well reaction plate six sets of standard dilutions for each Quantifiler kit were set up for real time PCR The experimental plate layout is shown in Figure 6 28 Three replicate plates were run consecutively The Cy slope and R values were compared to determine precision and accuracy Quantifiler Kits User s Manual 6 67 Chapter 6 Experiments and Results Reproducibility Sensitivity and Background Testing 6 68 O Quantifiler Y Human Male DNA Quantification Kit a Quantifiler Human DNA Quantification Kit rommoog 7000 System 7000 System SDS Software v1 0 SDS Software v1 2 3 Figure 6 28 Plate Layout Precision and accuracy testing on the 7000 System On each 96 well reaction plate the following were set up for real time PCR e Standard dilution series two replicates of each dilution point e Four replicate serial dilution sets of two sample DNAs 007 and 9948B e Sixteen no template controls N
122. n SDS Software v1 2 3 Figure 6 37 shows that there was a lt 6 quantity difference between results obtained with v1 0 and v1 2 3 software QTY differences between v1 0 and v1 2 3 analysis 8 6 A A gt ss 3 au e a S 4 I a A Fs a R X 5 gt 2 ro a a Quantifiler Human 007 5 i A R ae Es 3 ni m Quantifier Human 9948 S a po a A Quantifiler Y 007 gt 0 z iceman Sap e Quantifiler Y 9948 u s i g ate Dr 2 K ry A 5 A x ie S E 4 s 6 T 7 7 T 7 7 0 5 10 15 20 25 30 35 Well location Figure 6 37 Percent DNA quantity differences SDS Software v1 0 and v1 2 3 6 5 4 3 Background Figure 6 38 shows the background results for 16 NTCs and one positive control for both kits run on the 7000 System SDS Software v1 0 One out of 16 NTCs for the Quantifiler Human Kit resulted in a lt 40 Cy result 36 81 C7 Remaining NTCs resulted in gt 40 Cy values negative results f LA AEMT olin PMMA re ee eee ee ce ee Quantifiler Human Kit Quantifiler Y Human Male Kit Figure 6 38 Background amplification plots 7000 System
123. n Primer Mix or Quantifiler Y Human Male Materials Primer Mix e Quantifiler PCR Reaction Mix 10 mL polypropylene tube e 96 well reaction plate Extracted DNA samples e DNA quantification standards dilutions series Ty Ep Buffer with or without glycogen for negative controls e Optical Adhesive Cover Preparing the While preparing the reactions keep the 96 well reaction plate in its Reactions base and do not place it on the counter To prepare the reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below Volume Per Sempenent Reaction uL Quantifiler Human Primer Mix or Quantifiler Y 10 5 Human Male Primer Mix Quantifiler PCR Reaction Mix 12 5 Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers Neen CHEMICAL HAZARD Quantifiler PCR Reaction Mix may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Quantifiler Kits User s Manual 3 5 Chapter 3 PCR Amplification To prepare the reactions continued 2 Prepare the reagents Thaw the primer mix completely then vortex 3 to 5 seconds and centrifuge briefly before opening the tube e Swirl the Quantifiler PCR Reaction Mix gently befo
124. n Well Inspector 7000 SDS 2 15 2 17 selecting in Well Inspector 7900HT SDS 2 36 2 37 PCR inhibition 5 11 kinetic analysis of 1 10 phases of 1 11 process in 5 nuclease assay 1 4 reactions preparing 3 5 reactions running on the 7000 SDS 3 7 reactions running on the 7900HT SDS 3 9 standard preparing 3 2 plate document 7000 SDS analyzing 4 3 creating blank 2 10 creating from atemplate 2 24 detectors adding 2 14 detectors applying for standards 2 15 detectors applying for unknown samples 2 17 detectors creating 2 11 how used 2 7 sample names adding 2 18 saving 2 21 setup examples 2 8 template creating 2 22 template settingup 2 22 thermal cycler conditions setting 2 19 types 2 7 Index 3 plate document 7900HT SDS analyzing 4 7 creating blank 2 31 creating from a template 2 42 detectors applying for standards 2 35 detectors applying for unknown samples 2 36 detectors copying 2 34 detectors creating 2 32 how used 2 28 sample names adding 2 37 saving 2 39 setup examples 2 29 template creating 2 40 template setting up 2 40 thermal cycler conditions setting 2 38 types 2 28 plateau phase amplification plot 1 11 polymerization in 5 nuclease assay completion of 1 6 process 1 5 precision on the 7000 SDS 6 4 on the 7900HT SDS 6 37 printing results on the 7000 SDS 4 6 on the 7900HT SDS 4 10 probes about 1 4 pure dye spectra how used 1 9 Q Quantifiler Human detector creating 7000 SDS
125. nes Preparing the DNA Quantifica tion Standards While preparing the standards keep in mind that DNA quantification standards are critical for accurate analysis of run data Any mistakes or inaccuracies in making the dilutions directly affect the quality of results The quality of pipettors and tips and the care used in measuring and mixing dilutions affect accuracy If you use T E Buffer With glycogen you can store the prepared DNA quantification standards for up to 2 weeks at 2 to 8 C Without glycogen long term stability of the prepared DNA quantification standards may not be assured To prepare the DNA quantification standards dilution series 1 Label eight microcentrifuge tubes Std 1 Std 2 Std 3 and so on 2 Dispense the required amount of diluent T E 9 Buffer with or without glycogen to each tube 3 Prepare Std 1 a Vortex the Quantifiler Human DNA Standard 3 to 5 seconds b Using a new pipette tip add the calculated amount of Quantifiler Human DNA Standard to the tube for Std 1 c Mix the dilution thoroughly 4 Prepare Std 2 through 8 a Using a new pipette tip add the calculated amount of the prepared standard to the tube for the next standard b Mix the standard thoroughly c Repeat steps 4a and 4b until you complete the dilution series Quantifiler Kits User s Manual Preparing the Reactions Preparing the Reactions Required e Quantifiler Huma
126. nnen 2 10 Setting Up a Plate Document Template u c0a000 2 22 Section 2 2 7900HT SDS Software Setup 2 25 Ba AERO IOEO eee ee ec cae eee eee ee ae eee 2 26 Starting the 7900HT Real Time PCR System 2 27 About Plate Documents us 20a 2 28 Setting Up a Plate Document vs senden 2 31 Setting Up a Plate Document Template 2 40 Quantifiler Kits User s Manual 2 1 Chapter 2 Software Setup 2 5 3 Q i S ja 2 2 DO 7 r N 2 2 Quantifiler Kits User s Manual Section 2 1 Quantifiler Kits User s Manual 2 3 Section 2 1 7000 SDS Software Setup 7000 SDS Software Setup This section covers NEE ee 2 4 Stars the OOO SDr cscakevaveudeeue i badeursrakewye ne 2 5 About Plate Documents aan a a 2 7 Setting Up a Plate Document se eee ae ees 2 10 Setting Up a Plate Document Template 2 22 Q oO 2 g 7 n io n ke Chapter 2 Software Setup Overview Purpose During software setup you start up the ABI PRISM 7000 Sequence Detection System 7000 SDS and set up a plate document for DNA quantification using the Quantifiler kits Configuration The Quantifiler kits are supported using the 7000 SDS and Sequence Detection Systems SDS Software v1 0 for real time data collection and analysis a f 2 o 3 e 2 2 m 7 Q Q N
127. ntifiler Human Standard Curves e Software v1 0 e Software v1 2 3 0 20 40 60 80 100 120 140 160 Wells Figure 6 32 C Values per Sample v1 0 compared to v1 2 3 Quantifiler Human Kit Quantifiler Kits User s Manual 6 71 Chapter 6 Experiments and Results Slope Results Figure 6 33 shows the average slope values obtained using the SDS software v1 2 3 compared to v1 0 The slope values obtained for the 7000 System SDS Software v1 2 3 are within the established ranges Established Slope Kit Slope Range Quantifiler Human Kit 2 90 to 2 97 2 9 to 3 3 Quantifiler Y Human Male Kit 3 0 to 3 09 3 0 to 3 6 A 1 slope difference is observed between the v1 2 3 and v1 0 software Average Slopes Per Run 3 60 3 10 4 2 60 4 2 10 2 o Software v1 0 o D 460 m Software v1 2 3 1 10 4 0 60 0 10 Run 01 Run 02 Run 03 Run 01 Run 02 Run 03 Quantifiler Human Quantifiler Y Figure 6 33 Average slope values SDS Software v1 0 and v1 2 3 6 72 Quantifiler Kits User s Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 R Results Figure 6 34 shows that SDS software v1 2 3 yields data that are within the acceptable range of R values 0 98 to 1 for both kits lt 0 5 difference 1 0050 1 0000 0 9950 R2 0 9900 0
128. ntifiler Kits User s Manual 4 5 2 v D c lt fe m 2 a N o je N Chapter 4 Data Analysis and Results 4 6 Printing or For more information about exporting data see the ABI Pr sm 7000 Exporting the Sequence Detection System User Guide PN 4330228 Report P To print or export the report exported Report Orientation Portrait Landscape pr Data Columns r Graph s to Print in the Report 1 In the Report tab of the Results window select Tools gt Report Settings then set up how the report is printed and Report Settings x M Well Number Raw Spectra r M Amplification Plot M Sample Name Portrait Portrait Detector Landscape C Landscape Task va m W Dissociation r M Standard Curve Vv StaDev ct F Portrait Portrait M Quantity C Landscape C Landscape IV Mean and StdDev Oty Show detector results in detector color moona bots ic Enna Rer Thermal Profile Analysis Methods Show gray white rows Detector Setup of White rows 4 of Gray rows 4 Can 2 e Select File gt Print to print the report Select File gt Export to export the report as tab delimited text Note You can later open the exported file using spreadsheet software Quantifiler Kits User s Manual Section 4 2 7900HT SDS Data Analysis Section 4 2 7900HT SDS Data Analysis Analyzing the Plate Document Ana
129. ntifiler kits have been created they are listed in the Detector Manager Detector Manager J x r Detector List Detector Name Description Reporter QuenchelColor Last Quantifiler Human FAM none 2003 8 20 13 Quantifiler Y FAM none 2003 8 20 13 IPC VIC none 2003 8 20 13 2 5 3 Q 2 ku S O 2 a 7 N Fie Add To Plate Document Done 2 In the Detector Manager select the Quantifiler Human Quantifiler Y and the IPC detectors by clicking them while pressing the Ctrl key xl r Detector List Find al Description Reporter Guenche Color Notes Las F y File Add To Plate Document 3 Click Add To Plate Document 4 Click Done to close the Detector Manager 2 14 Quantifiler Kits User s Manual Setting Up a Plate Document Applying You need to apply detectors to the plate document for the wells on the Detectors for reaction plate that contain DNA quantification standards Repeat the Standards procedure until you complete applying detector tasks quantities and sample names for all quantification standards IMPORTANT Set up detectors for each quantity and for each kit separately For example set up detectors for quantification standard 1 for the Quantifiler Human kit first and then for quantification standard 2 for the Quantifiler Human kit and so on until you finish setting up t
130. oBaseline e Quantifiler Human Manual 1 5 A 0 5 0 0 5 1 1 5 2 Log Concentration Figure 6 40 Comparison of C values between Auto Baseline and Manual analysis Quantifiler Human Kit error bars indicate standard deviations Cr Average C values from AutoBaseline and Manual Analysis e Quantifiler Y AutoBaseline e Quantifiler Y Manual Hea 1 5 1 0 5 0 0 5 1 1 5 2 Log Concentration Figure 6 41 Comparison of C values between Auto Baseline and Manual analysis Quantifiler Y Human Male Kit error bars indicate standard deviations Quantifiler Kits User s Manual 6 77 Chapter 6 Experiments and Results C Figure 6 42 shows the C values obtained using the Auto Baseline Reproducibility and Sensitivity and Manual analysis modes with the Quantifiler Human Kit No significant differences were observed for Cy values generated using the Auto Baseline and Manual analysis modes with either ng ul 100 5 0 1 Quantifiler Human Auto Baseline and Manual Quantity values Quantifiler Human 007 Auto Baseline e Quantifiler Human 9948 Auto Baseline e Quantifiler Human 007 Manual ee0 a Quantifiler Human 9948 Manual eooo e008 gece ee e eee ers eeto 333 2 e HPE e o_a os e 5 10 15 20 25 30 35 Well locati
131. omputer attempts to establish communication with the 7000 instrument Ifthe connection is successful the software displays in the status bar 2 6 Quantifiler Kits User s Manual About Plate Documents About Plate Documents How Plate Running a reaction plate on the 7000 SDS requires creating and Documents Are setting up a plate document using the SDS software A plate Used document is a representation of the arrangement of samples standards and unknowns and detectors on the reaction plate The SDS software uses the plate document to e Coordinate the instrument operation such as thermal cycling and data collection 2 Organize and store the data gathered during the run gt e Analyze the data from the run u 7 Plate Document You can use the SDS software to create two types of plate document g files z Types 5 1 Plate Document Type File Extension Description 2 ke SDS document sds Primary file to use when performing a run Required for all experiments SDS template sdt File that already contains run parameters that are commonly used in plate documents such as detectors thermal cycler conditions and so on Streamlines the creation of the SDS document sds file Quantifiler Kits User s Manual 2 7 Chapter 2 Software Setup Example Plate You can arrange the reactions in any well of the reaction plate but Document Setup you need to set up the plate document so that it
132. on Figure 6 42 Average C values and average calculated quantities for 9948 and 007 Quantifiler Human Kit 30 ng uL to 0 1 ng uL Figure 6 43 shows the Cy values obtained using the Auto Baseline and Manual analysis modes with the Quantifiler Human Kit Quantifiler kit Quantifiler Human Auto Baseline and Manual C values e Quantifiler Human 007 Auto Baseline 34 e Quantifiler Human 9948 Auto Baseline e Quantifiler Human 007 Manual e Quantifiler Human 9948 Manual 32 e 30 tst te 28 130 8383 26 3338 3338 24 1n822 22 r r r 1 10 20 30 40 Well location 6 78 Quantifiler Kits User s Manual Section 6 5 ABI Prism 7000 Sequence Detection System Validation SDS Software v1 2 3 No significant differences were observed for Cy values generated using the Auto Baseline and Manual analysis modes with either Quantifiler kit Auto Baseline C values overlap the manual Cy values The corresponding quantities also overlap Quantifiler Y Auto Baseline and Manual Cr values Quantifiler Y Auto Baseline and Manual Quantity values e Quantifiler Y 007 Auto Baseline 34 Quantifiler Y 9948 Auto Baseline 100 5 Quantifiler Y 007 Auto Baseline e Quantifiler Y 007 Manual e Quantifiler Y 9948 Auto Baseline e Quantifiler Y 9948 Manual Quantifiler Y 007 Manual 32 O e Quantifiler Y 9948 Manual Te Bere 8 otoo e e 30 age 10 8008 e eye Ses e 3 e amp 28 Hees gt
133. on decreases the amount of variability in the quantification results increases This results from stochastic effects the statistical principles involved when testing DNA samples with low concentrations Stochastic effects may cause imbalance or dropouts of alleles when performing STR analysis of DNA samples with low concentrations 6 1 3 Specificity with a Human DNA Panel Purified genomic DNA samples from 500 human individuals were obtained from two different commercial sources Many of the samples were extracted from cell lines that provide distinct genotypes for forensic validation work other samples were extracted from blood specimens The sex of all samples was confirmed by genotypic analysis using the AmpF STR Identifiler PCR Amplification Kit amelogenin locus Experiment Approximately 20 to 40 ng of purified genomic DNA from the Human DNA Panel was used for each Quantifiler kit reaction Sequence Detection Systems SDS software was used to analyze the data and calculate the Cy pam value Cr FAM Value Result Cr ram lt 40 No amplification after 40 cycles 6 10 Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Results The results in Table 6 5 show that The Quantifiler Human kit detected all 500 human DNA samples The Quantifiler Y kit detected all 240 male DNA samples and none of the female DNA samples Table
134. or all reactions of each quantification standard dilution for the Quantifiler Human kit and the Quantifiler Y kit Table 6 15 Means and standard deviations for C results DNA Quantifiler Human Kit Quantifiler Y Kit Quantity Mall cy Meany Slander cy mean Sindara 50 23 83 0 13 24 50 0 09 16 7 25 36 0 08 26 08 0 09 5 56 26 79 0 08 27 50 0 06 1 85 28 14 0 08 29 03 0 08 0 62 29 56 0 14 30 68 0 30 0 21 31 00 0 06 32 54 0 42 0 068 32 51 0 25 34 41 0 56 0 023 33 86 0 49 35 59 0 58 Quantifiler Kits User s Manual Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 The following results are consistent with the 7000 SDS results e Cy vs sample concentration e Standard deviations of the Cy values e Cy value calculated using the Quantifiler Human kit was lower than that for the Quantifiler Y kit because there is only one copy of the Y chromosome target locus and two copies of the autosomal human target locus The Cy results for all quantification standard dilutions reactions using the Quantifiler Human kit and the Quantifiler Y kit are displayed in Figures 6 16 and 6 17 For each of the dilutions the mean and the standard deviation of Cy ram for the repeated runs is shown HAE l i an N LLI il J i 50 16 7 556 1 85 0 62 0 21 0 068 0 023 DNA quantity ng uL amp N ee Ti mam MIT i Figure 6 16 Precision
135. or the positive and negative controls are included for reference Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 E 90 120 150 180 210 2 mm Hematin 20 OuM Miia Aka i width alla dai 18 oum iata sou EE 1O Ou izt tza OuM iat OR iyi un tet tes OuM tet 1 Out ist tsa OuM tet zu 10 uM wits wm l uud Hha a ad BE Han nad n Be ee 12 uM E Ma U are ik i A A ahah Eee Lt Bee eee i 14 uM E Aka wadh Al d i e HE 1246 uM EES m 18 uM 1 20 uM 1 40 uM Positive a control Negative control Figure 6 8 Inhibition studies STR analysis 6 1 8 Mixture Studies The mixture studies in this section were designed to simulate circumstances in which a small component of male DNA must be discerned from a high background of female DNA Evidence samples may contain DNA from more than one individual and this should be considered when interpreting the results Applied Biosystems recommends that individual laboratories assign a minimum peak height threshold based on validation experiments performed in each laboratory Quantifiler Kits User s Manual 6 21 Chapter 6 Experiments and Results Experiment Results 6 22 Purified genomic DNA from the Raji male and K 562 female cell lines were mixed in rati
136. ors for the standards the incorrect Quantity was entered In the example shown above 0 062 was entered for the Quantity instead of 0 62 5 8 Quantifiler Kits User s Manual Troubleshooting the Standard Curve Well Inspector x Well s E1 Sample Name Use _Detector__ Reporter Quencha Task _Quantity _ Color M Quantifiler Human FAM none Standard 0 062 Incorrect Quantifiler Y FAM Unknown Quantity Vv PC vIC none Unknown entered for standard I Omit Well Passive sdd Detector Remove ROX Example 3 Observation At each concentration in the standard curve There are four replicates There is a large difference in the C between the replicates 40 n 38 gt 7 le 34 y a 2 D a 32 z lt a I 30 L 28 lt io 26 a ine L 24 Na a 22 nn ZZ nn Possible Cause The Quantifiler Human kit assay and the Quantifiler Y kit assay were performed on the same reaction plate and when applying detectors for standards the same detector was applied for Quantifiler Human kit standard reactions and for the Quantifiler Y kit standard reactions Quantifiler Kits User s Manual 5 9 Chapter 5 Interpretation of Results Using the Internal PCR Control System Purpose Components Interpreting IPC Results Use the Internal PCR Control IPC system to distinguish between true n
137. os of 1 1 1 4 1 16 1 64 1 256 and 1 1024 Raji K 562 The male DNA was added at a constant level of 0 05 ng uL in all samples and the female DNA was present at amounts ranging from 0 05 ng uL in the 1 1 sample to 50 ng uL in the 1 1024 sample The DNA amounts were calculated based only on the DNA concentrations provided by the suppliers and were not calibrated with the Quantifiler kits The mixtures were tested with the Quantifiler Human kit and the Quantifiler Y kit to determine the concentrations of total human genomic DNA Quantifiler Human kit and male DNA only Quantifiler Y kit For each sample three replicate reactions were performed for each assay Each assay used the same set of 8 human genomic DNA quantification standards run in duplicate reactions for each assay and both assays were run on the same reaction plate The reaction plates were run on a 7000 instrument The quantification results Figure 6 9 on page 6 23 from using the Quantifiler Human kit varied from an average of 0 16 ng uL for the 1 1 sample to 38 ng uL for the 1 1024 sample consistent with the increasing amounts of female DNA present The quantification results from using the Quantifiler Y kit varied from between 0 034 ng uL to 0 063 ng uL for all samples regardless of the amount of female DNA present For the 1 1024 sample the results showed a ratio of male DNA to total DNA of 1 974 Differences between target concentrations and actual measurements were
138. other dilutions 0 5 ng uL and 0 1 ng uL using the known dilution factors Dye intercalation The microplate assay mode was used and the plate was read on an ABI PRISM 7700 Sequence Detection System 7700 SDS All of the sample dilutions were within the detection range of the assay The assay was run using the A bacteriophage DNA quantification standard supplied with the kit and a quantification standard based on Raji human genomic DNA There were significant differences between the standard curves from the A DNA and Raji DNA The results obtained from using the Raji DNA standard were used in this experiment because the Raji DNA was considered to be more similar to the DNA measured in these experiments and because the results from using the Raji DNA standard were closer to the results obtained by the other methods Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 e Quantifiler kits DNA was quantified using the standard procedure The Quantifiler Human DNA standard provided with the kits was used as recommended with duplicate reactions for each of eight serial dilutions For each sample the percent differences between Quantifiler kit results and results from the other two methods were calculated The differences were expressed as a percentage of the reference method For each method the average percent differences from Quantifiler kit results were calculat
139. perimental Setup 0 000 eee eee 6 67 Data Collectionin 2 22 25 028 e eo is ot eae ee SE 2 6 69 Data Analysis une eee edna nee 6 69 DISCUSSION yi etne zu ae Hae back a ak Gee ede oh ng 6 79 GONClUSi n 2 rn eee eel abet ont See aes Bete 6 80 Bibliography Index Quantifiler Kits User s Manual v vi Quantifiler Kits User s Manual Preface This preface contains How to Use This Gilde esse ke a a ee vii A eine Hae ae in es ee lan aan How to Obtain More Information 0 00005 XIV How to Obtain Support o ci asia dr xiv How to Use This Guide Purpose of This Guide Text Conventions The Quantifiler Kits User s Manual provides information about and instructions for using the Quantifiler Human DNA Quantification Kit and the Quantifiler Y Human Male DNA Quantification Kit This guide uses the following conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix e A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter gt View All Runs Quantifiler Kits User s Manual vii Preface Vili User Attention Words Safety Alert Words
140. r 3 PCR Amplification 3 10 Quantifiler Kits User s Manual 03 2012 Part Number 4344790 Rev F Chapter 4 Data Analysis and Results Quantifiler Kits User s Manual Data Analysis and Results This chapter covers Section 4 1 7000 SDS Data Analysis Analyzing the Plate Document 0 000 eee Terme Ress ar era Section 4 2 7900HT SDS Data Analysis Analyzing the Plate Document 0 000000 Viewing Kell j05 4044 cbse Shoo Reees nen Quantifiler Kits User s Manual 4 1 Chapter 4 Data Analysis and Results 2 V 2 c lt Ss fa N ra N o N 4 2 Quantifiler Kits User s Manual Section 4 1 7000 SDS Data Analysis Section 4 1 7000 SDS Data Analysis Analyzing the Plate Document Analyze a run after it is complete and reanalyze after you make any changes to the plate document such as sample names To analyze a plate document 1 Open the plate document to analyze 2 Verify the analysis settings a Select Analysis gt Analysis Settings to open the Analysis Settings dialog box j 2 g 7 g D D gt 5 D lt o 7 b Verify that the settings are as shown below then click OK Analysis Settings x Detector all z Settings for Threshold 0 200000 Baseline Start cycle 6 Baseline End cycle 15 F Use System Calibration OK amp Reanalyze Cancel Apply IMPORTANT Ifth
141. r Lot Number Quantifiler Human Kit 4343895 0501020 Quantifiler Y Human Male Kit 4343906 0501018 6 4 1 2 Instruments Three 7500 systems SDS Software v1 2 3 and three 7000 systems SDS Software v1 0 were used for this study six instruments total Before the study each instrument was calibrated by an Applied Biosystems service engineer ROI calibration background calibration optical calibration pure dye calibration RNase P run The Biomek FX Laboratory Automation Workstation was used to set up the real time PCR reaction plates to minimize hand pipetting variations e The PCR master mixes PCR reagents with standard or sample DNA mixed together were aliquoted into a 96 well plate PCR master mix plate Six empty 96 well plates and the PCR master mix plate were placed on the Biomek FX work surface The Biomek FX aspirated 25 uL from the PCR master mix plate then slowly dispensed it into the corresponding well in an empty 96 well plate The plates were sealed spun down then quickly loaded onto a 7500 or 7000 system This process ensured timely and precise replication of real time PCR plates for six instruments at a time 6 4 2 Experimental Setup Precision and Accuracy Testing 6 52 On each 96 well reaction plate six sets of standard dilutions for each Quantifiler kit were set up for real time PCR Figure 6 19 shows the experimental plate layout For each instrument six replicate plates were
142. ranged from 50 ng uL to 23 pg uL in three fold increments Three different reaction plates were prepared and each plate contained duplicate reactions of the dilutions using the Quantifiler Human kit and the Quantifiler Y kit The three plates were run on three different 7000 SDS instruments using standard thermal cycler conditions for the Quantifiler kits The multiple runs were performed on two different days using the same three 7000 SDS instruments The Cy pam Values were recorded and the means and standard deviations of the Cy pam values were calculated for each of the eight dilutions using the Quantifiler Human kit and the Quantifiler Y kit Table 6 1 shows the means and standard deviations of the Cy fam values calculated for all 12 reactions of each quantification standard dilution for the Quantifiler Human and Quantifiler Y kits Table 6 1 Precision C values Quantification Quantifiler Human Kit Quantifiler Y Kit Standard Dilution Pall Cream Slander r mean Standard 50 23 09 0 10 23 94 0 21 16 7 24 64 0 17 25 38 0 17 5 56 26 19 0 16 26 91 0 13 1 85 27 67 0 17 28 35 0 15 0 62 29 09 0 17 29 84 0 26 0 21 30 31 0 19 31 38 0 31 Quantifiler Kits User s Manual Section 6 1 ABI Prism 7000 Sequence Detection System Validation SDS Software v1 0 Table 6 1 Precision C values continued Quantification Standard Dilution Quantifiler Human Kit Quantifiler Y Kit
143. re using Do not vortex it Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the PCR mix 3 to 5 seconds then centrifuge briefly Dispense 23 uL of the PCR mix into each reaction well Add 2 uL of sample standard or control to the appropriate wells For plate setup examples see page 2 8 page 2 9 page 2 29 and page 2 30 IMPORTANT Applied Biosystems recommends running duplicates of the eight DNA quantification standards for each assay and on each reaction plate see page 3 4 Seal the reaction plate with the Optical Adhesive Cover Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles Note Ifa tabletop centrifuge with 96 well plate adapters is not available this step can be omitted If you are using a 7000 or 7900HT instrument place the compression pad over the Optical Adhesive Cover with the gray side down and the brown side up and with the holes positioned directly over the reaction wells IMPORTANT Do not use a compression pad if you are using a 7500 instrument Quantifiler Kits User s Manual Running the Reactions Running the Reactions Before You Run the Reactions Running the Plate on the 7000 SDS Before you run the reactions make sure that you have Powered on the SDS instrument computer and software For 7000 SDS setup procedures see page
144. run consecutively The cycle threshold C R and slope values were compared statistically to determine precision and accuracy which established 95 confidence intervals for each instrument type Quantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 O Quantifiler Quantifiler Y Human Human DNA Male DNA Quantification Kit Quantification Kit rommooor 7500 Systems 3 7000 Systems 3 Figure 6 19 Plate layout Precision and accuracy testing on the 7500 System SDS Software v1 2 3 and 7000 System SDS Software v1 0 Quantifiler Kits User s Manual 6 53 Chapter 6 Experiments and Results Reproducibility On each 96 well reaction plate the following were set up for and Sensitivity real time PCR Testing e Standard dilution series two replicates of each dilution point Five replicate serial dilution sets of two sample DNAs Raji and 9948B The experimental plate layout is shown in Figure 6 20 01 02 03 04 05 06 07 08 09 10 11 12 AaLKAKIKIKL III B Do eee oie Secccsecosee Quantifiler Human E ee Kit or Quantifiler Y t IT F LITE Human Male Kit GIB DEE O00 50 0CEECS Standard DNA 1 DNA 2 Standard zen 7500 Systems 3 7000 Systems 3 Figure 6 20 Plate Layout Reproducibility and sensitivity testing on the 7500 System identical plate layout for both kits On each instrument si
145. s B5 D12 Ej Sample Name F Mixed Detector Reporter Color x IPC VIC Unknown O z Quantifiler Human FAM Unknown 0 _ E Quantifiler Y FAM Oo 2 36 Quantifiler Kits User s Manual Setting Up a Plate Document To apply detectors for unknown samples continued 3 Inthe Well Inspector make sure that ROX is selected for the Passive Reference Passive Reference Rox Adding Sample Repeat this procedure to enter the names for all unknown samples Names for Unknown To add sample names for unknown samples Samples 1 In the plate grid select a reaction well containing an unknown sample 2 In the Well Inspector panel enter a name in the Sample Name field Note Samples with identical sample names are treated as replicates by the SDS software Results for replicate reactions are grouped together automatically for data analysis Q I 2 g 7 n e D n O ke Quantifiler Kits User s Manual 2 37 a 2 g 3 fe 2 2 DO 7 Pr I Q f N Chapter 2 Software Setup Setting Thermal Before running a Quantifiler kit assay you need to make two changes Cycler Conditions to the default thermal cycler conditions Thermal profile Sample volume To set thermal cycler conditions l In the plate window select the Instrument tab 2 Delete the Stage 1 hol
146. s Inc All other trademarks are the sole property of their respective owners Part Number 4344790 Rev F 03 2012 Contents Preface How to Use This Guide 2 22 0 000000 eee vii DAISY nn sata oa Aidt erste dea tee a ba are Di ix How to Obtain More Information 0 0 0 0 cece eee ee xiii How to Obtain Support 0 0000 es xiii Chapter 1 Overview Product Overview 0 ccc eee eee ees 1 2 Chemistry Overview een tees 1 3 Instrument Overview 0 000 eee eee eens 1 7 SDS Software Overview 0 000 ccc nennen 1 8 Real Time Data Analysis 0 00 c cece eee eee 1 10 Procedural Overview n aaan nananana 1 15 Materials and Equipment 00 000 eee eee 1 16 Chapter 2 Software Setup Section 2 1 7000 SDS Software Setup 0 2 202055 2 3 OVEINVIEW dca debate Be ee en a Aaa ete Seer 2 4 Starting the 7000 SDS 1 ee 2 5 About Plate Documents 200 0c cece eee 2 7 Setting Up a Plate Document 0 000 eee 2 10 Setting Up a Plate Document Template 2 2055 2 22 Section 2 2 7900HT SDS Software Setup 2 25 OVEIVIEW bicep ne nn ele nate Ao ecb es ee 2 26 Starting the 7900HT Real Time PCR System 2 22 20 2 27 About Plate Documents 002 ccc eee 2 28 Setting Up a Plate Document 00 00 2 31 Setting Up a Plate Document Template 22 055 2 40 Quantifiler Kits User s Man
147. s the Task and Quantity were applied to the wrong detector see Example 1 on page 5 7 1 From the plate document double click a well containing a DNA quantification standard to view the Well Inspector 2 Verify that the Task and Quantity were applied to the correct detector and reanalyze When applying detectors for the standards the incorrect Quantity was entered see Example 2 on page 5 8 1 From the plate document double click a well containing a DNA quantification standard to view the Well Inspector 2 Verify that the correct Quantity was entered and reanalyze Stochastic effects when using the lowest concentration point with the Quantifiler Y kit Omit Std 8 of the DNA quantification standard 23 pg uL from analysis At each concentration in the standard curve e There are four replicates e There is a large difference in C between the replicates Note This observation applies only when Quantifiler Human kit reactions and Quantifiler Y kit reactions are run together on the same reaction plate The same detector was applied for the Quantifiler Human kit standard reactions and for the Quantifiler Y kit standard reactions see Example 3 on page 5 9 1 From the plate document double click a well containing a DNA quantification standard to view the Well Inspector 2 Verify that the correct detector is in use and that the Task and Quantity were
148. s which are normally insignificant are not expected to affect final sample quantification results because when samples and quantification standards are run on the same plate and instrument the C values are affected equally 6 6 Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 6 1 2 Reproducibility Experiment Six different human DNA samples were tested for reproducibility of the quantification results Table 6 2 Human DNA samples tested for reproducibility DNA Sex Extraction Source 007 Male Blood 9948 Male Cell line Human genomic Male Blood Raji Lot 1 Male Cell line Raji Lot 2 Male Cell line K 562 Female Cell line Using the concentrations provided by the supplier the DNA samples were diluted to 2 0 ng uL A 0 5 ng uL B and 0 1 ng uL C Note All dilutions were made in T E Buffer with 20 ug mL glycogen added as a carrier and stabilizer All samples and dilutions were tested in successive runs using the Quantifiler Human kit and the Quantifiler Y kit Three different runs were performed Each assay contained two reactions for each of the quantification standards and one reaction for each of the samples For each sample reaction the Cy pap values were obtained and the DNA quantity calculated The mean quantity and standard deviations were calculated for each sample The 95 confidence interval values were
149. s N in 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 6 5 Inhibition studies Quantifiler Human kit Hematin Delta Rn A 123456 7 8 9 1011121314 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 6 6 Inhibition studies Quantifiler Y kit Quantifiler Kits User s Manual 6 19 Chapter 6 Experiments and Results 6 20 Hematin 1 00 0 uM 10 uM LH 12 uM LTT H 14 uM Delta Rn EFH 18 uM oo iia da dtd dd 12345 67 8 910111213141516 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 6 7 Inhibition studies IPC detector The results of STR analysis using the Identifiler kit Figure 6 8 were consistent with the results from the Quantifiler kits as the concentration of hematin increased the overall STR peak profile decreased Complete STR profiles were obtained at hematin concentrations up to 20 uM The STR amplification reaction was completely inhibited by 40 uM hematin The results from the Quantifiler kits provided reasonable predictions of samples that would fail STR analysis because of the presence of the PCR inhibitor The STR profiles f
150. s reduced relative to IPC amplification for quantification standards the decreased IPC amplification may be interpreted as partial PCR inhibition The IPC results can help you decide the next step e Proceed directly to an STR assay of the sample Repeat the DNA extraction from the sample e Perform additional cleanup of the sample 5 11 On 1 a N jenueyy sasn Suy JejyNuend Troubleshooting Amplification Plots Table 5 4 Troubleshooting amplification plots Observation Possible Cause Recommended Action Deta Ra Sie 17 18 13 20 21 22 20 2T Z8 29 3031 32 33 34 39 36 37 33 39 40 Cycle Number AR and C values inconsistent with replicates Evaporation of reaction mixture from some wells because the Optical Adhesive Cover was not sealed to the reaction plate properly or the compression pad was not used during the run Confirm the cause 1 Select the Component tab Affected wells should generate significantly less fluorescence compared to unaffected replicates 2 Check the amount of solution in each well of the reaction plate Wells affected by evaporation should contain less solution compared to unaffected wells and should correspond with the inconsistent results For subsequent runs make sure that the Optical Adhesive Cover is sealed to the reaction plate properly and th
151. say used the same set of 8 human genomic DNA quantification standards run in duplicate reactions for each assay and both assays were run on the same reaction plate The reaction plates were run on a 7900HT instrument Quantifiler Kits User s Manual Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Results m Quantifiler Y kit o Quantifiler Human kit The quantification results from using the Quantifiler Human kit varied from an average of 0 12 ng uL for the 1 1 sample to 60 ng uL for the 1 1024 sample consistent with the increasing amounts of female DNA present The quantification results from using the Quantifiler Y kit varied from between 0 023 ng uL to 0 058 ng uL for all samples regardless of the amount of female DNA present For the 1 1024 sample the results showed a ratio of male DNA to total DNA of 1 1700 Differences between target concentrations and actual measurements were expected because the amounts of DNA added to the mixtures were based on the DNA concentrations provided by the suppliers and were not calibrated with the Quantifiler kits The results showed that the male DNA was detected and quantified accurately in all samples regardless of the amount of female DNA present 100 000 10 000 1 000 u an OC WA aa 1 1 1 4 1 16 1 64 1 256 1 1024 Male Female Mixture Ratio DNA Quantity ng uL Quantifiler Kits User s Manual 6 41 Chapter 6 Experiments and Results 6
152. sed on Raji human genomic DNA There were significant differences between the Quantifiler Kits User s Manual Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Table 6 17 Comparison with Azs and dye intercalation 7900HT SDS Results standard curves from the X DNA and Raji DNA The results obtained from using the Raji DNA standard were used in these experiments because the Raji DNA was considered to be more similar to the DNA measured and because the results from using the Raji DNA standard were closer to the results obtained by the other methods e Quantifiler kits DNA was quantified using the standard procedure The Quantifiler Human DNA standard provided with the kits was used as recommended with duplicate reactions for each of eight serial dilutions For each sample the percent differences between Quantifiler kit results and results from the other two methods were calculated The differences were expressed as a percentage of the reference method For each method the average percent differences from Quantifiler kit results were calculated For comparisons with the Quantifiler Y kit only results from male samples were used Table 6 17 shows the DNA concentrations calculated for all samples using the A method the dye intercalation method the Quantifiler Human kit and the Quantifiler Y kit It also shows the percent differences calculated for the comparisons
153. sis 6 46 CAUTION description ix chemical safety guidelines x chemical waste hazards xi safety guidelines xi cleavage in 5 nuclease assay 1 5 clipped results exporting 7900HT SDS 4 10 comparisons with other methods on the 7000 SDS 6 27 on the 7900HT SDS 6 42 composite spectrum 1 8 computer starting for the 7000 SDS 2 5 for the 7900HT SDS 2 27 contents ofkit 1 16 conventions bold text vii for describing menu commands vii IMPORTANTS viii in this guide vii italic text vii Notes viii user attention words viii C See threshold cycle customer feedback on Applied Biosystems documents xiv D DANGER description ix data collection 1 8 degraded DNA studies 6 23 Detector Manager 2 14 detectors 7000 SDS adding to plate document 2 14 analysis settings for 5 2 applying for standards 2 15 applying for unknown samples 2 17 creating 2 11 Index 2 detectors 7900HT SDS applying for standards 2 35 applying for unknown samples 2 36 copying to plate document 2 34 creating 2 32 DNA quantification standards dilution series example 3 3 dilution series guidelines for 3 2 guidelines for preparing 3 4 materials required to prepare 3 2 omitting Std 8 5 5 preparing 3 4 reaction recommendation 3 6 See also standards documentation related xiv E equipment not included with Quantifiler kits 1 17 exponential phase See geometric phase exporting results on the 7000 SDS 4 6 on the 7900HT SDS 4 10 F
154. ss e e 8085 ry 26 goes 1 oe e e e 2 33 08 o 24 4 e 8 22 0 1 T T T T T T 1 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 Well location Well location Figure 6 43 Average C values and average calculated quantities for 9948 and 007 Quantifiler Y Human Male Kit 6 5 5 Discussion 6 5 5 1 Precision and Accuracy The results from SDS Software v1 0 and v1 2 3 on a 7000 System slightly differ in C value 2 difference slope 1 and R lt 0 5 for both Quantifiler kits All v1 0 data and v1 2 3 data are within the Quantifiler User s Manual published parameter ranges 6 5 5 2 Reproducibility and Sensitivity For both Quantifiler kits there was a maximum difference of 6 when the calculated quantities using v1 0 and v1 2 3 were compared Such minor differences in calculated quantities should not affect the ability to obtain interpretable STR profiles using the optimal input amount determined by individual laboratories during validation of the Quantifiler kits Quantifiler Kits User s Manual 6 79 Chapter 6 Experiments and Results 6 5 5 3 Manual Analysis Versus Auto Baseline Analysis 6 5 6 Conclusion 6 80 Cry values and their corresponding calculated quantities showed a maximum 8 difference between Auto Baseline and Manual analysis modes on the 7000 System SDS Software v1 2 3 However the differences observed should have little effect on resulting STR amplification based on calcu
155. strument user guide for instructions on how to check ROI calibration and to perform instrument function tests S O q uoneoyydwuiy BunooysajgqnoL Chapter 5 Interpretation of Results Assessing Quantity Purpose Assay Sensitivity Stochastic Effects Validity If Insufficient DNA Is Present 5 16 After viewing the results and assessing the quality of the results the analyst should determine whether sufficient DNA is present to proceed with a short tandem repeat STR assay Quantifiler kit assays can detect lt 23 pg uL of human genomic DNA in samples For samples loaded at 2 0 uL per reaction this concentration corresponds to lt 13 copies of the Quantifiler Human target DNA and lt 7 copies of the Quantifiler Y target locus Y chromosome loci are haploid In the 23 pg uL concentration range stochastic effects or the statistical effect of sampling low copy loci may cause significant variability in assay results The detection and quantification of low copy DNA samples with the Quantifiler kits is valid However the amounts present in the sample may be below the working range of certain genotyping methods If the results from Quantifiler kit reactions indicate that insufficient DNA is present to perform an STR assay the analyst may decide to Extract the DNA again then repeat the test with the Quantifiler kit before performing STR analysis e Concentrate the sample then repeat the test with the Quant
156. tector for the Quantifiler Y kit New Detector x Name fQuantifiler Y Description Ee Reporter Dye FAM Enter Quantifiler Y Select FAM Make sure none is selected Quencher Dye none Color B Notes Click to select a color Create Another Cancel 6 Click Create Another to add the Quantifiler Y detector and to reset the New Detector dialog box 2 12 Quantifiler Kits User s Manual Setting Up a Plate Document To create detectors continued 7 Create a detector for the IPC assay New Detector x Name PCO Enter IPC Description Po Reporter Dye Select VIC Quencher Dye ake sure none is Color selected _Click to select a color Notes Create Another Cancel 8 Click OK to add the IPC detector and to return to the Detector Manager dialog box Detector Manager x r Detector List Find al M Detector Name Description Rep je Q oO 72 g 7 n e n xe Last Quantifiler Human FAM none 2003 8 20 13 Quantifiler Y FAM none 2003 8 20 13 IPC VIC none 2003 8 20 13 File Add To Plate Document Done Quantifiler Kits User s Manual 2 13 Chapter 2 Software Setup Adding Detectors To add detectors to the plate document to the Plate Document 1 In the SDS software select Tools gt Detector Manager If the detectors for the Qua
157. tes Created Jul 17 2003 3 07 55 PM Last Modified Jul 17 2003 3 07 55 PM Cancel b Click OK to return to the Detector Manager Quantifiler Kits User s Manual 2 33 y Q So I 2 g 7 n e D n O xe Chapter 2 Software Setup To create detectors continued 4 Create a detector for the IPC assay a In the Detector Manager click New then complete the Add Detector dialog box Add Detector x Name IPC Group Default z Description Reporter vIC Quencher Non Fluorescent Color E Notes Created Jul 17 2003 3 08 13 PM Last Modified Jul 17 2003 3 08 13 PM Cancel b Click OK to return to the Detector Manager Copying To copy detectors to the plate document Detectors to the Plate Document 1 If the Detector Manager is not already open select Tools gt Detector Manager 2 Select the Quantifiler Human Quantifiler Y and the IPC detectors by clicking them while pressing the Ctrl key Note Ifthe detectors are not available create them first see page 2 32 for the procedure a o 3 fe 2 2 DO 7 Pr I Q f N 3 With the three detectors selected click Copy To Plate Document 4 Click Done to close the Detector Manager and return to the plate window 2 34 Quantifiler Kits User s Manual Setting Up a Plate Docum
158. the Applied Biosystems 7900HT Real Time PCR System and set up a plate document for DNA quantification using the Quantifiler kits Configuration The Quantifiler kits are supported using the following configuration of the 7900HT Real Time PCR System for real time data collection and analysis e 96 well reaction plates e Manual setup Sequence Detection Systems SDS software v2 0 Note Use of the robotic microplate handler and or 384 well reaction plates is not supported a p o 3 0 2 a 7 Fr I Q oS O 2 26 Quantifiler Kits User s Manual Starting the 7900HT Real Time PCR System Starting the 7900HT Real Time PCR System Overview Starting the Applied Biosystems 7900HT Real Time PCR System involves 1 Powering on the computer 2 Powering on the instrument 3 Starting the SDS software Starting the To start the 7900HT System 7900HT System 1 Press the power buttons on the computer and on the monitor 2 In the login screen enter the User Name and Password 3 Press the power button below the status lights on the front of the instrument aos Red ij Orange Status lights fi Green Power button GR2010 At startup the instrument e Emits a high pitched tone indicating that the system is initialized e Cycles the status lights red gt orange gt green indicating that the instrument is active So
159. the Quantifiler Human kit complete inhibition occurred at 40 uM and for the Quantifiler Y kit complete inhibition occurred at 18 uM 20 uM and 40 uM The inhibition may be stronger with the Quantifiler Y kit because there is only one copy of the haploid Y chromosome target locus for the Quantifiler Y kit and two copies of the diploid autosomal target locus for the Quantifiler Human kit The IPC system is more sensitive to PCR inhibition For the Quantifiler Human kit in samples containing more than 16 uM hematin amplification of IPC detectors failed In samples containing less hematin amplification of IPC detectors was inhibited Figure 6 7 Although the Human detector amplified for the 16 uM 18 uM and 20 uM hematin samples the failure of IPC amplification Quantifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 in those reactions indicates that the presence of PCR inhibitors is likely Because the IPC system components are the same in both Quantifiler kits the IPC results for the Quantifiler Y kit were similar to those for the Quantifiler Human kit Hematin sft tt EE EEE EEE EET 14 0 uM 44 10 uM 3 AAT 12 uM 14 uM a JL Lote IIL LEE IVY B g 16 uM MT H 18 um peresa 20 uM 140 uM a ASG TR is pa
160. the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer F rster 1948 Lakowicz 1983 1 4 Quantifiler Kits User s Manual Chemistry Overview Forward TaqMan Primer MGB probe 5 3 3 5 5 3 amp 5 Reverse Primer Figure 1 2 Polymerization Forward d TaqMan Primer MGB probe 5 3 3 5 5 ee eee 3 P 5 Reverse Primer Figure 1 3 Strand displacement AmpliTaq Gold DNA polymerase cleaves only probes that are hybridized to the target Figure 1 4 Cleavage separates the reporter dye from the quencher dye which results in increased fluorescence by the reporter The increase in fluorescence signal occurs only if the target sequence is complementary to the probe and is amplified during PCR Because of these requirements nonspecific amplification is not detected Forward sane TaqMan Primer MGB probe 5 3 3 5 5 3 P 5 Reverse Primer Figure 1 4 Cleavage Quantifiler Kits User s Manual 1 5 Chapter 1 1 6 Overview Polymerization of the strand continues but because the 3 end of the probe is blocked there is no extension of the probe during PCR Figure 1 5 TaqMan Forward gt MGB probe Primer 3 3 5 5 g 5 Reverse Primer Figure 1 5 Completion of polymerization Quantifiler Kits User s Man
161. tifiler Kits User s Manual Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Table 6 4 Reproducibility using the Quantifiler Y kit DNA Quantity ng uL Sample Baviction Comes Run 1 Run 2 Run 3 Mean 007 A 3 760 3 600 3 840 3 733 0 122 6 55 007 B 1 180 0 898 1 040 1 039 0 141 27 13 007 C 0 238 0 185 0 172 0 198 0 035 35 26 9948 A 2 590 2 540 2 670 2 600 0 066 5 04 9948 B 0 810 0 612 0 709 0 710 0 099 27 88 9948 C 0 146 0 130 0 151 0 142 0 011 15 41 Human genomic A 2 010 1 770 1 760 1 847 0 142 15 33 Human genomic B 0 577 0 462 0 591 0 543 0 071 26 06 Human genomic C 0 081 0 053 0 052 0 062 0 017 54 04 K 562 A n d n d n d K 562 B n d n d n d K 562 C n d n d n d Raji 1 A 2 500 2 090 2 400 2 330 0 214 18 35 Raji 1 B 0 679 0 481 0 565 0 575 0 099 34 57 Raji 1 C 0 123 0 096 0 148 0 122 0 026 42 80 Raji 2 A 2 630 2 050 2 190 2 290 0 303 26 43 Raji 2 B 0 574 0 536 0 612 0 574 0 038 13 24 Raji 2 C 0 091 0 123 0 160 0 125 0 034 55 02 a n d not determined Quantifiler Kits User s Manual 6 9 Chapter 6 Experiments and Results The 95 confidence interval shows the approximate range expected for results when using the Quantifiler kits The average 95 confidence interval for each kit e Quantifiler Human kit 18 5 e Quantifiler Y kit 26 9 The data show that as the DNA concentrati
162. tion that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices B VAGONE Indicates a potentially hazardous situation that f not avoided could result in death or serious injury bied Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations gt Nem CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Quantifiler Kits User s Manual ix Preface Chemical Safety To minimize the hazards of chemicals Guidelines About MSDSs Obtaining MSDSs Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufac
163. turer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain MSDSs l 2 Go to https docs appliedbiosystems com msdssearch html In the Search field of the MSDS Search page a Type in the chemical name part number or other information that you expect to appear in the MSDS of interest b Select the language of your choice c Click Search Quantifiler Kits User s Manual Safety 3 To view download or print the document of interest a Right click the document title b Select Open To view the document Save Target As To download a PDF version of the document to a destination that you choose Print Target To print the document 4 To have a copy of an MSDS sent by fax or e mail in the Searc
164. ual Instrument Overview Instrument Overview Fluorescence Detection on the ABI PRISM 7000 Sequence Detection System Detection l A tungsten halogen lamp directs light to each well on the reaction plate The light passes through the ABI PRISM Optical Adhesive Cover and excites the fluorescent dyes in each well of the plate A system of lenses filters and a dichroic mirror focuses the fluorescence emission into a charge coupled device CCD camera Based on wavelength the filters separate the light into a predictably spaced pattern across the CCD camera During the run the CCD camera detects the fluorescence emission between 500 nm and 660 nm from each well The SDS software obtains the fluorescence emission data from the CCD camera and applies data analysis algorithms Detection on the ABI PRism 7900HT Sequence Detection System l Quantifiler Kits User s Manual An argon ion laser directs light to each well on the microplate The light passes through the ABI PRISM Optical Adhesive Cover and excites the fluorescent dyes in each well of the plate A system of lenses filters and a dichroic mirror focuses the fluorescence emission into a grating Based on wavelength the grating separates the light into a predictably spaced pattern across the CCD camera During the run the CCD camera detects the fluorescence emission between 500 nm and 660 nm from each well The SDS software obtains the fluorescence emiss
165. ual iii Chapter 3 Chapter 4 Chapter 5 Chapter 6 PCR Amplification Preparing the DNA Quantification Standard 3 2 Preparing the Reactions 0 0c eee eee 3 5 Running the Reactions 2 222 een eee 3 7 Data Analysis and Results Section 4 1 7000 SDS Data Analysis 0 00 eee 4 3 Analyzing the Plate Document 0 0c nennen nen 4 3 Viewing Results sudasegh ecg naher hear 4 4 Section 4 2 7900HT SDS Data Analysis 0000 e eee eee 4 7 Analyzing the Plate Document 0 0c eee eee 4 7 Viewing Results 0 00 tee 4 8 Interpretation of Results Checking Analysis Settings 0 000 e eee ee 5 2 Examining the Standard Curve 0 naaar aana 5 4 Troubleshooting the Standard Curve 0000 eee eee 5 6 Using the Internal PCR Control System 222222 5 10 Troubleshooting Amplification Plots 0 2 00 eee eee 5 12 Assessing Quantity 0 00 00 cece eee ene 5 16 Experiments and Results OVERVIEW sti ioe BOO cis tie aes ate RO thea AID Mat RN ae 6 2 Section 6 1 ABI PrRism 7000 Sequence Detection System Validation SDS Software v1 0 cc ee eee 6 3 Preeision s twee ici tne en aio neater Whe ate ated 6 4 Reproducibility 0 0 0 eee 6 7 Specificity with a Human DNA Panel 222222 ernennen 6 10 Specificity with a Non Human Panel 0000000ee eee 6 11 Specificity with a Bacterial Poo
166. uantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Slope Results Figure 6 21 shows the average slope values obtained for replicate standard curves run on each instrument The slope values obtained for the 7500 System SDS Software v1 2 3 are listed below and are within the ranges previously established on the 7000 System SDS Software v1 0 Established Slope Kit Slope Range Quantifiler Human Kit 2 93 to 3 18 2 9 to 3 3 Quantifiler Y Human Male Kit 3 05 to 3 36 3 0 to 3 6 Average Slope per Instrument Quantifiler Human kit EB Quantifiler Y kit 4 50 4 00 3 50 5 3 00 2 50 2 00 1 50 5 1 00 0 50 0 00 Ave Slope Value 7000 Instrument 7500 System Instrument Figure 6 21 Average slope values Replicate standard curves Quantifiler Kits User s Manual 6 57 Chapter 6 Experiments and Results R Results Figure 6 22 shows the average R values obtained for replicate standard curves on each instrument All R values were greater than 0 98 and are within the established range R Average per Instrument Quantifiler Human kit E Quantifiler Y kit 1 005 5 1 000 z S 0 995 N amp 0 990 g 0 985 0 980 o 02 o3 o1 02 03 7000 Instrument 7500 System Instrument
167. uence Detection System Validation SDS Software v1 0 Results The Quantifiler Human Kit and the Quantifiler Y kit did not detect DNA from any of the bacterial or yeast species tested Table 6 7 Specificity with bacterial pools panel Result Species Composition ze Quantifiler Quantifiler Human Kit Y Kit Lactobacillus acidophilus Lactobacillus delbrueckii 2 Lactobacillus rhamnosus Lactobacillus casei Brochothrix thermosphacta Brochothrix campestris Aerococcus viridians Kurthia gibsonii Alcaligenes faecalis Bacillus subtilis Bacillus cereus Bacillus licheniformis Bacillus mycoides Bacillus stearothermophilus Pseudomonas fluorescens Flavobacterium odoratum Clostridium sporogenes Candida kefyr yeast Deinococcus radiodurans Lactococcus lactis Bordetella bronchiseptica Acinetobacter baumannii Aeromonas caviae Corynebacterium varibile Nocardia asteroides Stenotrophomonas maltophilia Bacillus coagulans Rhodococcus equi Acinetobacter calcoaceticus Propionibacterium acnes Clostridium difficile Fusebacterium necrophorum Burkholderia cepacia Delftia acidovorans Micrococcus luteus Streptomyces rimosus Gordonia sputi Legionella ansia Pasteurella aerogenes Citrobacter freundii Klebsiella pneumoniae Escherichia hermanii Enterobacter cloacae Escherichia coli 0157 H7 Salmonella enteritidis Shigella dysenteriae
168. uments and software e ABI PRISM 7000 Sequence Detection System and SDS Software v1 0 e ABI PRISM 7900HT Sequence Detection System no automation module and SDS Software v2 0 See Chapter 6 Experiments and Results for validation studies performed using the Applied Biosystems 7500 Real Time PCR System with SDS Software v1 2 3 and the ABI PRISM 7000 Sequence Detection System with SDS Software v1 2 3 Quantifiler Kits User s Manual Chemistry Overview Chemistry Overview Assay Overview The DNA quantification assay combines two 5 nuclease assays e A target specific human DNA or human male DNA assay e An internal PCR control IPC assay The target specific assay consists of Target Specific Com o Two primers for amplifying human DNA or human male DNA P One TaqMan MGB probe labeled with FAM dye for detecting the amplified sequence About the Targets Table 1 1 provides information about the targets of PCR amplification in the Quantifiler Human kit and the Quantifiler Y kit Table 1 1 Targets of Quantifiler kits Kit Gene Target Location Amplicon Region Ploidy Length Amplified Quantifiler Human kit Human telomerase 5p15 33 62 bases Nontranslated Diploid reverse region intron transcriptase gene hTERT Quantifiler Y kit Sex determining Yp11 3 64 bases Nontranslated Haploid region Y gene SRY region a Single copy target IPC Assay The IPC assay consists
169. us for the Quantifiler Y kit Because of stochastic effects when using the lowest concentration point with Quantifiler Y kits the C values are more variable at the lowest concentration point and may affect the closeness of fit between the standard curve regression line and the individual data points of the quantification standard To omit Std 8 from analysis for Quantifiler Y kits only 1 Select the wells in the plate document that correspond to Std 8 and open the Well Inspector 2 Change the Task assignment for the Quantifiler Y detector from Standard to Unknown 3 Reanalyze the plate to incorporate the change Slope A slope close to 3 3 indicates optimal 100 PCR amplification efficiency Table 5 1 Range and average of standard curve slope values Kit Typical Slope range Average Slope Quantifiler Human 2 9 to 3 3 3 1 Quantifiler Y 3 0 to 3 6 3 3 Ifthe slope varies beyond the typical range indicated in Table 5 1 check the following e Assay setup e Software setup e Reagents Instrument Quantifiler Kits User s Manual 5 5 Chapter 5 Interpretation of Results Troubleshooting the Standard Curve Table 5 2 Troubleshooting the standard curve Observation Possible Cause Recommended Action Slope for the standard curve differs significantly from 3 33 or R value significantly less than 0 98 to 0 99 When applying detectors for standard
170. ware is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 Select File gt Open Locate the plate document for the assay run of interest select it then click Open Select Analysis gt Analysis Settings and confirm that the settings are as shown below Automatic Ct Manual Ct Threshold 0 20 C Automatic Baseline Manual Baseline sarf 4 stop 15 If the analysis settings differ from those shown in step 4 a Change the settings to match those in step 4 b Click OK c Select Analysis gt Analyze for the software to reanalyze the data d View the results using Chapter 4 Data Analysis and Results Quantifiler Kits User s Manual Chapter 5 Interpretation of Results Examining the Standard Curve About Standard Curve Results R2 Value Examine the standard curve results to evaluate the quality of the results from the quantification standard reactions The standard curve is a graph of the Cy of quantification standard reactions plotted against the starting quantity of the standards The software calculates the regression line by calculating the best fit with the quantification standard data points The regression line formula has the form Cr m log Qty b where m is the slope b is the y intercept and Qty is the starting DNA quantity The values associated with the regression analysis can be interpreted as follows e
171. x replicate plates were run consecutively with each Quantifiler kit for a total of 18 plates on 7500 systems and 18 plates on 7000 systems To demonstrate reproducibility and sensitivity the replicate DNA samples were quantitated and the results were compared statistically between instrument types 6 54 Quantifiler Kits User s Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Background Ninety five no template controls NTCs and one positive control Testing the 50 ng uL standard DNA dilution sample were set up on a 96 well plate One plate from each Quantifiler kit was run on each instrument for a total of 12 plates 6 4 3 Data Collection The standard thermal cycling protocol 9600 Emulation mode described in the Chapter 3 PCR Amplification was used for all instrument runs 6 4 4 Data Analysis Initial Data Allruns were analyzed initially using Manual analysis mode with the Compiling and baseline set to 3 to 15 and the threshold set at 0 2 Analysis Average values and standard deviations for C slope and R were calculated for all replicate samples in a run For Auto Baseline to Manual analysis comparisons the run files from the 7500 System SDS Software v1 2 3 were reanalyzed using Auto Baseline mode and a threshold of 0 2 Statistical Data For statistical analysis the Stat Ease Design Expert Software was Analysis used for all ANOVA analysis of variance
172. y systems performed normally These data show that there is no inherent false positive background associated with the Quantifiler kits However the assays are extremely sensitive and achieving clean results requires care in assay setup and good contamination control for reagents instruments and laboratory work surfaces Quantifiler Kits User s Manual 6 35 Chapter 6 Experiments and Results 108001 py Ue eee nn IPC amplification 1 0e 000 1 08 001 z 4 ld A Op 7 1 0e 002 ty Ye n gf My PAA KRASNI Human DNA i N AKG iy i i N amplification dl ui aT ii 1 00 003 W li P 1 02 004 1234567 ih N Cycle Number Figure 6 14 Assay background with the Quantifiler Human kit Toso I 1 17 E sas S tO OOOO nee m HPC amplification 1 08 000 ai 1 02 001 j ii il 5 Re if A 1 amp a AP AS APRs 1 08 02 A Hate dai Seay T Shi amplification N 7 i IN ER Nu Human Y DNA IR Ei i ae il ia iid 123456 7 8 9101112431415161718192021 222324 252627 28293031 3233 Cycle Number Sa I 11 37383940 41 424344454647 434950 Figure 6 15 Assay background with the Quantifiler Y kit 6 36 Quantifiler Kits User s Manual Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Section 6 2 Applied Biosystems Overview Experiments 7900HT Real Time PCR System Validation SDS Software v2 0 Cert

Quantifiler Kits User`s Manual (PN 4344790F)

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