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Quantifiler Kits User Manual (PN 4344790D)

1. 6 16 Stability 6 17 Mixture Studies 6 21 Degraded DNA Studies 6 23 Comparisons with Other Methods 6 27 Comparison with A260 and Quantiblot Kit 6 27 Comparison with A260 and Dye Intercalation 6 31 Assay Background 6 35 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 6 37 Precision 7900HT SDS 6 37 Mixture Studies 7900HT SDS 6 40 Comparisons with Other Methods 7900HT SDS 6 41 Section 6 3 Casework Sample Analysis 6 45 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 6 50 Materials and Methods 6 50 E
2. xiii Chapter 1 Overview Product Overview 1 2 Chemistry Overview 1 3 Instrument Overview 1 7 SDS Software Overview 1 8 Real Time Data Analysis 1 10 Procedural Overview 1 15 Materials and Equipment 1 16 Chapter 2 Software Setup Section 2 1 7000 SDS Software Setup 2 3 Overview 2 4 Starting the 7000 SDS 2 5 About Plate Documents 2 7 Setting Up a Plate Document 2 10 Setting Up a Plate Document Template 2 22 Section 2 2 7900HT SDS Software Setup
3. 2 3 Overview 2 4 Starting the 7000 SDS 2 5 About Plate Documents 2 7 Setting Up a Plate Document 2 10 Setting Up a Plate Document Template 2 22 Section 2 2 7900HT SDS Software Setup 2 25 Overview 2 26 Starting the 7900HT Real Time PCR System 2 27 About Plate Documents 2 28 Setting Up a Plate Document 2 31 Setting Up a Plate Document Template 2 40 Chapter 2 Software Setup 2 2 Quantifiler Kits User s Manual 7000 SDS Software Setup Section 2 1 7000 SDS Software Setup Quantifiler Kits User s Manual 2 3 7000 SDS Software Setup Section 2 1 7000 SDS Software Setup This section covers Overview 2 4 Starting the 7000 SDS 2 5 About Plat
4. 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 O157 H7 Salmonella enteritidis Shigella dysenteriae Proteus vulgaris Pseudomonas aeruginosa Hafnia alvei Yersinia enterocolitica Campylobacter coli Providencia stuartii Vibrio parahaemolyticus Alcaligenes faecalis Chapter 6 Experiments and Results 6 16 Quantifiler Kits User s Manual 6 1 6 Sensitivity 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 DNA Samples Tested Five different human DNA samples were tested Experiment Using the concentrations provided by the suppliers five fold serial dilutions of th
5. Plate Document Type File Extension Description 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 Chapter 2 Software Setup 2 8 Quantifiler Kits User s Manual 7000 SDS Software Setup Example Plate Document Setup You can arrange the reactions in any well of the reaction plate but 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 1 shows one example of arranging reactions when running two Quantifiler kits on one 96 well reaction plate Wells A1 through D12 gray correspond to reactions using the Quantifiler Human kit Wells E1 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 Figure 2 1 Example plate setup of reactions with two kits 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std 5 Std 6 Std 6 B Std 7
6. 1 Select View gt Well Inspector to open the dialog box 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 Make sure that ROX is selected Wells selected Chapter 2 Software Setup 2 16 Quantifiler Kits User s Manual 7000 SDS Software Setup 3 With the wells selected go to the Well Inspector and a Select the Use boxes for the applicable detectors IPC 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 IMPORTANT Although you do not enter units for Quantity you must use a consistent unit for example ng L 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 Quantity values are not needed for IPC detectors d Enter the Sample Name for example Std 1 Std 2 and so on For example To apply detectors for quantification standards continued Task for IPC set to Unknown default Setting Up a Plate Document Quantifiler Kits User
7. CT Quantifiler Human 007 Auto Baseline Quantifiler Human 9948 Auto Baseline Quantifiler Human 007 Manual Quantifiler Human 9948 Manual Quantifiler Human Auto Baseline and Manual Quantity values 0 1 1 10 100 0 5 10 15 20 25 30 35 Well location ng ul Quantifiler Human 007 Auto Baseline Quantifiler Human 9948 Auto Baseline Quantifiler Human 007 Manual Quantifiler Human 9948 Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 79 No significant differences were observed for CT values generated using the Auto Baseline and Manual analysis modes with either Quantifiler kit Auto Baseline CT values overlap the manual CT values The corresponding quantities also overlap Figure 6 43 Average CT 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 CT value 2 difference slope 1 and R2 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 affe
8. 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 Obtaining MSDSs The MSDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain MSDSs 1 Go to https docs appliedbiosystems com msdssearch html 2 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 Safety Quantifiler Kits User s Manual xi 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 Search 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 Fo
9. Quantifiler Human Kit SDS Software v1 0 and v1 2 3 error bars indicate standard deviations Average CT values fromSoftware v1 0 and v1 2 3 22 24 26 28 30 32 34 2 1 5 1 0 5 0 0 5 1 1 5 2 Log Concentration CT Quantifiler Human v1 0 Quantifiler Human v1 2 3 Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 71 Figure 6 31 Average CT values Quantifiler Y Human Male Kit SDS Software v1 0 and v1 2 3 error bars indicate standard deviations Figure 6 32 CT Values per Sample v1 0 compared to v1 2 3 Quantifiler Human Kit Average CT values from Software v1 0 and v1 2 3 22 24 26 28 30 32 34 36 2 1 5 1 0 5 0 0 5 1 1 5 2 Log Concentration CT Quantifiler Y v1 0 Quantifiler Y v1 2 3 Software v1 0 and v1 2 3 analysis CT values from 3 plates of Quantifiler Human Standard Curves 20 22 24 26 28 30 32 34 36 0 20 40 60 80 100 120 140 160 Wells CT Software v1 0 Software v1 2 3 Chapter 6 Experiments and Results 6 72 Quantifiler Kits User s Manual 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 A 1 slope difference is observed between the v1
10. 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 Chapter 6 Experiments and Results 6 62 Quantifiler Kits User s Manual 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 CT values gt 40 with both Quantifiler kits Figure 6 24 Background amplification plots 7000 System SDS Software v1 0 Figure 6 25 Background amplification plots 7500 System SDS Software v1 2 3 Quantifiler Human Kit Quantifiler Y Human Male Kit Quantifiler Human Kit Quantifiler Y Human Male Kit Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 63 6 4 4 4 Auto Baseline Analysis Versus Manual Analysis CT Precision and Accuracy 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 The CT values were compared to each other Figure 6 2
11. 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 b 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 Table 6 10 Comparison with A260 and Quantiblot kit continued Sample Sex A260 Result ng L QBa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from QB Result ng L Diff from A260 Diff from QB Chapter 6 Experiments and Results 6 30 Quantifiler Kits User s Manual 29 F 11 2 20 10 45 6 3 47 8 n d n d 30 F
12. Experiments and Results 6 50 Quantifiler Kits User s Manual Figure 6 18 STR profiles of casework samples 17 20 19 18 21 22 23 24 25 26 27 28 29 16 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 51 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Overview 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 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 Validation Experiments Performed Precision and Accuracy Reproducibility and Sensitivity Background 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 fo
13. 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 No significant difference is observed between CT values and calculated quantities derived by using Auto Baseline and Manual analysis modes Chapter 6 Experiments and Results 6 66 Quantifiler Kits User s Manual Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Overview 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 the 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 Validation Experiments Performed Precision and Accuracy Reproducibility and Sensitivity Background Auto Baseline versus Manual analysis 6 5 1 Materials and Methods 6 5 1 1 Reagen
14. Possible Cause Recommended Action Rn and CT 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 that the compression pad is used Rn and CT 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 Troubleshooting Amplification Plots Quantifiler Kits User s Manual 5 13 Jagged amplification plots Weak lamp or improper replacement Replace the lamp or make sure that the lamp was replaced
15. 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 Chapter 2 Software Setup 2 22 Quantifiler Kits User s Manual 7000 SDS Software Setup 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 for running Quantifiler kit assays Template Settings In addition to plate document settings assay and container templates can contain Assay specific detectors 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 Creating a Plate Document Template This procedure assumes that you have created the detectors for running reactions using the Quantifiler kits page 2 11 To create a plate document template 1 If the 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 3 Apply the desired template settings to the plate document Add detectors to the plate document page 2 14 Apply detectors for standa
16. 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 instruments and software ABI PRISM 7000 Sequence Detection System and SDS Software v1 0 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 Chemistry Overview Quantifiler Kits User s Manual 1 3 Chemistry Overview Assay Overview The DNA quantification assay combines two 5 nuclease assays A target specific human DNA or human male DNA assay An internal PCR control IPC assay Target Specific Assay Components The target specific assay consists of Two primers for amplifying human DNA or human male DNA 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 IPC Assay Components The IPC assay consists of 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 det
17. pH 8 0 0 1 mM Na2EDTA 20 g mL glycogen optional Note If you use T10E0 1 buffer with glycogen you can store the DNA quantification standards for up to 2 weeks at 2 to 8 C Guidelines for Calculating the Standards Dilution Series The standard dilution series example shown in Table 3 1 on page 3 3 is suitable for general use IMPORTANT Applied Biosystems recommends Three fold dilution series with eight concentration points in the standard series for each assay Minimum input volume of 10 L DNA for dilutions to ensure accuracy of pipetting Preparing the DNA Quantification Standard Quantifiler Kits User s Manual 3 3 Standards Dilution Series Example Table 3 1 shows an example of one standards dilution series with the concentrations ranging from 50 ng L Std 1 to 0 023 ng L or 23 pg L Std 8 A sample at the lowest concentration 2 L 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 Standard Concentration ng L Example Amounts Minimum Amounts Dilution Factor Std 1 50 000 50 L 200 ng L stock 150 L T10E0 1 glycogen buffer 10 L 200 ng L stock 30 L T10E0 1 buffer 4 Std 2 16 700 50 L Std 1 100 L T10E0 1 glycogen buffer 10 L Std 1 20 L T10E0 1 buffer 3 Std 3 5 560 50 L Std 2 100 L
18. page 2 31 2 Creating Detectors page 2 32 3 Copying Detectors to the Plate Document page 2 34 4 Applying Detectors for Standards page 2 35 5 Applying Detectors for Unknown Samples page 2 36 6 Adding Sample Names for Unknown Samples page 2 37 7 Setting Thermal Cycler Conditions page 2 38 8 Saving the Plate Document page 2 39 Creating a Blank Plate Document To create a blank plate document 1 If the 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 Chapter 2 Software Setup 2 32 Quantifiler Kits User s Manual 7900HT SDS Software Setup Creating Detectors Before you set up the plate document you need to create detectors in 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 b Click OK to return to the Detector Manager Setting Up a Plate Document Quantifiler Kits User s Manual 2 33 7900HT SDS Software Setup 3 Cr
19. s Manual Normalization of Reporter Signals The SDS software displays cycle by cycle changes in normalized reporter signal Rn 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 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 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 Amplification Plot Example When using TaqMan probes with the 7000 SDS or 7900HT SDS the fluorescent signal or normalized reporter Rn increases as the amount of specific amplified product increases Figure 1 8 shows the amplification of PCR product in a plot of Rn vs cycle number during PCR This amplification plot contains three distinct phases that characterize the progression of the PCR Figure 1 8 Phases of PCR amplification Phase 1 geometric Phase 2 linear Phase 3 plateau Cycle Number Rn 40 30 20 10 0 0 0 1 0 2 0 8 0 Real Time Data Ana
20. transfers 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 Component Volume Per Reaction L Quantifiler Human Primer Mix or Quantifiler Y Human Male Primer Mix 10 5 Quantifiler PCR Reaction Mix 12 5 Chapter 3 PCR Amplification 3 6 Quantifiler Kits User s Manual 2 Prepare the reagents Thaw the primer mix completely then vortex 3 to 5 seconds and centrifuge briefly before opening the tube Swirl the Quantifiler PCR Reaction Mix gently before using Do not vortex it 3 Pipette the required volumes of components into an appropriately sized polypropylene tube 4 Vortex the PCR mix 3 to 5 seconds then centrifuge briefly 5 Dispense 23 L of the PCR mix into each reaction well 6 Add 2 L 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 7 Seal the reaction plate with the Optical Adhesive Cover 8 Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles Note If a tabletop centrifuge w
21. 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 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 9 Table 6 4 Reproducibility using the Quantifiler Y kit Sample DNA Quantity ng L Standard Deviation 95 Confidence 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 a n d n d K 56
22. 2 Femalea b Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 13 Cat Unknown Dog Unknown Pig Unknown Cow Unknown Mouse Unknown Rabbit Unknown Hamster Unknown Rat Unknown Chicken Unknown Fish Unknown Gorilla Male Cat Male Dog 2 Male Mouse Male Rabbit Male Rat Male Horse 2 Male Bovine Male Sheep Male Pig Male Deer Male Table 6 6 Specificity with non human panel continued Organism Sex Result Quantifiler Human Kit Quantifiler Y Kit Chapter 6 Experiments and Results 6 14 Quantifiler Kits User s Manual 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 Species associated with the human gut for example Proteus Providencia Alcaligenes Species associated with food Lactobacillus spp Species associated with spoilage and decomposition for example Pseudomonas Flavobacterium Clostridium Candida Species associated with human enteric disease for example Salmonella E
23. 31 0 40 80 Sample CT 7000 Instruments 7500 Systems DNA Calculated Quantitation with Quantifiler Human kit 2 3 0 40 80 Sample Calculated Quantities ng ul 7000 Instruments 7500 Systems Chapter 6 Experiments and Results 6 60 Quantifiler Kits User s Manual Table 6 21 shows the average calculated quantities for each DNA sample obtained with the Quantifiler Human Kit For sample concentrations between 2 ng L and 0 5 ng L 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 DNA Sample 7000 Avg Calculated Qty ng L 7000 Std Dev 7500 Avg Calculated Qty ng L 7500 Std Dev Difference Between 7000 amp 7500 Calculated Qty ng L Difference of 7000 Qty Value from 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
24. 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 of kit 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 CT 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 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 fluorescence detection of 1 7 G geometric phase amplificat
25. 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 genomic A 2 2 2 270 1 810 17 7 20 3 2 010 8 6 11 5 Human genomic B 0 55 0 584 0 495 10 0 15 2 0 577 4 9 1 1 Human genomic C 0 11 0 134 0 128 16 4 4 8 0 081 26 2 39 6 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 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 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 35 The different methods produced similar quantification results 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
26. 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 Figure 6 13 STR analysis using degraded DNA Minutes 0 1 2 3 4 5 15 60 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 27 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 6 1 11 Comparison with A260 and Quantiblot Kit The concentration of DNA was measured for 50 human genomic DNA samples using a A260 method the Quantiblot kit and the Quantifiler kits The DNA quantification results were compa
27. Keep protected from direct exposure to light Excessive exposure to light may affect the fluorescent probes Table 1 2 Quantifiler kit contents Reagent Contents Quantity Storage Quantifiler Human Primer Mix or Quantifiler Y Human Male Primer Mix Forward and reverse primers to amplify human DNA or human male DNA target Probe to detect human DNA or human male DNA target IPC system primers template and probe 3 tubes 1 4 mL each 15 to 25 C Quantifiler Human DNA Standard 200 ng L purified DNA standard 1 tube 120 L 15 to 25 C Quantifiler PCR Reaction Mix AmpliTaq Gold DNA Polymerase dNTPs with dUTP Passive Reference and optimized buffer components 1 tube 5 mL 2 to 8 C Materials and Equipment Quantifiler Kits User s Manual 1 17 Equipment and Materials Not Included Tables 1 3 through 1 5 list required and optional equipment and materials not supplied with the Quantifiler kits Unless otherwise 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 Contact your local Applied Biosystems sales representative ABI PRISM 7000 Sequence Detection System Tabletop centrifuge with 96 well plate adapters optional MLS Table 1 4 User supplied materials Material Source Quantifiler Human DNA Quantification Kit App
28. Kits User s Manual 6 1 6 Experiments and Results 6 This chapter covers Overview 6 2 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 6 3 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 6 37 Section 6 3 Casework Sample Analysis 6 46 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 6 50 Section 6 5 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 Chapter 6 Experiments and Results 6 2 Quantifiler Kits User s Manual Overview About This Chapter 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 Importance of Validation Although the Quantifiler kits are not DNA genotyping assays they are intended for use before p
29. Source Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 33 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 Results Table 6 13 shows the DNA concentrations calculated for all samples using the A260 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 A260 method and the dye intercalation method Chapter 6 Experiments and Results 6 34 Quantifiler Kits User s Manual Table 6 13 Comparison with A260 and dye intercalation Sample A260 Result ng L DIa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from DI Result ng L Diff from A260 Diff from DI 007 A 2
30. Std 8 of the DNA quantification standard 23 pg L if using the Quantifiler Y kit Examining the Standard Curve Quantifiler Kits User s Manual 5 5 R2 Value lt 0 98 for Quantifiler Y Kit Only If the R2 value is lt 0 98 for the Quantifiler Y kit only you may choose to omit Std 8 of the DNA quantification standard 23 pg L from analysis At the lowest concentration point there are only 7 to 8 copies per 2 L reaction of the haploid target locus for the Quantifiler Y kit Because of stochastic effects when using the lowest concentration point with Quantifiler Y kits the CT 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 Slope A slope close to 3 3 indicates optimal 100 PCR amplification efficiency If the slope varies beyond the typical range indicated in Table 5 1 check the following Assay setup Software setup Reagents Instrument 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 Table 5 1 Range and average of standard curve slope values Kit Typical Slope range Average Slope Qua
31. gpo gov nara cfr waisidx_01 29cfr1910a_01 html Additional information about biohazard guidelines is available at http www cdc gov Preface xiv Quantifiler Kits User s Manual How to Obtain More Information Related Documentation ABI PRISM 7000 Sequence Detection System User Guide Describes the 7000 SDS hardware and software and provides information on preparing maintaining and troubleshooting the system 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 Send Us Your Comments 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 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 Search 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 informat
32. plots continued Observation Possible Cause Recommended Action Chapter 5 Interpretation of Results 5 16 Quantifiler Kits User s Manual Assessing Quantity Purpose 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 Assay Sensitivity Quantifiler kit assays can detect lt 23 pg L of human genomic DNA in samples For samples loaded at 2 0 L 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 Stochastic Effects In the 23 pg L concentration range stochastic effects or the statistical effect of sampling low copy loci may cause significant variability in assay results Validity 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 Insufficient DNA Is Present 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 Concentrate the sample then repeat the test with the Quantifiler kit before performing STR analysis Quantifiler
33. 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 L of T10E0 1 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 assay 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 Table 6 14 Average differences from A260 and dye intercalation Method Average Difference Quantifiler Human Kit Quantifiler Y Kit A260 23 3 34 9 Dye intercalation 19 0 48 0 Chapter 6 Experiments and Results 6 36 Quantifiler Kits User s Manual Figure 6 14 Assay background with the Quantifiler Human kit Figure 6 15 Assay background with the Quantifiler Y kit IPC amplification Human DNA amplification IPC amplification Human Y DNA amplification Section 6 2 A
34. 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 CT values are shown Chapter 4 Data Analysis and Results 4 6 Quantifiler Kits User s Manual 7000 SDS Data Analysis Printing or Exporting the Report For more information about exporting data see the ABI PRISM 7000 Sequence Detection System User Guide PN 4330228 To print or export 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 exported 2 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 Section 4 2 7900HT SDS Data Analysis Quantifiler Kits User s Manual 4 7 7900HT SDS Data Analysis Section 4 2 7900HT SDS Data Analysis Analyzing the Plate Document Analyze 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 After you make changes to the plate document such as sample names To analyze the plate document
35. the carriage to unlatch it The carriage automatically slides forward into position over the sample plate 4 After the door moves to the front pull the handle down into place to close the cover Do not pull the door handle to move the carriage forward This may cause serious damage to the door or the door mechanism 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 the procedure keep hands away until the heated cover and sample block reach room temperature 5 In the SDS software open the plate document that you set up for the run 6 Select the Instrument tab then click Start To run the plate on the 7000 SDS continued Do not pull the door forward by the handle Gently push carriage back and release Running the Reactions Quantifiler Kits User s Manual 3 9 Running the Plate on the 7900HT SDS To run the plate on the 7900HT SDS 1 In the SDS software select the Instrument tab for the plate document 2 In the Real Time tab click Open Close to rotate the instrument tray to the OUT position 3 Place the plate in the instrument tray so that Well A1 is in the upper left corner The notched corner is in the upper right corner 4 Click Start to rotate the instrument tray to the IN position and to start the run Note The instrument may
36. the standards the incorrect Quantity was entered In the example shown above 0 062 was entered for the Quantity instead of 0 62 Task and Quantity applied to wrong detector Outlier Troubleshooting the Standard Curve Quantifiler Kits User s Manual 5 9 Example 3 Observation At each concentration in the standard curve There are four replicates There is a large difference in the CT between the replicates 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 Incorrect Quantity entered for standard Chapter 5 Interpretation of Results 5 10 Quantifiler Kits User s Manual Using the Internal PCR Control System Purpose Use the Internal PCR Control IPC system to distinguish between true negative sample results and reactions affected by The presence of PCR inhibitors Assay setup A chemistry or instrument failure Components The following components of the IPC system are present in the Quantifiler PCR mix Synthetic DNA template Primers that hybridize specifically to the synthetic DNA template Probe labeled with VIC dye Interpreting IPC Results In the amplification plot window of the SDS software observe amplification of the FA
37. 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 007 9948 Human genomic Raji K 562 0 1 1 10 100 0 01 DNA Quantity ng L 40 00 36 00 34 00 32 00 30 00 28 00 26 00 24 00 22 00 20 00 38 00 CT FAM 007 9948 Human genomic Raji 0 1 1 10 100 0 01 DNA Quantity ng L 40 00 36 00 34 00 32 00 30 00 28 00 26 00 24 00 22 00 20 00 38 00 CT FAM Chapter 6 Experiments and Results 6 18 Quantifiler Kits User s Manual 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 DNA 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 of the PCR reaction most likely acting as a chelating agent with many inhibitors BSA is added to the Quantifiler kit and AmpFlSTR kit reaction mixes specifically to counteract the presence of PCR inhibitors Experiment Human genomic DNA was mixed with varying concentrations of hematin 0 M 10 M 12 M 14 M 16 M 18 M 20 M and 40 M 2 0 L of each DNA hematin
38. 0 06 0 060 0 087 0 8 1 2 3 4 5 6 7 8 9 10 11 12 Chapter 6 Experiments and Results 6 48 Quantifiler Kits User s Manual Cutting from denim 0 16 0 10 0 13 1 3 Cutting from sock 0 04 0 11 0 15 1 1 Cutting from sweatshirt 1 2 2 61 3 75 1 4 Cutting from cotton 0 4 0 52 0 87 1 1 Cutting from sweatshirt 0 4 0 94 0 97 1 0 Cutting from cloth 0 4 0 31 0 56 1 1 Cutting from fabric 0 04 0 23 0 34 1 1 Cutting from leather 0 08 0 10 0 18 1 2 Cutting from carpet 0 4 0 76 0 95 1 3 Cutting from cloth 1 6 1 89 2 95 1 1 Cutting from shirt 1 2 2 29 3 10 1 2 Swab from hammer 0 6 0 47 0 58 1 1 Cutting from cloth 0 4 0 45 0 58 1 1 Cutting from fabric 0 08 0 16 0 18 1 3 Cutting from carpet 0 4 1 45 1 62 1 3 Cutting from cap 0 4 0 45 0 46 1 0 Cutting from shirt 1 2 2 29 3 10 1 3 a See Figure 6 18 for STR profiles Table 6 19 Input for STR analysis of casework samples continued STRa Sample Quantity ng L Input Amount for Identifiler Kit ng QuantiBlot Kit Quantifiler Human Kit and 7000 SDS Quantifiler Human Kit and 7900HT SDS 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Section 6 3 Casework Sample Analysis Quantifiler Kits User s Manual 6 49 1 2 5 4 3 6 7 8 9 10 11 12 13 15 14 6 Chapter 6
39. 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 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 Chapter 4 Data Analysis and Results 4 8 Quantifiler Kits User s Manual 7900HT SDS Data Analysis Viewing Results Overview Viewing the results of data analysis can involve one or more of the following Viewing the Standard Curve page 4 8 Viewing the Amplification Plot page 4 9 Viewing the Results Table page 4 9 Printing the Results page 4 10 Exporting the Results page 4 10 Viewing the Standard Curve 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 Amplification Plot Results The amplification plot can display one of the following Plot of normalized reporter signal Rn versus cycle number for each reaction CT versus well position on the assay plate For more information about the amplification plot see Real Time Data Analysis on page 1 10 To view the stand
40. 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 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 Table 6 10 Comparison with A260 and Quantiblot kit continued Sample Sex A260 Result ng L QBa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from QB Result ng L Diff from A260 Diff from QB Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 31 The different methods produced similar q
41. 2 25 Overview 2 26 Starting the 7900HT Real Time PCR System 2 27 About Plate Documents 2 28 Setting Up a Plate Document 2 31 Setting Up a Plate Document Template 2 40 iv Quantifiler Kits User s Manual Chapter 3 PCR Amplification Preparing the DNA Quantification Standard 3 2 Preparing the Reactions 3 5 Running the Reactions 3 7 Chapter 4 Data Analysis and Results Section 4 1 7000 SDS Data Analysis 4 3 Analyzing the Plate Document 4 3 Viewing Results 4 4 Section 4 2 7900HT SDS Data Analysis 4 7 Analyzing the Plate Document 4 7 Viewing Results 4 8 Chapter
42. 2 3 and v1 0 software Figure 6 33 Average slope values SDS Software v1 0 and v1 2 3 Kit Slope Established 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 Average Slopes Per Run 3 60 3 10 2 60 2 10 1 60 1 10 0 60 0 10 Run 01 Run 02 Run 03 Run 01 Run 02 Run 03 Quantifiler Human Quantifiler Y Slope Software v1 0 Software v1 2 3 Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 73 R2 Results Figure 6 34 shows that SDS software v1 2 3 yields data that are within the acceptable range of R2 values 0 98 to 1 for both kits lt 0 5 difference Figure 6 34 Average R2 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 CT values were generated in Manual analysis mode then the quantities were calculated using the standard curve on each plate Figure 6 35 shows the CT values for 007 and 9948B across a set of eight serial dilutions 30 ng L to 0 1 ng L with the Quantifiler Human Kit and the corresponding quantitated concentrations As the data show differen
43. 2 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 Chapter 6 Experiments and Results 6 10 Quantifiler Kits User s Manual The 95 confidence interval shows the approximate range expected for results when using the Quantifiler kits The average 95 confidence interval for each kit Quantifiler Human kit 18 5 Quantifiler Y kit 26 9 The data show that as the DNA concentration 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 ge
44. 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 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 57 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 Figure 6 21 Average slope values Replicate standard curves Kit Slope Established 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 4 50 4 00 3 50 3 00 2 50 2 00 1 50 1 00 0 50 0 00 01 02 03 01 02 03 7000 Instrument 7500 System Instrument Ave Slope Value Quantifiler Human kit Quantifiler Y kit Chapter 6 Experiments and Results 6 58 Quantifiler Kits User s Manual R2 Results Figure 6 22 shows the average R2 values obtained for replicate standard curves on each instrument All R2 values were greater than 0 98 and are within the established range Figu
45. 5 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 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 of assay 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 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 CT 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 stra
46. 5 Interpretation of Results Checking Analysis Settings 5 2 Examining the Standard Curve 5 4 Troubleshooting the Standard Curve 5 6 Using the Internal PCR Control System 5 10 Troubleshooting Amplification Plots 5 12 Assessing Quantity 5 16 Chapter 6 Experiments and Results Overview 6 2 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 6 3 Precision 6 4 Reproducibility 6 7 Specificity with a Human DNA Panel 6 10 Specificity with a Non Human Panel 6 11 Specificity with a Bacterial Pools Panel 6 14 Quantifiler Kits User s Manual v Sensitivity
47. 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 00 0 01 18 14 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 61 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 L to 0 5 ng L 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 DNA Sample 7000 Ave Calculated Qty ng L 7000 Std Dev 7500 Ave Calculated Qty ng L 7500 Std Dev Difference Between 7000 amp 7500 Calculated Qty ng L Difference of 7000 Qty Value from 7500 Qty 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
48. 6 shows the CT 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 CT values generated using the Auto Baseline and Manual analysis modes with either Quantifiler kit Figure 6 26 Comparison of CT values between Auto Baseline and Manual analysis modes CT Reproducibility and Sensitivity Figure 6 27 shows the CT 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 CT values and calculated quantities derived using the Auto Baseline and Manual analysis modes with either Quantifiler kit Raji DNA CT values with Quantifiler Human 23 00 26 00 29 00 32 00 0 40 80 120 160 Sample CT Auto Baseline Manual Chapter 6 Experiments and Results 6 64 Quantifiler Kits User s Manual Figure 6 27 Comparison of CT 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 CT slope and R2 values between replicate samples run on the 7500 System SDS Software v1 2 3 usi
49. 60 CT FAM Value Result CT FAM lt 40 No amplification after 40 cycles Chapter 6 Experiments and Results 6 12 Quantifiler Kits User s Manual 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 between 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 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 Organism Sex Result Quantifiler Human Kit Quantifiler Y Kit Gorillla 2 Femalea Chimpanzee 2 Unknown Orangutan 2 Femalea Macaque
50. Amplification Plot tab 2 In the Detector drop down list select the detector Quantifiler Human or Quantifiler Y 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 Threshold is set to 0 20 the default setting Chapter 4 Data Analysis and Results 4 10 Quantifiler Kits User s Manual 7900HT SDS Data Analysis Printing the Results Exporting the Results You can export the results in tab delimited txt format and later open the exported files using spreadsheet software To print the results 1 Select File gt Print Report 2 Select the data to include in the report by selecting the corresponding boxes for Document Information Thermal Cycler Conditions Detector Information Well Status Summary Raw Data Plot Multicomponent Data Plot Amplification Plot 3 Click Page Setup then select Header footer information and placement Layout orientation and size 4 Click Print to print the report To export the results 1 Select File gt Export 2 Select the results to export Setup Table Results Table Multicomponent Clipped 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 result
51. Assay select Absolute Quantitation For Container select 96 Well Clear Plate For Template select an appropriate template from the list Note If the template is not available in the list click Browse to locate and select an appropriate template 3 Complete the plate document setup Copy detectors page 2 34 Apply detectors for standards page 2 35 Apply detectors for unknown samples page 2 36 Set thermal cycler conditions page 2 38 Note The tasks that you perform vary according to which settings were defined in the template 4 Save the plate document page 2 39 Note For Files of Type select ABI PRISM SDS Single Plate sds 04 2006 Part Number 4344790 Rev D Quantifiler Kits User s Manual Chapter 3 PCR Amplification I Quantifiler Kits User s Manual 3 1 3 PCR Amplification 3 This chapter covers Preparing the DNA Quantification Standard 3 2 Preparing the Reactions 3 5 Running the Reactions 3 7 Chapter 3 PCR Amplification 3 2 Quantifiler Kits User s Manual Preparing the DNA Quantification Standard Required Materials Pipettors Pipette tips Quantifiler Human DNA Standard Note The same standard can be used for both Quantifiler kits T10E0 1 buffer 10 mM Tris HCl
52. 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 96 well reaction plates 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 Starting the 7900HT Real Time PCR System Quantifiler Kits User s Manual 2 27 7900HT SDS Software Setup 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 7900HT System To start the 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 At startup the instrument Emits a high pitched tone indicating that the system is initialized Cycles the status lights red gt orange gt green indicating that the instrument is active 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
53. C detectors was inhibited Figure 6 7 Although the Human detector amplified for the 16 M 18 M and 20 M hematin samples the failure of IPC amplification Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 19 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 Figure 6 5 Inhibition studies Quantifiler Human kit Figure 6 6 Inhibition studies Quantifiler Y kit 0 M 10 M 12 M 14 M 16 M 18 M 20 M 40 M Hematin 0 M 10 M 12 M 14 M 16 M 18 M 20 M 40 M Hematin Chapter 6 Experiments and Results 6 20 Quantifiler Kits User s Manual 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 M The STR amplification reaction was completely inhibited by 40 M 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 for th
54. Data Analysis Quantifiler Kits User s Manual 4 3 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 b Verify that the settings are as shown below then click OK IMPORTANT If the analysis settings differ from those shown here change them to match the settings before clicking OK 3 Select Analysis gt Analyze Chapter 4 Data Analysis and Results 4 4 Quantifiler Kits User s Manual 7000 SDS Data Analysis Viewing Results Overview Viewing the results of data analysis can involve one or more of the following Viewing the Standard Curve page 4 4 Viewing the Amplification Plot page 4 5 Viewing the Report page 4 5 Printing or Exporting the Report page 4 6 Viewing the Standard Curve 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 Amplification Plot Results The amplification plot can display one of the following Plot of normalized reporter signal Rn versus cycle number for each reaction C
55. KN UNKN D Std 4 Std 4 UNKN UNKN UNKN UNKN Std 4 Std 4 UNKN UNKN UNKN UNKN E Std 5 Std 5 UNKN UNKN UNKN UNKN Std 5 Std 5 UNKN UNKN UNKN UNKN F Std 6 Std 6 UNKN UNKN UNKN UNKN Std 6 Std 6 UNKN UNKN UNKN UNKN G Std 7 Std 7 UNKN UNKN UNKN UNKN Std 7 Std 7 UNKN UNKN UNKN UNKN H Std 8 Std 8 UNKN UNKN UNKN UNKN Std 8 Std 8 UNKN UNKN UNKN UNKN Chapter 2 Software Setup 2 10 Quantifiler Kits User s Manual 7000 SDS 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 2 Creating Detectors the first time only page 2 11 3 Adding Detectors to the Plate Document page 2 14 4 Applying Detectors for Standards page 2 15 5 Applying Detectors for Unknown Samples page 2 17 6 Adding Sample Names for Unknown Samples page 2 18 7 Setting Thermal Cycler Conditions page 2 19 8 Saving the Plate Document page 2 21 Creating a Blank Plate Document To create a blank plate document 1 If the 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 Default settings Setting Up a Plate Document Quantifiler Kits User s Manual 2 11 7000 SDS Software Setup Creating Detectors Before you set up the plate document you n
56. M dye Quantifiler Human detector or Quantifiler Y detector and the VIC dye IPC detector then use Table 5 3 to interpret the IPC results Note Positive amplification is when the CT value for the detector is lt 40 Because samples contain unknown amounts of DNA a large range of CT values is possible Because the IPC system template DNA is added to the reaction at a fixed concentration the CT VIC should range from 20 to 30 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 CT and high Rn No amplification Disregard IPC result Amplification high CT and low Rn No amplification Partial PCR inhibition Using the Internal PCR Control System Quantifiler Kits User s Manual 5 11 True Negative Results With a true negative result FAM dye signal indicates that the human specific target failed to amplify VIC dye signal CT VIC between 20 and 30 indicates that the IPC target was amplified so the PCR was not inhibited Invalid IPC Results 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 Disregarding IPC Results With extremely high concentrations of human genomic DNA gt 10 ng L competition between the hu
57. Quantifiler Kits Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit User s Manual DRAFT April 12 2006 3 32 pm 7x9_Title fm Copyright 2006 Applied Biosystems All rights reserved For Research Forensic or Paternity Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems 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 NOTICE TO PURCHASER LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 079 352 6 127 155 5 677 152 claims 1 23 5 773 258 claims 1 10 5 210 015 5 487 972 5 804 375 5 538 848 5 723 591 5 876 930 6 030 787 and 6 258 569 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product sol
58. 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 L to 0 5 ng L 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 No statistically significant difference was observed for CT values and calculated quantities derived using the Auto Baseline and Manual analysis modes on the 7500 System SDS Software v1 2 3 6 4 6 Conclusion This validation study demonstrates that the Applied Biosystems 7500 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 CT slope and R2 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
59. 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 1 In the plate grid press the Ctrl key while you select the wells that correspond to a specific quantification standard for one kit 2 Complete the Well Inspector a Select the Use boxes for the applicable detectors IPC 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 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 L 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 Quantity values are not needed for IPC detectors Chapter 2 Software Setup 2 36 Quantifiler Kits User s Manual 7900HT SDS Software Setup Applying Detectors for Unknown Samples You need to apply detectors to the plate document for the wells on the reaction plate that contain unknown samples IMPORTAN
60. Std 7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN C UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN D UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN E Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std 5 Std 6 Std 6 F Std 7 Std 7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN G UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN H UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN About Plate Documents Quantifiler Kits User s Manual 2 9 7000 SDS Software Setup 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 Wells A1 through D6 gray correspond to reactions using the Quantifiler Human kit 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 Figure 2 2 Example plate setup of reactions using repeat pipettors 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Std 1 UNKN UNKN UNKN UNKN Std 1 Std 1 UNKN UNKN UNKN UNKN B Std 2 Std 2 UNKN UNKN UNKN UNKN Std 2 Std 2 UNKN UNKN UNKN UNKN C Std 3 Std 3 UNKN UNKN UNKN UNKN Std 3 Std 3 UNKN UNKN UN
61. T If you run reactions for the Quantifiler Human kit and the Quantifiler Y kit on the same plate apply detectors for unknown samples for each kit separately 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 To apply detectors for quantification standards continued Task for IPC set to Unknown default 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 IPC Quantifiler Human or Quantifiler Y For example Setting Up a Plate Document Quantifiler Kits User s Manual 2 37 7900HT SDS Software Setup Adding Sample Names for Unknown Samples Repeat this procedure to enter the names for all unknown samples 3 In the Well Inspector make sure that ROX is selected for the Passive Reference To apply detectors for unknown samples continued To add sample names for unknown 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 Chap
62. T versus well position on the assay plate For more information about the amplification plot see Real Time Data Analysis on page 1 10 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 Quantifiler Human or Quantifiler Y 3 View the CT values for the quantification standard reactions and the calculated regression line slope y intercept and R2 values Viewing Results Quantifiler Kits User s Manual 4 5 7000 SDS Data Analysis Viewing the Amplification Plot For troubleshooting information see Troubleshooting Amplification Plots on page 5 12 Viewing the Report The report summarizes the quantity of DNA present in the samples For information about the quantities reported see Assessing Quantity on page 5 16 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 Quantifiler Human or Quantifiler Y 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 To view the report 1 In the analyzed plate document
63. T10E0 1 glycogen buffer 10 L Std 2 20 L T10E0 1 buffer 3 Std 4 1 850 50 L Std 3 100 L T10E0 1 glycogen buffer 10 L Std 3 20 L T10E0 1 buffer 3 Std 5 0 620 50 L Std 4 100 L T10E0 1 glycogen buffer 10 L Std 4 20 L T10E0 1 buffer 3 Std 6 0 210 50 L Std 5 100 L T10E0 1 glycogen buffer 10 L Std 5 20 L T10E0 1 buffer 3 Std 7 0 068 50 L Std 6 100 L T10E0 1 glycogen buffer 10 L Std 6 20 L T10E0 1 buffer 3 Std 8 0 023 50 L Std 7 100 L T10E0 1 glycogen buffer 10 L Std 7 20 L T10E0 1 buffer 3 Chapter 3 PCR Amplification 3 4 Quantifiler Kits User s Manual Preparation Guidelines 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 Preparing the DNA Quantifica tion Standards If you use T10E0 1 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 tube
64. a grating 3 Based on wavelength the grating separates the light into a predictably spaced pattern across the CCD camera 4 During the run the CCD camera detects the fluorescence emission between 500 nm and 660 nm from each well 5 The SDS software obtains the fluorescence emission data from the CCD camera and applies data analysis algorithms Chapter 1 Overview 1 8 Quantifiler Kits User s Manual SDS Software Overview 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 Composite Spectrum 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 Figure 1 6 Example of a composite spectrum Wavelength FAM dye ROX dye 500 VIC dye 550 600 650 0 000 0 027 0 055 0 083 0 110 Composite spectrum all dyes Background MSE Pure Dye components Background from background calibration run SDS Software Overview Quantifiler Kits User s Manual 1 9 Processing Multicomponent Data During the multicomponent transformation the SDS software uses
65. aji 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 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 Table 6 17 Comparison with A260 and dye intercalation 7900HT SDS Sample A260 Result ng L DIa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from DI Result ng L Diff from A260 Diff from DI Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Quantifiler Kits User s Manual 6 45 The different methods produced similar quantification results Table 6 18 Average differences from A260 and dye intercalation 7900HT Method Average Difference Quantifiler Human Kit Quantifiler Y Kit A260 34 8 23 5 Dye intercalation 29 8 34 9 Chapter 6 Experiments and Results 6 46 Quantifiler Kits User s Manual Section 6 3 Casework Sample Analysis Case Type Studies There is a recommended optimal DNA concentration range for using AmpFlSTR PCR Amplification kits The recommended amount of DNA input for the AmpFlSTR Identifiler PCR Amplification Kit is 0 5 to 1 25 ng human DNA total per reaction and for four dye assays such as the AmpFlSTR Profiler Plus PCR Am
66. algorithms to determine the contribution of each dye 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 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 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 Figure 1 7 Typical component contributions in a multiplex reaction Measured spectrum a FAM b VIC c ROX d Background MSE Background component MSE VIC dye component ROX dye component FAM dye component Time Temperature 0 4000 83 73 63 53 8000 12000 6978 93 Fluorescence 0 10 0 30 0 50 1 10 1 30 Chapter 1 Overview 1 10 Quantifiler Kits User
67. antifiler 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 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 7900HT instrument 1 2 Mean 3 Day 16 7 5 56 0 62 0 068 50 DNA quantity ng L 40 35 30 25 20 15 10 CT 5 0 0 023 1 85 0 21 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Quantifiler Kits User s Manual 6 41 Results The quantification results from using the Quantifiler Human kit varied from an average of 0 12 ng L for the 1 1 sample to 60 ng L 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 L to 0 058 ng L 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 showe
68. ard 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 Quantifiler Human or Quantifiler Y 3 View the CT values for the quantification standard reactions and the calculated regression line slope intercept and R2 values Viewing Results Quantifiler Kits User s Manual 4 9 7900HT SDS Data Analysis Viewing the Amplification Plot For troubleshooting information see Troubleshooting Amplification Plots on page 5 12 Results Table The results table displays Well position of samples Sample names Detector assignments Task assignments CT values Quantity Mean and standard deviation for CT values and Quantity if replicate groups were defined in assay setup Viewing the Results Table View the Qty column to determine the quantity of DNA present in 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 Note Quantities are calculated only if quantification standards were run and set up correctly in the software Otherwise only CT values are shown For more information about the quantities reported see Assessing Quantity on page 5 16 To view the amplification plot 1 After the run is finished select the Results tab then select the
69. assay contained two reactions for each of the quantification standards and one reaction for each of the samples For each sample reaction the CT FAM 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 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 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 Chapter 6 Experiments and Results 6 8 Quantifiler Kits User s Manual 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 Sample DNA Quantity ng L Standard Deviation 95 Confidence percent Run 1 Run 2 Run 3 Mean 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
70. at the 5 minute time point and to 0 11 ng L at the 15 minute time point At the 60 minute time point no amplifiable DNA was detected 1000 bp 700 bp 525 500 bp 400 bp 300 bp 200 bp 100 bp 50 bp 0 1 2 3 4 5 15 60 Minutes degraded High molecular weight DNA Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 25 Figure 6 11 Degraded DNA Quantifiler Human kit amplification plot Figure 6 12 Degraded DNA Quantifiler Y kit amplification plot Using the DNA quantification results from the Quantifiler 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 L 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 Minutes 0 1 2 3 4 5 15 60 Minutes 0 1 2 3 4 5 15 60 Chapter 6 Experiments and Results 6 26 Quantifiler Kits User s Manual amplifiable DNA and produced only a partial STR profile of mostly smaller molecular weight loci
71. ces in CT values do not affect calculated quantities calculated quantities were normalized resulting in comparable concentrations from results generated with both software versions Average R2 Per Run 0 9800 0 9850 0 9900 0 9950 1 0000 1 0050 Run 01 Run 02 Run 03 Run 01 Run 02 Run 03 Quantifiler Human Quantifiler Y R2 Software v1 0 Software v1 2 3 Chapter 6 Experiments and Results 6 74 Quantifiler Kits User s Manual Figure 6 35 Average CT values and quantitated DNA concentrations 007 and 9948B Quantifiler Human Kit Figure 6 36 shows CT 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 CT values do not affect calculated quantities calculated quantities were normalized resulting in comparable concentrations from results generated with both software versions Figure 6 36 Average CT values and quantitated DNA concentrations 007 and 9948B Quantifiler Y Human Male Kit Software v1 0 and Software v1 2 3 CT values 22 24 26 28 30 32 34 0 5 10 15 20 25 30 35 Well location CT Quantifiler Human 007 v1 0 Quantifiler Human 9948 v1 0 Quantifiler Human 007 v1 2 3 Quantifiler Human 9948 v1 2 3 Quantity Calculations between software v1 0 and software v1 2 3 analysis 0 1 1 10 100 0 5 10 15 20 25 30 35 Well location ng ul Quantifiler H
72. complete the New Document dialog box 3 Apply the desired template settings to the plate document Copy detectors page 2 34 Apply detectors for standards page 2 35 Apply detectors for unknown samples page 2 36 Set thermal cycler conditions page 2 38 4 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 To create a plate document template continued Chapter 2 Software Setup 2 42 Quantifiler Kits User s Manual 7900HT SDS Software Setup Creating a Plate Document from a Template After you create a template you can use it to create a plate document To create a plate document from a template 1 If the SDS software is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 2 Select File gt New and in the New Document dialog box and make the following selections For
73. ct the ability to obtain interpretable STR profiles using the optimal input amount determined by individual laboratories during validation of the Quantifiler kits Quantifiler Y Auto Baseline and Manual Quantity values 0 1 1 10 100 0 5 10 15 20 25 30 35 Well location ng ul Quantifiler Y 007 Auto Baseline Quantifiler Y 9948 Auto Baseline Quantifiler Y 007 Manual Quantifiler Y 9948 Manual Quantifiler Y Auto Baseline and Manual CT values 22 24 26 28 30 32 34 0 5 10 15 20 25 30 35 Well location CT Quantifiler Y 007 Auto Baseline Quantifiler Y 9948 Auto Baseline Quantifiler Y 007 Manual Quantifiler Y 9948 Manual Chapter 6 Experiments and Results 6 80 Quantifiler Kits User s Manual 6 5 5 3 Manual Analysis Versus Auto Baseline Analysis CT 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 calculated DNA quantities 6 5 6 Conclusion 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 CT slope and R2 values to results obtained u
74. ctions For Assay select Absolute Quantitation For Container select 96 Well Clear For Template select an appropriate template from the list Note If the template is not available in the list click Browse to locate and select an appropriate template 3 Complete the plate document setup Add detectors to the plate document page 2 14 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 Section 2 2 7900HT SDS Software Setup Quantifiler Kits User s Manual 2 25 7900HT SDS Software Setup Section 2 2 7900HT SDS Software Setup This section covers Overview 2 26 Starting the 7900HT Real Time PCR System 2 27 About Plate Documents 2 28 Setting Up a Plate Document 2 31 Setting Up a Plate Document Template 2 40 Chapter 2 Software Setup 2 26 Quantifiler Kits User s Manual 7900HT SDS Software Setup Overview Purpose During software setup you start up the Applied
75. d that the male DNA was detected and quantified accurately in all samples regardless of the amount of female DNA present Quantifiler Y kit Quantifiler Human kit 100 000 1 000 10 000 0 100 0 010 1 1 1 4 1 16 1 64 1 256 1 1024 Male Female Mixture Ratio DNA Quantity ng L Chapter 6 Experiments and Results 6 42 Quantifiler Kits User s Manual 6 2 3 Comparisons with Other Methods 7900HT SDS Experiment Six human genomic DNA samples were obtained from different commercial sources Using the concentrations provided by the supplier the DNA samples were diluted to 2 0 ng L A 0 5 ng L B and 0 1 ng L C Note All dilutions were made in T10E0 1 Buffer with 20 g mL glycogen added as a carrier and stabilizer All sample dilutions were quantified using the following methods A260 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 g mL 50 A260 The results calculated for the 2 0 ng L dilutions were then extrapolated for the higher dilutions 0 5 ng L and 0 1 ng L 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 r
76. detectors continued Enter IPC Select VIC Make sure none is selected Click to select a color Chapter 2 Software Setup 2 14 Quantifiler Kits User s Manual 7000 SDS Software Setup Adding Detectors to the Plate Document To add detectors to the plate document 1 In the SDS software select Tools gt Detector Manager If the detectors for the Quantifiler kits have been created they are listed in the Detector Manager 2 In the Detector Manager select the Quantifiler Human Quantifiler Y and the IPC detectors by clicking them while pressing the Ctrl key 3 Click Add To Plate Document 4 Click Done to close the Detector Manager Setting Up a Plate Document Quantifiler Kits User s Manual 2 15 7000 SDS Software Setup Applying Detectors for Standards You need to apply detectors to the plate document for the wells on the reaction plate that contain DNA quantification standards Repeat 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 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 the detectors for all wells containing quantification standards To apply detectors for quantification standards
77. e DNA samples were made Concentrations ranged from 10 ng L to 0 016 ng L 16 pg L Note All dilutions were made in T10E0 1 Buffer with 20 g mL glycogen added as a carrier and stabilizer For each 25 L reaction 2 0 L of DNA sample was used Results A plot of the CT 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 L gave positive results for the Quantifiler Human kit and the Quantifiler Y kit For each dilution series the data points formed an acceptable standard curve The small differences in CT values among the dilutions of different DNA samples likely reflect differences in the quantification measurements made by each supplier 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 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 17 Figure 6 3 Sensitivity using the Quantifiler Human kit Figure 6 4 Sensitivity using the Quantifiler Y kit 6 1 7 Stability 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
78. e Documents 2 7 Setting Up a Plate Document 2 10 Setting Up a Plate Document Template 2 22 Chapter 2 Software Setup 2 4 Quantifiler Kits User s Manual 7000 SDS 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 Starting the 7000 SDS Quantifiler Kits User s Manual 2 5 7000 SDS Software Setup 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 If you 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 3 In the 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 GR2078 Instrument power button Laptop power button Chapter 2 Software Setup 2 6 Quantifiler Kits User
79. e 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 Wells A1 through D12 gray correspond to reactions using the Quantifiler Human kit Wells E1 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 Figure 2 3 Example arrangement of reactions with two kits 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std 5 Std 6 Std 6 B Std 7 Std 7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN C UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN D UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN E Std 1 Std 1 Std 2 Std 2 Std 3 Std 3 Std 4 Std 4 Std 5 Std 5 Std 6 Std 6 F Std 7 Std 7 Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN G UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN H UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN Chapter 2 Software Setup 2 30 Quantifiler Kits User s Manual 7900HT SDS Software Setup Figure 2 4 shows ano
80. e positive and negative controls are included for reference 0 M 10 M 12 M 14 M 16 M 18 M 20 M 40 M Hematin Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 21 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 0 M 10 M 12 M 14 M 16 M 18 M 20 M 40 M Hematin Positive control Negative control Chapter 6 Experiments and Results 6 22 Quantifiler Kits User s Manual Experiment Purified genomic DNA from the Raji male and K 562 female cell lines were mixed 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 L in all samples and the female DNA was present at amounts ranging from 0 05 ng L in the 1 1 sample to 50 ng L in the 1 1024 sample The DNA amounts were calculated based only on the DNA concentrations provided by the suppliers and were no
81. eate a detector for the Quantifiler Y Human Male kit a In the Detector Manager click New and complete the dialog box b Click OK to return to the Detector Manager To create detectors continued Chapter 2 Software Setup 2 34 Quantifiler Kits User s Manual 7900HT SDS Software Setup Copying Detectors to the Plate Document 4 Create a detector for the IPC assay a In the Detector Manager click New then complete the Add Detector dialog box b Click OK to return to the Detector Manager To create detectors continued To copy 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 If the detectors are not available create them first see page 2 32 for the procedure 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 Setting Up a Plate Document Quantifiler Kits User s Manual 2 35 7900HT SDS Software Setup Applying Detectors for Standards You need to apply the detectors to the plate document for the wells on the reaction plate that contain DNA quantification standards Repeat the procedure until you complete applying detector tasks quantities and sample names for all quantification standards IMPORTANT
82. ecting the amplified IPC DNA Table 1 1 Targets of Quantifiler kits Kit Gene Target Location Amplicon Length Region Amplified Ploidy Quantifiler Human kit Human telomerase reverse transcriptase gene hTERT 5p15 33 62 bases Nontranslated region intron Diploida Quantifiler Y kit Sex determining region Y gene SRY Yp11 3 64 bases Nontranslated region Haploida a Single copy target Chapter 1 Overview 1 4 Quantifiler Kits User s Manual 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 Tm 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 Assay Process The 5 nuclease assay process Figures 1 1 through 1 5 takes place during PCR amplification This process occurs in every cycle and does not interfere with the exponential accumulation of product 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 pr
83. eed to create detectors in 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 3 Click OK to use the default settings and to view a blank plate document To create a blank plate document continued 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 Chapter 2 Software Setup 2 12 Quantifiler Kits User s Manual 7000 SDS Software Setup 3 Create a detector for the Quantifiler Human kit 4 Click Create Another to add the Quantifiler Human detector and to reset the New Detector dialog box 5 Create a detector for the Quantifiler Y kit 6 Click Create Another to add the Quantifiler Y detector and to reset the New Detector dialog box To create detectors continued Enter Quantifiler Human Select FAM Make sure none is selected Click to select a color Enter Quantifiler Y Select FAM Make sure none is selected Click to select a color Setting Up a Plate Document Quantifiler Kits User s Manual 2 13 7000 SDS Software Setup 7 Create a detector for the IPC assay 8 Click OK to add the IPC detector and to return to the Detector Manager dialog box To create
84. ely in forensic and paternity testing including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal research No right under any other patent claims such as apparatus or system claims in U S Patent No 6 814 934 is conveyed expressly by implication or by estoppel Further information on purchasing licenses may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA TRADEMARKS Applied Biosystems AB Design ABI PRISM AmpFlSTR Genotyper GeneScan Identifiler MicroAmp Profiler Plus QuantiBlot Quantifiler and VIC are registered trademarks and Applera FAM MicroAmp and ROX are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Part Number 4344790 Rev D 04 2006 Contents Quantifiler Kits User s Manual iii Preface How to Use This Guide vii Safety ix How to Obtain More Information xiii How to Obtain Support
85. erforming genotyping assays such as the AmpFlSTR PCR Amplification 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 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 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 3 Section 6 1 ABI PRISM 7000 Sequence Detection Sy
86. esults 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 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 R R2 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 Rn 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 Index
87. esults 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 CT slope and R2 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 Standard curve DNA 1 DNA 2 NTC Quantifiler Human Kit Quantifiler Y Human Male Kit Standard curve DNA 1 DNA 2 NTC Standard Chapter 6 Experiments and Results 6 70 Quantifiler Kits User s Manual 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 CT average slope and average R2 values were determined CT Results Figures 6 30 to 6 32 show CT values obtained using the SDS Software v1 0 and v1 2 3 The data consistently show that SDS Software v1 2 3 yields lower CT values 2 difference Figure 6 30 Average CT values
88. from all time points were run on a 2 agarose gel for 25 minutes and visualized by staining with ethidium bromide The Quantifiler Y kit Quantifiler Human kit 100 000 1 000 10 000 0 100 0 010 1 1 1 5 1 16 1 64 1 256 1 1024 Male Female Mixture Ratio DNA Quantity ng L Chapter 6 Experiments and Results 6 24 Quantifiler Kits User s Manual 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 AmpFlSTR Identifiler kit reactions were calculated so that 1 0 ng L was added for each reaction The PCR products were run on an ABI PRISM 3100 Genetic Analyzer Results 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 Figure 6 10 DNase I degradation of human genomic DNA The results from the Quantifiler kits Figures 6 11 and 6 12 showed higher CT 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 L to 1 2 ng L
89. g 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 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 2 Quantifiler Kits User s Manual C casework sample analysis 6 46 CAUTION description ix chemical safety guidelines x chemical waste hazards xi safety guidelines xi cleavage in 5 nuclease assay 1
90. h A260 and Dye Intercalation 6 31 6 1 13 Assay Background 6 35 Chapter 6 Experiments and Results 6 4 Quantifiler Kits User s Manual 6 1 1 Precision The precision of the Quantifiler Human kit and the Quantifiler Y kit was tested by performing runs on different instruments and on different days Experiment One set of eight serial dilutions of the Quantifiler Human DNA Standard was prepared The dilutions ranged from 50 ng L to 23 pg L 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 CT FAM values were recorded and the means and standard deviations of the CT FAM values were calculated for each of the eight dilutions using the Quantifiler Human kit and the Quantifiler Y kit Results Table 6 1 shows the means and standard deviations of the CT FAM values calculated for all 12 reactions of each quantification standard dilution for the Quantifiler Human and Quantifiler Y kits Table 6 1 Precision CT values Quantification Standard Dilution ng L Quantifiler Human K
91. he Quantifiler Y kit are displayed in Figures 6 16 and 6 17 For each of the dilutions the mean and the standard deviation of CT FAM for the repeated runs is shown Figure 6 16 Precision Quantifiler Human kit CT results 7900HT SDS 1 2 Mean 3 Day 16 7 5 56 0 62 0 068 50 DNA quantity ng L 40 35 30 25 20 15 10 CT 5 0 0 023 1 85 0 21 Chapter 6 Experiments and Results 6 40 Quantifiler Kits User s Manual Figure 6 17 Precision Quantifiler Y kit CT results 7900HT SDS 6 2 2 Mixture Studies 7900HT SDS 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 Experiment 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 L in all samples and the female DNA was present at amounts ranging from 0 05 ng L in the 1 1 sample to 50 ng L 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 Qu
92. ification 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 CT 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 R2 value Measure of the closeness of fit between the standard curve regression line and the individual CT data points of quantification standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points 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 CT value for a sample with Qty 1 for example 1 ng L R2 Value An R2 value 0 99 indicates a close fit between the standard curve regression line and the individual CT data points of quantification standard reactions If the R2 value is lt 0 98 check the following Quantity values entered for quantification standards in the Well Inspector during plate document setup Making of serial dilutions of quantification standards Loading of reactions for quantification standards Failure of reactions containing quantification standards CT value for
93. ifiler 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 applied to the correct detector and reanalyze Troubleshooting the Standard Curve Quantifiler Kits User s Manual 5 7 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 Page 2 15 7000 SDS 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 CT values for the DNA quantification standard reactions lie outside of the standard curve and form a straight horizontal line Chapter 5 Interpretation of Results 5 8 Quantifiler Kits User s Manual 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 Example 2 Observation One point lies outside of the standard curve Possible Cause When applying detectors for
94. ight 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 5 T T10E0 1 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 6 Quantifiler Kits User s Manual 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 s
95. imer sites Figure 1 2 When the probe is intact Figures 1 2 and 1 3 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 MGB P NFQ R Nonfluorescent quencher Minor groove binder Reporter AmpliTaq Gold DNA Polymerase Chemistry Overview Quantifiler Kits User s Manual 1 5 Figure 1 2 Polymerization 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 Figure 1 4 Cleavage 5 3 5 5 3 3 5 Forward Primer TaqMan MGB probe Reverse Primer MGB P P NFQ R R 5 3 5 5 3 3 5 Forward Primer TaqMan MGB probe Reverse Primer MGB P P NFQ 5 Forward Primer P R 5 3 5 3 3 5 TaqMan MGB probe Reverse Primer MGB P NFQ Chapter 1 Overview 1 6 Quantifiler Kits User s Manual Polymerization of the strand continues but becau
96. ion System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 23 Figure 6 9 DNA quantities determined in mixture studies 6 1 9 Degraded DNA Studies 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 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 Experiment 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
97. ion about customer training Download software updates and patches Quantifiler Kits User s Manual 1 1 1 Overview 1 This chapter covers Product Overview 1 2 Chemistry Overview 1 3 Instrument Overview 1 7 SDS Software Overview 1 8 Real Time Data Analysis 1 10 Procedural Overview 1 15 Materials and Equipment 1 16 Chapter 1 Overview 1 2 Quantifiler Kits User s Manual Product Overview Purpose 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 If sufficient human DNA or human male DNA is present to proceed with short tandem repeat STR analysis How much sample to use in STR analysis applications Product Description The Quantifiler kits contain all the necessary reagents for the amplification detection
98. ion plot 1 11 glycogen adding to T10E0 1 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 Index 3 I 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 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 use in 1 9 normalization using 1 10 selecting in Well I
99. is successful the software displays in the status bar GR2010 Power button Green Orange Red Status lights Chapter 2 Software Setup 2 28 Quantifiler Kits User s Manual 7900HT SDS Software Setup About Plate Documents How Plate Documents Are Used Running a reaction plate on the 7900HT Real Time PCR System requires creating and setting up a plate document using the SDS 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 Coordinate the instrument operation such as thermal cycling and data collection Organize and store the data gathered during the run Analyze the data from the run Plate Document Types You can use SDS software to create two types of plate document 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 document 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 About Plate Documents Quantifiler Kits User s Manual 2 29 7900HT SDS Software Setup Example Plate Document Setup You can arrange the reactions in any well of the reaction plate but you need to set up the plat
100. it Quantifiler Y Kit CT Mean Standard Deviation CT Mean Standard Deviation 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 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 5 Figure 6 1 and Figure 6 2 show the CT FAM results for all 8 quantification standard dilutions reactions using the Quantifiler Human kit and the Quantifiler Y kit Figure 6 1 Precision using the Quantifiler Human kit 0 068 31 90 0 28 33 38 0 44 0 023 33 45 0 48 35 19 0 73 Table 6 1 Precision CT values continued Quantification Standard Dilution ng L Quantifiler Human Kit Quantifiler Y Kit CT Mean Standard Deviation CT Mean Standard Deviation Day 1 Day 2 Instruments 1 2 3 1 2 3 Mean 16 7 5 56 0 62 0 068 50 DNA quantity ng L 40 35 30 25 20 15 10 CT 5 0 0 023 1 85 0 21 Chapter 6 Experiments and Results 6 6 Quantifiler Kits User s Manual Figure 6 2 Precision using the Quantifiler Y kit The data show that at lower DNA concentrations the CT values increased and the standard deviation increased most likely because of stochastic effects For each sample the CT values obtained using the Quantifiler Human kit are lower than those obtained
101. ith 96 well plate adapters is not available this step can be omitted 9 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 To prepare the reactions continued Running the Reactions Quantifiler Kits User s Manual 3 7 Running the Reactions Before You Run the Reactions 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 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 Running the Plate on the 7000 SDS To run the plate on the 7000 SDS 1 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 2 Position the plate in the instrument thermal block so that Well A1 is in the upper left corner The notched corner of the plate is in the upper right corner Lift handle to open Do not push here to open Chapter 3 PCR Amplification 3 8 Quantifiler Kits User s Manual 3 Gently push then release
102. lications 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 Applera Corporation is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Printed in the USA 04 2006 Part Number 4344790 Rev D
103. lied 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 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 Chapter 1 Overview 1 18 Quantifiler Kits User s Manual Mid to Low Throughput Setup MicroAmp Optical Tubes 8 tubes strip 125 strips Applied Biosystems 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 Covers Starter Kit Note Not necessary if using Optical Caps Applied Biosystems PN 4313663 Table 1 5 Documents Document Applied Biosystems Part Number ABI PRISM 7000 Sequence Detection System User Guide 4330228 ABI PRISM 7900HT Sequence Detection System User Guide 4317596 Table 1 4 User supplied materials continued Material Source Quantifiler Kits User s Manual Chapter 2 Software Setup I Quantifiler Kits User s Manual 2 1 2 Software Setup 2 This chapter covers Section 2 1 7000 SDS Software Setup
104. ll location Difference QTYv1 0 QTYv1 2 3 QTYv1 0 Quantifiler Human 007 Quantifiler Human 9948 Quantifiler Y 007 Quantifiler Y 9948 Quantifiler Human Kit Quantifiler Y Human Male Kit Chapter 6 Experiments and Results 6 76 Quantifiler Kits User s Manual 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 CT Overall the NTC results do not change when analyzed with version 1 2 3 Figure 6 39 Background amplification plots 7000 System SDS Software v1 2 3 6 5 4 4 Auto Baseline Analysis Versus Manual Analysis CT Precision and Accuracy For Manual to Auto Baseline analysis comparisons 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 The CT values were compared to each other Figures 6 40 and 6 41 show the average CT values between Auto Baseline analysis and Manual analysis There is a lt 2 difference between the two analysis methods for both kits Quantifiler Human Kit Quantifiler Y Human Male Kit Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 77 Figure 6 40 Comparison of CT values between Auto Baseline and Ma
105. llowing 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 L DNA and 1 000 L 10 mM Tris HCl pH 8 0 and 0 1 mM Na2EDTA T10E0 1 buffer Chapter 6 Experiments and Results 6 52 Quantifiler Kits User s Manual One manufactured lot of each kit was used for all validation studies 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 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 L 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 pla
106. lysis Quantifiler Kits User s Manual 1 11 Phases of Amplification Initially Rn appears as a flat line because the fluorescent signal is below the detection limit of the sequence detector Phase 1 Geometric 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 10 8 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 e
107. man specific and IPC PCR reactions appears to suppress IPC amplification for that sample If the target amplifies with low CT and high Rn results it is unlikely that PCR inhibitors are present In these cases appearance of suppression or failure of IPC amplification can be disregarded Partial PCR Inhibition Weak amplification high CT value and low Rn value of the human target and no amplification of the IPC may indicate partial PCR inhibition in the sample Determining the Normal Range for IPC To determine the normal range of CT 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 Evaluating PCR Inhibition If the IPC amplification for certain samples appears 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 Proceed directly to an STR assay of the sample Repeat the DNA extraction from the sample Perform additional cleanup of the sample Chapter 5 Interpretation of Results 5 12 Quantifiler Kits User s Manual Troubleshooting Amplification Plots Table 5 4 Troubleshooting amplification plots Observation
108. ment 1 Select File gt Save As 2 For Files of Type select ABI PRISM SDS Single Plate sds Chapter 2 Software Setup 2 40 Quantifiler Kits User s Manual 7900HT SDS Software Setup 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 Assay specific detectors 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 Creating a Plate Document Template This procedure assumes that you have created the detectors for running reactions using the Quantifiler kits page 2 32 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 To save the plate document continued To create a plate document template 1 If the SDS software is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 Setting Up a Plate Document Template Quantifiler Kits User s Manual 2 41 7900HT SDS Software Setup 2 Select File gt New then
109. ment double click a well to view the Well Inspector b Verify that the detector settings are correct and reanalyze Abnormal Rn values and some negative Rn 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 Table 5 4 Troubleshooting amplification plots continued Observation Possible Cause Recommended Action Troubleshooting Amplification Plots Quantifiler Kits User s Manual 5 15 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 If a 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 check ROI calibration and to perform instrument function tests Table 5 4 Troubleshooting amplification
110. 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 AmpFlSTR 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 Results Amplification plots Figures 6 5 and 6 6 showed lower Rn values and higher CT values as the concentration of hematin increased CT results and corresponding quantification results were relatively stable up to 14 M 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 the Quantifiler Human kit complete inhibition occurred at 40 M and for the Quantifiler Y kit complete inhibition occurred at 18 M 20 M and 40 M 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 M hematin amplification of IPC detectors failed In samples containing less hematin amplification of IP
111. ng both Quantifiler kits 7500 to 7000 System Comparison Statistically significant differences in CT slope and R2 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 CT 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 DNA CT values with Quantifiler Human 23 26 29 32 0 40 80 Sample CT Auto Baseline Manual DNA Calculated Quantitation with Quantifiler Human 0 5 10 0 40 80 Sample ng ul Auto Baseline Manual Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 65 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
112. 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 Safety alert words also appear in user documentation For more information see Safety Alert Words on page ix Safety Quantifiler Kits User s Manual ix Safety Safety Alert Words 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 Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices Indicates a potentially hazardous situation that if not avoided could result in death or serious inju
113. notypic analysis using the AmpFlSTR 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 CT FAM value CT FAM Value Result CT FAM lt 40 No amplification after 40 cycles Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 11 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 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 CT FAM value Table 6 5 Specificity with human DNA panel Sex Result Quantifiler Human Kit Quantifiler Y Kit Male 240 Female 2
114. nspector 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 a template 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 setting up 2 22 thermal cycler conditions setting 2 19 types 2 7 Index 4 Quantifiler Kits User s Manual 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 r
115. ntifiler Human 2 9 to 3 3 3 1 Quantifiler Y 3 0 to 3 6 3 3 Chapter 5 Interpretation of Results 5 6 Quantifiler Kits User s Manual 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 R2 value significantly less than 0 98 to 0 99 When applying detectors for standards 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 L from analysis At each concentration in the standard curve There are four replicates There is a large difference in CT between the replicates Note This observation applies only when Quantifiler Human kit reactions and Quant
116. nual analysis Quantifiler Human Kit error bars indicate standard deviations Figure 6 41 Comparison of CT values between Auto Baseline and Manual analysis Quantifiler Y Human Male Kit error bars indicate standard deviations Average CT values from AutoBaseline and Manual Analysis 22 24 26 28 30 32 34 2 1 5 1 0 5 0 0 5 1 1 5 2 Log Concentration CT Quantifiler Human AutoBaseline Quantifiler Human Manual Average CT values from AutoBaseline and Manual Analysis 22 24 26 28 30 32 34 36 2 1 5 1 0 5 0 0 5 1 1 5 2 Log Concentration CT Quantifiler Y AutoBaseline Quantifiler Y Manual Chapter 6 Experiments and Results 6 78 Quantifiler Kits User s Manual CT Reproducibility and Sensitivity Figure 6 42 shows the CT values obtained using the Auto Baseline and Manual analysis modes with the Quantifiler Human Kit No significant differences were observed for CT values generated using the Auto Baseline and Manual analysis modes with either Quantifiler kit Figure 6 42 Average CT values and average calculated quantities for 9948 and 007 Quantifiler Human Kit 30 ng L to 0 1 ng L Figure 6 43 shows the CT values obtained using the Auto Baseline and Manual analysis modes with the Quantifiler Human Kit Quantifiler Human Auto Baseline and Manual CT values 22 24 26 28 30 32 34 0 10 20 30 40 Well location
117. nual 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 Analysis For statistical analysis the Stat Ease Design Expert Software was used for all ANOVA analysis of variance 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 Average CT Average Slope Average R2 95 confidence intervals CI by ANOVA analysis Chapter 6 Experiments and Results 6 56 Quantifiler Kits User s Manual CT Results Table 6 20 shows the average CT 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 Statistically the two instrument types resulted in significantly different CT values p lt 0 0001 when compared with the ANOVA analysis No significant difference in CT values was observed when comparing results from instruments of the same type Table 6 20 CT Values 95 CI Standard Curve Dilution ng L 7500 System 7000 System Average CT Value 95 CI CT Value Range 95 CI Average CT Value 95 CI CT Value Range 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
118. of the CT FAM values were calculated for each of the eight dilutions using the Quantifiler Human kit and the Quantifiler Y kit Chapter 6 Experiments and Results 6 38 Quantifiler Kits User s Manual Results Table 6 15 shows the means and standard deviations of the CT FAM values calculated for 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 CT results DNA Quantity ng L Quantifiler Human Kit Quantifiler Y Kit CT Mean Standard Deviation CT Mean Standard Deviation 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 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Quantifiler Kits User s Manual 6 39 The following results are consistent with the 7000 SDS results CT vs sample concentration Standard deviations of the CT values CT 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 CT results for all quantification standard dilutions reactions using the Quantifiler Human kit and t
119. on of the PCR where Xn number of target molecules at cycle n so that n gt m Xm number of target molecules at cycle m EX efficiency 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 EX 1 so that To define the significance in amplified product of one thermal cycle set n m 1 so that Therefore each cycle in the PCR reaction corresponds to a two fold increase in product Likewise a difference in CT values of 1 equates to a two fold difference in initial template amount Xn Xm 1 EX n m Xn Xm 1 1 n m Xm 2 n m Xn Xm 2 1 2Xm Procedural Overview Quantifiler Kits User s Manual 1 15 Procedural Overview Use of the Quantifiler kits involves the following workflow Software Setup PCR Amplification Data Analysis Interpretation of Results Chapter 1 Overview 1 16 Quantifiler Kits User s Manual Materials and Equipment Kit Contents and Storage Each Quantifiler kit contains materials sufficient to perform 400 reactions at a 25 L reaction volume Additional Storage Guidelines For Primer Mixes Follow the additional guidelines for storing the primer mixes Minimize freeze thaw cycles
120. onjugated 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 Bibliography 2 Quantifiler Kits User s Manual Quantifiler Kits User s Manual Index 1 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 validation 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 startin
121. ontains How to Use This Guide vii Safety ix How to Obtain More Information xiv How to Obtain Support xiv How to Use This Guide Purpose of This Guide 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 Text Conventions This guide uses the following conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix 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 Preface viii Quantifiler Kits User s Manual User Attention Words 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
122. opy Number Real Time Data Analysis Quantifiler Kits User s Manual 1 13 About the Threshold 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 About the Threshold Cycle The threshold cycle CT for a specified amplification plot occurs when the fluorescent signal increases beyond the value of the threshold setting The CT value depends on Starting template copy number Efficiency of DNA amplification by the PCR system How CT Values Are Determined To determine the CT value the SDS software uses the Rn 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 Rn versus cycle number 2 An algorithm defines the cycle where the Rn value crosses the threshold setting the default threshold setting is 0 2 as the threshold cycle CT Chapter 1 Overview 1 14 Quantifiler Kits User s Manual Relationship of Threshold Cycles to Initial Template Amount The following equation describes the exponential amplificati
123. 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 5 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 04 2006 Part Number 4344790 Rev D Quantifiler Kits User s Manual Chapter 4 Data Analysis and Results I Quantifiler Kits User s Manual 4 1 4 Data Analysis and Results 4 This chapter covers Section 4 1 7000 SDS Data Analysis 4 3 Analyzing the Plate Document 4 3 Viewing Results 4 4 Section 4 2 7900HT SDS Data Analysis 4 7 Analyzing the Plate Document 4 7 Viewing Results 4 8 Chapter 4 Data Analysis and Results 4 2 Quantifiler Kits User s Manual 7000 SDS Data Analysis Section 4 1 7000 SDS
124. plification 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 Experiment 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 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 p
125. pplied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Quantifiler Kits User s Manual 6 37 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Overview Certain 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 Experiments 6 2 1 Precision 7900HT SDS 6 37 6 2 2 Mixture Studies 7900HT SDS 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 L to 23 pg L 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 CT FAM values were recorded and the means and standard deviations
126. properly 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 If a 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 Table 5 4 Troubleshooting amplification plots continued Observation Possible Cause Recommended Action Chapter 5 Interpretation of Results 5 14 Quantifiler Kits User s Manual Abnormal amplification plots in one column of reactions Uncalibrated pure dyes damage to the lens or dust on the filters and or mirror If the pure dyes are not calibrated run the pure dyes and recalibrate Note See your instrument user guide for instructions on how to run pure dyes and recalibrate 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 docu
127. r the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical Waste Hazard CHEMICAL WASTE HAZARD Some wastes produced by the operation of the instrument or system are potentially hazardous and can cause injury illness or death Chemical Waste Safety Guidelines To minimize the hazards of chemical waste 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 Preface xii Quantifiler Kits User s Manual 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 Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the conten
128. rds and for unknown samples page 2 15 and page 2 17 Set thermal cycler conditions page 2 19 Setting Up a Plate Document Template Quantifiler Kits User s Manual 2 23 7000 SDS Software Setup 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 d Click Save To create a plate document template continued Save the template file in the Templates folder Enter a name for the template Chapter 2 Software Setup 2 24 Quantifiler Kits User s Manual 7000 SDS Software Setup Creating a Plate Document from a Template After you create a template you can use it to create a plate document To create a plate document from a template 1 If the 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 and in the New Document dialog box and make the following sele
129. re 6 22 Average R2 values Replicate standard curves 6 4 4 2 Reproducibility and Sensitivity 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 CT values were generated in Manual analysis mode then the quantities were calculated using the standard curve on each plate Figure 6 23 shows average CT values each point n 90 replicates across a set of four serial dilutions 2 ng L to 0 5 ng L 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 CT values do not affect calculated quantities calculated quantities were normalized resulting in comparable concentrations on both instrument types R2 Average per Instrument 0 980 0 985 0 990 0 995 1 000 1 005 01 02 03 01 02 03 7000 Instrument 7500 System Instrument Ave R 2 value Quantifiler Human kit Quantifiler Y kit Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 59 Figure 6 23 CT values and quantitated concentrations Quantifiler Human Kit comparable data were obtained for the Quantifiler Y Human Male Kit DNA CT values with Quantifiler Human kit 25 28
130. red Resolution Panel 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 l All samples were within the range of sensitivity for the A260 method Table 6 9 Comparison sensitivity and specificity of methods Method Sensitivity Specificity A260 Cannot detect DNA in the picogram range Not specific for human genomic DNA Detects single stranded DNA double stranded DNA and RNA Dye intercalation 25 pg mLa Not specific for human genomic DNA Quantiblot kit 2 ng L to 0 03125 ng L Specific for human genomic DNA a Value obtained from the manufacturer s documentation Chapter 6 Experiments and Results 6 28 Quantifiler Kits User s Manual Experiment Each DNA sample was quantified using A260 method Absorbance at 260 nm was measured DNA concentration was calculated using the formula Concentration g mL 50 A260 Quantiblot kit DNA was quantified using a protocol for chemiluminescence detection with film autoradiography 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 me
131. results from male samples were used Results Table 6 17 shows the DNA concentrations calculated for all samples using the A260 method the dye intercalation method the Quantifiler Human kit and the Quantifiler Y kit It also shows the percent differences calculated for the comparisons Table 6 17 Comparison with A260 and dye intercalation 7900HT SDS Sample A260 Result ng L DIa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from DI Result ng L Diff from A260 Diff from DI 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 genomic A 2 2 2 270 2 887 31 2 27 2 1 792 18 6 21 1 Chapter 6 Experiments and Results 6 44 Quantifiler Kits User s Manual Human genomic B 0 55 0 584 0 805 46 3 37 9 0 379 31 0 35 0 Human genomic C 0 11 0 134 0 184 67 4 36 9 0 105 4 3 21 7 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 R
132. rofiles produced complete profiles with peak heights between 200 and 4000 relative fluorescence units RFU Section 6 3 Casework Sample Analysis Quantifiler Kits User s Manual 6 47 Results According to the results from the Quantifiler Human kit reactions run on the 7000 SDS the range of DNA concentrations was 0 06 ng L to 2 61 ng L Table 6 19 Successful STR profiles were obtained for the 28 samples that were analyzed Figure 6 18 These samples contained the minimum amount of DNA recommended for optimal Identifiler kit results 50 pg L in a 10 L 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 STRa Sample Quantity ng L Input Amount for Identifiler Kit ng QuantiBlot Kit Quantifiler Human Kit and 7000 SDS Quantifiler Human Kit and 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
133. ry 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 Chemical Hazard Warning CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Preface x Quantifiler Kits User s Manual Chemical Safety Guidelines To minimize the hazards of chemicals 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 manufacturer 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 About MSDSs Chemical manufacturers supply current Material Safety Data Sheets
134. s Viewing Results Quantifiler Kits User s Manual 4 11 7900HT SDS Data Analysis 6 Locate then select the folder where you want to save the exported results file 7 Enter the File Name then click Export To export the results continued 04 2006 Part Number 4344790 Rev D Quantifiler Kits User s Manual 5 1 5 Interpretation of Results 5 This chapter covers Checking Analysis Settings 5 2 Examining the Standard Curve 5 4 Troubleshooting the Standard Curve 5 6 Using the Internal PCR Control System 5 10 Troubleshooting Amplification Plots 5 12 Assessing Quantity 5 16 Chapter 5 Interpretation of Results 5 2 Quantifiler Kits User s Manual Checking Analysis Settings The validity of the results requires correct analysis settings Checking Analysis Settings on the 7000 SDS To check analysis settings on the 7000 SDS 1 If the 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 Anal
135. s Std 1 Std 2 Std 3 and so on 2 Dispense the required amount of diluent T10E0 1 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 Preparing the Reactions Quantifiler Kits User s Manual 3 5 Preparing the Reactions Required Materials Quantifiler Human Primer Mix or Quantifiler Y Human Male Primer Mix Quantifiler PCR Reaction Mix 10 mL polypropylene tube 96 well reaction plate Extracted DNA samples DNA quantification standards dilutions series T10E0 1 Buffer with or without glycogen for negative controls Optical Adhesive Cover Preparing the Reactions While preparing the reactions keep the 96 well reaction plate in its 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 Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent
136. s Manual 7000 SDS Software Setup Powering On the Instrument Note Wait for the computer to finish starting up before powering on the 7000 instrument 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 Starting SDS Software Select Start gt ABI Prism 7000 gt ABI Prism 7000 SDS Software The software attempts to initialize the instrument and displays a message in the status bar for a few seconds Then the computer attempts to establish communication with the 7000 instrument If the connection is successful the software displays in the status bar Power button About Plate Documents Quantifiler Kits User s Manual 2 7 7000 SDS Software Setup About Plate Documents How Plate Documents Are Used Running a reaction plate on the 7000 SDS requires creating and setting up a plate document using the SDS software A plate 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 Coordinate the instrument operation such as thermal cycling and data collection Organize and store the data gathered during the run Analyze the data from the run Plate Document Types You can use the SDS software to create two types of plate document files
137. s Manual 2 17 7000 SDS Software Setup Applying Detectors for Unknown Samples You need to apply detectors to the plate document for the wells on the reaction plate that contain unknown samples IMPORTANT If you run reactions for the Quantifiler Human kit and 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 View gt Well Inspector and check the Use boxes for the applicable detectors Quantifiler Human or Quantifiler Y IPC For example 3 If you 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 Make sure that ROX is selected Chapter 2 Software Setup 2 18 Quantifiler Kits User s Manual 7000 SDS Software Setup Adding Sample Names for Unknown Samples Repeat the procedure to add sample names for all 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 Note Samples with identical sample names are treated as replicates by the SDS software Results for replicate reac
138. s were quantified using the following methods A260 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 L DNA concentration was calculated from the formula Concentration g mL 50 A260 The results calculated for the 2 0 ng L dilutions were then extrapolated for the other dilutions 0 5 ng L and 0 1 ng L 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 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 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 Human genomic Male Blood Raji 1 Male Cell line Raji 2 Male Cell line K 562 Female Cell line Table 6 12 Human DNA samples tested with A260 and dye intercalation DNA Sex Extraction
139. scherichia coli Yersinia Several species of Bacillus a common and pervasive bacterial genus Experiment There were approximately 1 105 genome copies of each species in each reaction SDS software was used to analyze the data and calculate the CT FAM value Chicken Male Human Female Human Male a Sex confirmed by STR analysis b Weak but positive amplification with higher CT values and lower Rn values than normal for the input amount of DNA in the reaction Table 6 6 Specificity with non human panel continued Organism Sex Result Quantifiler Human Kit Quantifiler Y Kit CT FAM Value Result CT FAM lt 40 No amplification after 40 cycles Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 15 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 Species Composition Result Quantifiler Human Kit Quantifiler 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
140. se the 3 end of the probe is blocked there is no extension of the probe during PCR Figure 1 5 Figure 1 5 Completion of polymerization 3 5 Reverse Primer 3 5 5 5 Forward Primer R 3 TaqMan MGB probe MGB NFQ Instrument Overview Quantifiler Kits User s Manual 1 7 Instrument Overview Fluorescence Detection Detection on the ABI PRISM 7000 Sequence Detection System 1 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 2 A system of lenses filters and a dichroic mirror focuses the fluorescence emission into a charge coupled device CCD camera 3 Based on wavelength the filters separate the light into a predictably spaced pattern across the CCD camera 4 During the run the CCD camera detects the fluorescence emission between 500 nm and 660 nm from each well 5 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 1 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 2 A system of lenses filters and a dichroic mirror focuses the fluorescence emission into
141. sing the previously validated 7000 System SDS Software v1 0 Small percentage differences are observed in CT slope and R2 values 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 No significant difference is observed between CT values and calculated quantities derived using Auto Baseline and Manual analysis modes Quantifiler Kits User s Manual Bibliography 1 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 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 c
142. stem Validation SDS Software v1 0 DAB DNA Advisory Board Guideline 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 6 1 1 Precision 6 4 6 1 2 Reproducibility 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 6 1 6 Sensitivity 6 16 6 1 7 Stability 6 17 6 1 8 Mixture Studies 6 21 6 1 9 Degraded DNA Studies 6 23 6 1 10 Comparisons with Other Methods 6 27 6 1 11 Comparison with A260 and Quantiblot Kit 6 27 6 1 12 Comparison wit
143. t 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 Results The quantification results Figure 6 9 on page 6 23 from using the Quantifiler Human kit varied from an average of 0 16 ng L for the 1 1 sample to 38 ng L 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 L to 0 063 ng L 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 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 Section 6 1 ABI PRISM 7000 Sequence Detect
144. tandard 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 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 software 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 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 app
145. te Layout Reproducibility and sensitivity testing on the 7500 System identical plate layout for both kits On each instrument six 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 7000 Systems 3 7500 Systems 3 Standard curve DNA 1 DNA 2 Quantifiler Human Kit or Quantifiler Y Human Male Kit Standard Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 55 Background Testing Ninety five no template controls NTCs and one positive control the 50 ng L 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 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 CT slope and R2 were calculated for all replicate samples in a run For Auto Baseline to Ma
146. ter 2 Software Setup 2 38 Quantifiler Kits User s Manual 7900HT SDS Software Setup Setting Thermal Cycler Conditions Before running a Quantifiler kit assay you need to make two changes to the default thermal cycler conditions Thermal profile Sample volume To set thermal cycler conditions 1 In the plate window select the Instrument tab 2 Delete the Stage 1 hold step 50 C for 2 minutes a Press the Shift key and click within the Stage 1 hold step b After the hold step is selected press the Delete key 3 Make sure that the thermal profile appears as follows Press the Shift key and click within the Stage 1 hold step Hold step is selected Setting Up a Plate Document Quantifiler Kits User s Manual 2 39 7900HT SDS Software Setup Saving the Plate Document Before running the reaction plate save the plate document as an ABI 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 4 Set the Sample Volume to 25 L and make sure that the 9600 Emulation box is selected Note Selecting the 9600 Emulation box reduces the ramp rate 5 Make sure that the default settings are kept on the remaining tabs Auto Increment Ramp Rate Data Collection To set thermal cycler conditions continued Set the volume to 25 L Make sure that this box is selected To save the plate docu
147. tes for six instruments at a time 6 4 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 Figure 6 19 shows the experimental plate layout For each instrument six replicate plates were run consecutively The cycle threshold CT R2 and slope values were compared statistically to determine precision and accuracy which established 95 confidence intervals for each instrument type Kit Part Number Lot Number Quantifiler Human Kit 4343895 0501020 Quantifiler Y Human Male Kit 4343906 0501018 Section 6 4 Applied Biosystems 7500 Real Time PCR System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 53 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 7500 Systems 3 7000 Systems 3 Quantifiler Human DNA Quantification Kit Quantifiler Y Human Male DNA Quantification Kit Chapter 6 Experiments and Results 6 54 Quantifiler Kits User s Manual Reproducibility and Sensitivity Testing On each 96 well reaction plate the following were set up for real time PCR 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 Figure 6 20 Pla
148. ther example of arranging reactions when running two Quantifiler kits on one 96 well reaction plate if you are using repeat pipettors Wells A1 through D6 gray correspond to reactions using the Quantifiler Human kit 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 Figure 2 4 Example arrangement of reactions using repeat pipettors 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Std 1 UNKN UNKN UNKN UNKN Std 1 Std 1 UNKN UNKN UNKN UNKN B Std 2 Std 2 UNKN UNKN UNKN UNKN Std 2 Std 2 UNKN UNKN UNKN UNKN C Std 3 Std 3 UNKN UNKN UNKN UNKN Std 3 Std 3 UNKN UNKN UNKN UNKN D Std 4 Std 4 UNKN UNKN UNKN UNKN Std 4 Std 4 UNKN UNKN UNKN UNKN E Std 5 Std 5 UNKN UNKN UNKN UNKN Std 5 Std 5 UNKN UNKN UNKN UNKN F Std 6 Std 6 UNKN UNKN UNKN UNKN Std 6 Std 6 UNKN UNKN UNKN UNKN G Std 7 Std 7 UNKN UNKN UNKN UNKN Std 7 Std 7 UNKN UNKN UNKN UNKN H Std 8 Std 8 UNKN UNKN UNKN UNKN Std 8 Std 8 UNKN UNKN UNKN UNKN Setting Up a Plate Document Quantifiler Kits User s Manual 2 31 7900HT SDS Software Setup Setting Up a Plate Document Overview Setting up a plate document involves 1 Creating a Blank Plate Document
149. thod 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 Results 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 A260 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 A260 and Quantiblot kit Sample Sex A260 Result ng L QBa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from QB Result ng L Diff from A260 Diff from 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 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 29 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
150. tifiler 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 CT slope and R2 values were compared to determine precision and accuracy Kit Part Number Lot Number Quantifiler Human Kit 4343895 0501022 Quantifiler Y Human Male Kit 4343906 0501020 Chapter 6 Experiments and Results 6 68 Quantifiler Kits User s Manual Figure 6 28 Plate Layout Precision and accuracy testing on the 7000 System Reproducibility Sensitivity and Background Testing On each 96 well reaction plate the following were set up for real time PCR Standard dilution series two replicates of each dilution point Four replicate serial dilution sets of two sample DNAs 007 and 9948B Sixteen no template controls NTCs which served as background samples Figure 6 29 shows the experimental plate layout 7000 System SDS Software v1 2 3 7000 System SDS Software v1 0 Quantifiler Human DNA Quantification Kit Quantifiler Y Human Male DNA Quantification Kit Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 69 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 r
151. tions are grouped together automatically for data analysis Setting Up a Plate Document Quantifiler Kits User s Manual 2 19 7000 SDS Software Setup Setting Thermal Cycler Conditions Before running a Quantifiler kit assay you need to make two changes to the default thermal cycler conditions Thermal profile 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 50 C for 2 minutes to select it 3 After the hold step is selected press the Delete key Press the Shift key while you click within the Stage 1 hold step Stage 1 hold step is selected Chapter 2 Software Setup 2 20 Quantifiler Kits User s Manual 7000 SDS Software Setup 4 Make sure that the thermal profile appears as follows 5 Change the Sample Volume to 25 L and make sure that the 9600 Emulation box is selected Note Selecting the 9600 Emulation box reduces the ramp rate To set thermal cycler conditions continued Set the volume to 25 L Make sure that this box is selected Setting Up a Plate Document Quantifiler Kits User s Manual 2 21 7000 SDS Software Setup Saving the Plate Document Before running the reaction plate save the plate document as an SDS Document sds file Note To save the plate document as a template see Setting Up a Plate Document Template on page 2 22
152. ts 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 L DNA and 1 000 L 10mM Tris HCl pH 8 0 and 0 1 mM Na2EDTA T10E0 1 buffer Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 67 One manufactured lot of each kit was used for all validation studies 6 5 1 2 Instruments 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 Biosystems 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 Quan
153. ts of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste Disposal If potentially hazardous waste is generated when you operate the instrument you must 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 Biological Hazard Safety 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 Microbiological and Biomedical Laboratories stock no 017 040 00547 4 http bmbl od nih gov Safety Quantifiler Kits User s Manual xiii Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access
154. uantification results 6 1 12 Comparison with A260 and Dye Intercalation The concentration of DNA was measured for 13 human genomic DNA samples using the A260 method a dye intercalation method and the Quantifiler kits DNA Samples Tested Six human genomic DNA samples were obtained from different commercial sources 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 Table 6 10 Comparison with A260 and Quantiblot kit continued Sample Sex A260 Result ng L QBa Result ng L Quantifiler Human Kit Quantifiler Y Kit Result ng L Diff from A260 Diff from QB Result ng L Diff from A260 Diff from QB Table 6 11 Average differences from A260 and Quantiblot kit Method Average Difference Quantifiler Human Kit Quantifiler Y Kit A260 16 9 15 1 Quantiblot 42 0 35 5 Table 6 12 Human DNA samples tested with A260 and dye intercalation DNA Sex Extraction Source 007 Male Blood 9948 Male Cell line Chapter 6 Experiments and Results 6 32 Quantifiler Kits User s Manual Experiment Using the concentrations provided by the supplier the DNA samples were diluted to 2 0 ng L A 0 5 ng L B and 0 1 ng L C Note All dilutions were made in T10E0 1 Buffer with 20 g mL glycogen added as a carrier and stabilizer All sample dilution
155. uman 007 v1 0 Quantifiler Human 9948 v1 0 Quantifiler Human 007 v1 2 3 Quantifiler Human 9948 v1 2 3 Software v1 0 and Software v1 2 3 CT values 22 24 26 28 30 32 34 0 5 10 15 20 25 30 35 Well location CT Quantifiler Y 007 v1 0 Quantifiler Y 9948 v1 0 Quantifiler Y 007 v1 2 3 Quantifiler Y 9948 v1 2 3 Quantity Calculations between software v1 0 and software v1 2 3 analysis 0 1 1 10 100 0 5 10 15 20 25 30 35 Well location ng ul Quantifiler Y 007 v1 0 Quantifiler Y 9948 v1 0 Quantifiler Y 007 v1 2 3 Quantifiler Y 9948 v1 2 3 Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 Quantifiler Kits User s Manual 6 75 Figure 6 37 shows that there was a 6 quantity difference between results obtained with v1 0 and v1 2 3 software 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 CT result 36 81 CT Remaining NTCs resulted in gt 40 CT values negative results Figure 6 38 Background amplification plots 7000 System SDS Software v1 0 QTY differences between v1 0 and v1 2 3 analysis 6 4 2 0 2 4 6 8 0 5 10 15 20 25 30 35 We
156. un using the bacteriophage DNA quantification standard supplied with the kit and a quantification standard based on Raji human genomic DNA There were significant differences between the Table 6 16 DNA samples tested with A260 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 Section 6 2 Applied Biosystems 7900HT Real Time PCR System Validation SDS Software v2 0 Quantifiler Kits User s Manual 6 43 standard curves from the 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 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
157. 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 CT values do not vary significantly from run to run or from instrument to instrument The CT value from one sample run on three different 7000 instruments varies with an average standard deviation of 0 3 Systematic differences between instruments 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 CT values are affected equally Day 1 Day 2 Instruments 1 2 3 1 2 3 Mean 16 7 5 56 0 62 0 068 50 DNA quantity ng L 40 35 30 25 20 15 10 CT 5 0 0 023 1 85 0 21 Section 6 1 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 0 Quantifiler Kits User s Manual 6 7 6 1 2 Reproducibility Experiment Six different human DNA samples were tested for reproducibility of the quantification results Using the concentrations provided by the supplier the DNA samples were diluted to 2 0 ng L A 0 5 ng L B and 0 1 ng L C Note All dilutions were made in T10E0 1 Buffer with 20 g 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
158. xhibits low precision Phase 3 Plateau The amplification plot achieves the plateau phase when the PCR stops the Rn signal remains relatively constant and the template concentration reaches a plateau at about 10 7 M Martens and Naes 1989 Chapter 1 Overview 1 12 Quantifiler Kits User s Manual Relationship of Amplified PCR Product to Initial Template Concentration 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 Rn value The following equation describes the relationship of amplified PCR product to initial template during the geometric phase where Nc 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 Figure 1 9 Amplification plot from a real time run of an RNase P Instrument Verification Plate Nc N 1 E c 2500 10000 20000 Cycle Number Rn 40 30 20 10 0 0 0 1 0 2 0 8 0 5000 1250 C
159. xperimental Setup 6 51 Data Collection 6 54 Data Analysis 6 54 Discussion 6 63 Conclusion 6 64 Section 6 5 ABI PRISM 7000 Sequence Detection System Validation SDS Software v1 2 3 6 66 Materials and Methods 6 66 Experimental Setup 6 67 Data Collection 6 69 Data Analysis 6 69 Discussion 6 79 Conclusion 6 80 Bibliography Index vi Quantifiler Kits User s Manual Quantifiler Kits User s Manual vii Preface This preface c
160. ysis Settings 5 For all detectors confirm that the settings are as shown below 6 If the 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 Checking Analysis Settings Quantifiler Kits User s Manual 5 3 Checking Analysis Settings on the 7900HT SDS To check analysis settings on the 7900HT SDS 1 If the SDS software is not already started select Start gt Programs gt Applied Biosystems gt SDS 2 0 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 and confirm that the settings are as shown below 5 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 Chapter 5 Interpretation of Results 5 4 Quantifiler Kits User s Manual Examining the Standard Curve Examine the standard curve results to evaluate the quality of the results from the quantification standard reactions About Standard Curve Results The standard curve is a graph of the CT of quant

Quantifiler Kits User Manual (PN 4344790D)

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