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Quantifiler® Duo DNA Quantification Kit User`s Manual (PN

1. 1 Ifthe SDS software is not already started select Start gt Programs gt 7500 System gt 7500 System Software 2 Select File gt Open 3 Locate the plate document for the assay run of interest select it then click Open Alternatively navigate to the folder containing the run file and double click the run file to skip the previous steps 4 Select Analysis gt Analysis Settings 5 For all detectors confirm that the settings are set as shown Select All Detectors Select Manual Ct and enter 0 2 as the Threshold Select Manual Baseline and enter 3 to 15 as cycle range Analysis Settings Absolute Quantific x Ct Analysis Detector NEIN Auto Ct Manual Ct Threshold 0 2000000 Automatic Baseline Manual Baseline Start cycle 3 End cycle 15 r OK amp Reanalyze Cancel Apply If the analysis settings differ from those shown set as noted above and click Apply then click OK amp Reanalyze to reanalyze and close the plate document View the results using Chapter 4 Data Analysis and Results Quantifiler Duo DNA Quantification Kit User s Manual Examining the Standard Curve Examining the Standard Curve Examine the standard curve results to evaluate the quality of the results from the quantification standard reactions About Standard The standard curve is a graph of the C of quantification standard Curve Results reactions p
2. Table 5 4 Troubleshooting amplification plots continued o Observation Possible Cause Recommended Action Dus Ens Cice Weak lamp or incorrect Replace the lamp or make sure the existing TOH PTT TTT NAH replacement replacement is correct gee HUHUHUHU AHH i Wa ABE cea ne EE d e d EE NR cc T Jagged amplification plots Incorrect detector selected on 1 Make sure that the correct detector is o NEREEEEEEEEHEEEEEREHRENER EE the amplification plot or selected on the amplification plot 77 EE incorrect detector applied fo 2 If the amplification plots are still not defined E EREB Cad tert the reactions when setting up make sure settings are correct 3 AN H N Pal the plate document In the g CG NTT ANT example a detector that uses a From the plate document double click a 1000s ANN Cy5 as reporter dye was well to view the Well Inspector ES tN H selected and applied in the b Verify that the detector settings are correct Emm HEB LL MEN amplification plot and reanalyze No defined amplification plots enue sasn NA uoneo nueno YN ONG sJelumueno G 19 dey sjjnseg Jo uoiejeJdieju enue s Jesf 4y uoneoynueno YNA ONG g elllnueno 11 9 Table 5 4 Troubleshooting amplification plots continued Observation Possible Cause Recommended Action Delta Rn Delta Rn Delta Rn 2001 K 12345678 9101112131415161718192021 2223 24
3. 4 7 0 oO m o B jn m m m m c m m m jul n Im m m jul mn jm m m m Ul m m mU jv o ol m m m mn n m m m n lm m m m n Im m m m mn ul m m jul o F jr m c m m ol m jn m D m m o m jn m Ready Disconnected NU Figure 2 1 7500 System Software Plate Setup tab 2 18 Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document Adding Detectors To alternatively add detectors to an open plate document to an Open Plate Within an open plate document select Tools gt Detector Manager to open the Detector Manager window Detector Manager Detector List Find a Detector Name Description Reporter Quencher Color Hotes Last Mod D an VIC none Add the detectors to the plate document File v Add To Plate Document Help In the Detector Manager window Ctrl click the Duo Human Duo Male and Duo IPC detectors then click Add to plate Document If the detectors for the Duo DNA Quantification Kit are not listed in the Detector Name field see Creating Detectors on page 2 10 Document 1 Alternative 2 3 Click Done to close the Detector Manager When you are finished in the 7500 System Software Plate Setup tab shown previously in Figure 2 1 assign sample names tasks and quantities to standards as necessary see page 2 20 for procedure Quantifiler Duo DNA Quantification Kit User s Manual 2 19 Chapter 2 Software S
4. kk kk kK kk kk kk kk kk kk kk kk kk kk 1 3 Instrument Overview S a n ye yan Wad ss 1 7 SDS Software Overview kk kk kk kK KK eee teens 1 8 Real Time Data Analysis ka aka kk KERR KE kK kk kk a 1 9 Quantifiler Duo Kit Workflow dll kK KEK ikv 1 15 Materials and Equipment kk kk kk kk KK KK KK KK KI eee 1 16 Software Setup OVEINIEW hi ea nd oda haha aaah Dh Ay aden 2 2 Starting the 7500 Real Time PCR System aa 2 3 About Plate Documents kk kk KK KK KK KK ee eee 2 6 Setting Up a Plate Document kk kk 0 0 0 eee ee ee 2 8 Setting Up a Plate Document Template 0 RR KK KK J 2 26 PCR Amplification Preparing the DNA Quantification Standards 3 2 Preparing the Reactions ananuna rnaar RR KK KK KK KK 3 5 Running the Reactions lt i i lt as ik ka Wa Xw kwa kl eee 3 7 Quantifiler Duo DNA Quantification Kit User s Manual iii Chapter 4 Chapter 5 Chapter 6 Bibliography Index Data Analysis and Results Analyzing the Plate Document kk kk 000 KIRR eee 4 2 Viewing Results orsi 2 xa xa 6d Waya K tee 4 4 Interpretation of Results Checking Analysis Settings 0 0 e eee eee ee 5 2 Examining the Standard Curve WWW kk kk kK KK KK KK ee 5 3 Troubleshooting the Standard Curve uk kK KK RR RR KK KK 5 5 Using the Internal PCR Control System kk kK KK KK 5 13 Troubleshooting Amplification Plots kk KK RR KK RR KK 5 15
5. Quantifiler Duo DNA Quantification Kit User s Manual 2 11 Chapter 2 Software Setup To create detectors continued 2 In the New Detector dialog box create a detector for the RPPH1 human target a Enter Duo Human in the name field b Select VIC for the Reporter Dye c Select none for the Quencher Dye d Click and select a color IMPORTANT Make sure that you select the VIC dye for Duo Human New Detector Name Duo Human mg Enter Duo Human Description EU Reporter Dye Select VIC Quencher Dye Select none Color Click to select a color Notes Create Another Cancel 3 Click Create Another to add Duo Human to the Detector Name list and to reset the New Detector dialog box 2 12 Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document To create detectors continued 4 Create a detector for the SRY Male target a Enter Duo Male in the name field b Select FAM for the Reporter Dye c Select none for the Quencher Dye d Click and select a color IMPORTANT Make sure that you select the FAM dye for Duo Male New Detector Name Duo Male Enter Duo Male Description Reporter Dye Select FAM Quencher Dye Select none Color Click to select a color Notes Create Another Cancel Click Create Another to add Duo Male to the Detector Name list and to reset the New Detector dialog box
6. Mds H ns Hla an Eh Partial Profile 2000 4006 D a ao mo awo oao ow o aso oao O o O ao O ao oo oao oo oo Oa O ao ee 30 c0 se se aa ao and NE No Profile 2000 4000 D Figure 6 6 Identifiler Kit profiles obtained for samples containing a range of hematin concentrations 0 Q0 120 100 4 180 s 470 deo 190 200 20 220 29 20 280 26o 270 30 290 90 sto 30 sao aw ssa ao Ho 120 9 e 48o deo 470 deo 190 29 oao 220 220 2m 280 26o 20 200 290 90 3o 220 soo so asa Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation IPC Crin humic acid inhibited samples IPC Cr 0 1 2 3 3 75 75 11 25 15 30 Humic Acid Concentration ng uL Figure 6 7 IPC C values obtained for samples containing a range of humic acid concentrations Quantifiler Duo DNA Quantification Kit User s Manual 6 15 Chapter 6 Experiments and Results T e e 20 ae 20 39 ae ze ae zm ae ze o se o e 30 a 0 ng ul 1 ng uL Full Profile m e X 2 20 280 08 mw 2o 20 20 280 800 se xm sao 3 so Full Profile 2 ng uL Te e we ae 2 e ze 9 2e ze we ze ae z a 3 a se 3 6 Full Profile 0 we ae 2 e 0 m 2e mne ze me ze w w AU a 3 ng uL 3 75 ng uL 7 5 ng uL 11 25 ng uL Full Profile T mw ae ae mw m 09 ae 9
7. o 123485678 9 101112191415 1617 1819 20 21 2223 24 2528 27 28 29 90 31 3231 34 35 3637 38 3940 C values 40 are detected in NTC wells for the Duo Human and or Duo Male detectors Observation Possible Cause Recommended Action ee Spectral crosstalk into the From the plate document select the NTC j Duo IPC FAM and or VIC channels wells click on the Amplification Plot tab then d change the AR scale to linear Duo Human If the AR values for the Duo Human VIC reporter dye and or the Duo Male FAMTM reporter dye detectors slowly increase until the curves cross the threshold at a late threshold cycle as shown perform an instrument Pure Dye Spectra calibration see the Applied Biosystems 7300 7500 Real Time PCR System Installation and Maintenance Guide for more details on the maintenance procedures and repeat the experiment If the problem persists contact Applied Biosystems Technical Support im Duo IPC 2 Duo Human and or Duo Male 010 C4 values 40 are detected in NTC wells for the Duo Human and or Duo Male detectors 123459678 9 1011121314151617 181920212223 24 2526 27 28 2030 31 3233 34 35 3637 38 39 4 Contamination of reagents due to DNA or amplified PCR products From the plate document select the NTC wells click on the Amplification Plot tab then change the AR scale to linear If the AR values for the Duo Human VIC reporter dye and or the Duo M
8. zi al I Note 0 25 ng of human DNA is used for amplification of the sample in panel A 0 1 ng of human DNA is used for amplification of samples in panels B C and D 10 uL of the diluted extract as indicated is used for amplification of samples in panels E through J Label corresponds to inhibitor concentration in the Quantifiler Duo reaction Figure 6 12 MiniFiler Kit analysis of hematin inhibited samples after dilution of highly inhibited samples 6 24 Quantifiler Duo DNA Quantification Kit User s Manual 0 ng uL 0 25 ng 1 ng uL 0 1 ng 2 ng uL 0 1 ng 3 ng uL 0 05 ng 3 75 ng L 0 03 ng 7 5 ng uL 10 uL 11 25 ng LL 10 uL 15 ng LL 10 uL 30 ng uL 10 uL Developmental Validation UIT EUETERUE COE TIC UEUYETOOEUTYI EESTI ESTE mE E se A NG au J dun aha id a a 080707 minitier ihi ip Jumic Ingul 0 Ing DOGS humic Ingul Ing MiniFllr G 500 1 mm na B AA Manila 5500 v mm a C i all sahi m 00707 initia hi i humic gud Hx20 05 f humic ngu Ho20 Mini GS5t0 vl mm a a D UU uita hip emi 375ngul N70 Ct fumi Fangu M Mime 6550 HN ai MG E AA mM i F a a RACER Mini G5500 vt mm n H N i a a Ti P al 1 i Note 0 25 ng of human DNA is used for amplification of the sample in panel A 0 1 ng of human DNA is used for amplification of samples in panels B and C 0 05
9. IPC C i 1 di HEHE HIN 0 25 5 75 10 125 15 175 20 40 Hematin Concentration uM Figure 6 5 IPC C values obtained for samples containing a range of hematin concentrations Quantifiler Duo DNA Quantification Kit User s Manual 6 13 Chapter 6 Experiments and Results 0 uM 2 5 uM 5 uM 7 5 uM 10 uM 12 5 uM 15 uM 17 5 uM 20 uM 40 uM 6 14 067 uibs validation Soir corel AL CU iir von ul eiii sl Hm z 3000 H Full Profile MEN Aa AAA E IN DAA AA cnm jl Ni A ox 70607 Rainbow validation nain 2 Fu 2ul DU malin 2 SAPA mm 3000 H Full Profile 070007 Juinbew validation Jatin Suh Ful KO fsa hematin Suh Jul mm 3000 1 Full Profile AER RAT aT kaa a 3000 H Full Profile E LJ AAAA Ja an ah hu A d L j JM EN a ganas Up PA VERNI a F Full Profile n i AL bk laha An nan A Ji MA noh al i i j76607 Rxinbewyalidation_ amain 12 SuM Jul HIT erat 12 SuM Pu arang HN ES 070607 Rainbew validation hematin 15uM 2ul A05 fs henaiim 15uM 2ul Tetite vi m m E Partial Profile 7067 Jin vidio aman 17 t Pl OO hein 17 Sul Pu DET mm 070607 Juinbew validation hematin 201M 2ul CO s nanatin 20W Put Menit v1 mm 070607 Rainbow validation henatin 40uM 2ul DOS fs hematin dou Jul denier vi mm a Full Profile with Low Peak a E E AA RA Heights 4000 dejame E AND egere eau dn kan Partial Profile 2000 4000 alle ee md
10. Table 6 2 Human DNA samples tested for sensitivity Sample Source 1 Human male blood pool 2 Human male blood single source Results The quantities of DNA obtained from the Quantifiler Duo DNA Quantification Kit were very similar to the expected quantities across a range of concentrations from 20 ng uL to 23 pg uL as shown in Table 6 3 Furthermore quantities as low as 11 5 pg uL of human DNA were reproducibly detected across all replicates using the Quantifiler Duo DNA Quantification Kit as shown in Table 6 3 At concentrations of 5 75 pg uL and below human DNA cannot be reproducibly detected across all replicates due to stochastic variation in the amplification efficiency resulting from low DNA input amounts Table 6 3 Stochastic effects provide greater variation in the quantification results from samples containing lower quantities of DNA In general for samples containing DNA at concentrations of 0 1 ng uL or less it is necessary to add the maximum volume of DNA extract to the AmpFZSTR kit STR reaction A plot of the C values versus the known DNA quantities see Figure 6 1 and Figure 6 2 showed the expected log linear relationship between the two quantities from 20 ng uL to 23 pg uL For each dilution series the data points formed an acceptable standard curve Quantifiler Duo DNA Quantification Kit User s Manual 6 7 Chapter 6 Experiments and Results 6 8 Table 6 3 Sensitivity using th
11. Experiment The precision of the Quantifiler Duo DNA Quantification Kit was tested by performing two runs on different days one run per day on three different instruments One set of eight serial dilutions was prepared containing 50 16 7 5 56 1 85 0 62 0 21 0 068 and 0 023 ng uL of the human male DNA standard included in the Quantifiler Duo DNA Quantification Kit Six reaction plates were set up and each of them contained 10 replicates of the 8 dilutions Two plates per instrument were run on three different 7500 Real time PCR System instruments using recommended thermal cycler conditions for the Quantifiler Duo DNA Quantification Kit 6 49 Chapter 6 Experiments and Results The two runs were performed on two different days using the same three 7500 Real Time PCR System instruments For each dilution the Cy values for RPPHI VIC SRY FAM and IPC NED signals were recorded for all 60 reactions Slope R and Y intercept values were also computed Results Table 6 15 shows the means and standard deviations of the C values for RPPHI SRY and IPC targets calculated for each quantification standard dilution across all 6 plates Table 6 15 Precision C values Quantification Human Male IPC Standard Dilution C Standard Gr Standard Cr Standard ng uL Mean Deviation Mean Deviation Mean Deviation 50 23 36 0 26 23 92 0 19 29 80 0 35 16 7 24 98 0 23 25 55 0 16 29 61 0 18 5 56 26
12. Quantifiler Duo DNA Quantification Kit User s Manual 2 13 Chapter 2 Software Setup To create detectors continued 6 Create a detector for the IPC target a Enter Duo IPC in the name field b Select NED for the Reporter Dye c Select none for the Quencher Dye d Click and select a color IMPORTANT Make sure that you select the NED dye for Duo IPC New Detector Name Duo IPC Enter Duo IPC Description Reporter Dye Select NED Quencher Dye Select none Color Click to select a color Notes Create Another Cancel 2 14 Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document To create detectors continued 7 Click OK to add Duo IPC to the Detector Name list and to return to the Select Detectors window New Document Wizard Select Detectors Select the detectors you will be using in the document none none none none none New Detector lt Back Finish Cancel When you are finished add detectors to the plate document IMPORTANT Make sure that the appropriate reporter dye is selected for the three detectors VIC for Duo Human FAM for Duo Male and NED for Duo IPC Adding Detectors There are two methods for adding detectors to the Plate Document You can add the detectors from the New Document Wizard Select Detector Window or You can add the detectors from an open plate docum
13. UNKN UNKN UNKN UNKN UNKN B Std2 Std2 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN e Std 3 Std 3 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN D Std 4 Std 4 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN E Std5 Std5 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN F Std 6 Std 6 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN G Std 7 Std 7 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN H Std 8 Std 8 UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN UNKN NTC Quantifiler Duo DNA Quantification Kit User s Manual 2 7 Chapter 2 Software Setup Setting Up a Plate Document Overview Setting up a plate document to run the Quantifiler Duo DNA Quantification Kit assay involves 1 Creating a Blank Plate Document page 2 9 2 Creating Detectors page 2 10 3 Adding Detectors to the Plate Document page 2 15 4 Adding Detectors to an Open Plate Document Alternative page 2 19 5 Assigning Sample Name Task and Quantity to Standards page 2 20 6 Assigning Sample Name and Task to Unknown Samples and Non Template Controls page 2 22 7 Setting Thermal Cycler Conditions page 2 24 8 Saving the Plate Document and Starting the Run page 2 25 2 8 Quantifiler Duo DNA Quantification Kit User s Manual Creating a Blank To create a blank plate document Pla
14. Batzer M A and Sinha S K 2007 Human genomic DNA quantitation system H Quant Development and validation for use in forensic casework J Forensic Sci 52 364 370 Swango K L Hudlow W R Timken M D and Buoncristiani M R 2006 Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples Forensic Sci Intl 170 35 45 Timken M D Swango K L Orrego C and Buoncristiani M R 2005 A duplex real time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples Implications for quantifying DNA in degraded samples J Forensic Sci 50 1044 1060 Tringali G Barbaro A Insirello E Cormaci P and Roccazzello A M 2004 Rapid and efficacious real time quantative PCR assay for quantitation of human DNA in forensic samples Forensic Sci Intl 146 S177 181 Walker J A Kilroy G E Xing J Shewale J Sinha S K and Batzer M A 2003 Human DNA quantitation using Alu element based polymerase chain reaction Anal Biochem 315 122 128 Quantifiler Duo DNA Quantification Kit User s Manual Walker J A Hedges D J Perdeau B P Landry K E Stoilova N Laborde M E Shewale J Sinha S K and Batzer M A 2005 Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear mitochondrial and male Y chromosome DNA application in human identification Anal Biochem 337 89 97 Qu
15. IM e ze xo se x9 m o x o Full Profile with Low Peak Heights m gw m 20 w 20 m 09 ze 2o 20 09 xo o hw m Me 90 Partial Profile m ww f 20 o ze 20 O8 280 39 20 20 290 w mw 30 m 3w 0 15 ng uL xe Partial Profile m 0 ae ee mm 0 ae w zm s z 9 o 3b s lm 30 ng uL Figure 6 8 ie Mc ae No Profile Identifiler Kit profiles obtained for samples containing a range of humic acid concentrations 6 16 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Experiment 2 In this experiment the same samples described in Experiment 1 Human genomic DNA mixed with varying concentrations of hematin and humic acid were analyzed However in this experiment 2 uL of each sample containing approximately 1 0 ng of DNA was quantified in triplicate using the Quantifiler Duo DNA Quantification Kit and the quantification results from the RPPHI human target were used to determine the sample volume necessary for the autosomal STR reaction In this way the impact of the inhibitor concentration on the quantification results was evaluated Further the effect of the altered quantification results on the quality of the STR profile was studied for both the AmpF STR Identifiler Kit and the AmpF STR MiniFiler Kit 1 0 ng of template DNA was used in the PCR amplification reaction using the AmpF STR Identifiler Kit
16. To view the standard curve continued 4 In the Detector drop down list select ALL to view both standard curves at the same time Only C values can be viewed with this selection Note The figure below shows an example of the standard curve plots The gap between the Duo Human and the Duo Male C4 values may vary depending on the relative slopes of the two targets and instrument performance e E t Setup Y Instrument it ults Plate Y Spectra Y Component Y Amplification Plot Y standard Curve E i Ez 34 Su e l gt A NA SE ONE Duo Male Md NG E SB SR H Duo Human ATs 28 De SK 26 Ng j T NG 3 The amplification plot can display one of the following Plot of normalized reporter signal R versus cycle log view Cr versus well position view Plot of normalized reporter signal R versus cycle linear View For more information about the amplification plot see Real Time Data Analysis on page 1 9 or the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute Quantification Getting Started Guide PN 4378658 Quantifiler Duo DNA Quantification Kit User s Manual 4 5 Chapter 4 Data Analysis and Results Viewing the Amplification Plot Viewing the Report For troubleshooting information see Troubleshooting Amplification Plots on page 5 15 To view the amplification plot
17. quantification results obtained Experiment Four male and one female genomic DNA samples were tested to assess the reproducibility of the quantification results Table 6 6 The DNA samples were diluted from initial estimated concentrations to 20 0 10 0 1 0 0 1 and 0 05 ng L All dilutions were made in T 9Eo Buffer All samples and dilutions were tested in triplicate using the Quantifiler Duo DNA Quantification Kit Three different runs were performed For each sample reaction the Cy values were obtained and the DNA quantities calculated The mean quantity and standard deviation were calculated for each sample dilution The 9596 confidence interval values were calculated as the mean of the DNA quantity 2 standard deviation units for each sample and expressed as a percentage of the mean quantification result Table 6 6 Human DNA samples tested for reproducibility Sample Name Source Sex A Human blood single source Male B Human blood single source Male C Human blood pool Male D Human blood single source Male E Human blood single source Female Results The following table shows the DNA quantity calculated for all samples and dilutions tested for all three runs using the Quantifiler Duo DNA Quantification Kit Table 6 7 Quantifiler Duo DNA Quantification Kit User s Manual 6 33 gt Co enue s Jesf 11x uoneoynueno YN ONG e linueno Table 6 7 Reproducibility using the
18. values for the Duo Male detector When creating detectors for the standards FAM was selected as reporter dye for Duo Human and VIC was selected as reporter dye for Duo Male see Example 4 on page 5 11 From the plate document double click a well containing a DNA quantification standard to view the Well Inspector Verify that at each concentration the correct reporter dye is selected for each detector add new detectors if necessary see Creating Detectors on page 2 10 select the correct Task and Quantity then reanalyze Do the same for the unknown sample wells The following examples illustrate the observations referenced in the table above Example 1 Observation All of the Cy values for the DNA quantification standard reactions for the Duo Human detector lie outside of the standard curve and form a horizontal line when All detectors are selected in the Standard Curve tab Figure 5 1 Note that the affected detector disappears from the detector list and Duo IPC appears instead as shown in the pull down menu in Figure 5 2 As a result the slope for the Duo Human standard curve was outside the typical range and the R value 1s significantly less than 0 98 5 6 Quantifiler Duo DNA Quantification Kit User s Manual Troubleshooting the Standard Curve Setup Y Instrument YResults Y Plate Y Spectra Y Component Y Amplification Plot Y standard Curve Y Dissociation Y Report V
19. 0 31 ng uL to 14 66 ng uL for the SRY male specific target and from 0 27 ng uL to 13 28 ng uL for the RPPH1 human target Table 6 9 Table 6 9 Quantifiler Duo Quantification Kit reactions performed on case type samples xis u paraan Vendor ean PERI TAN RPPHI ng uL ng pL IPC difference 1 Organic In House 2 060 2 010 29 74 2 5 2 Organic In House 11 000 11 350 29 77 3 1 3 Organic In House 0 820 0 908 29 84 9 7 4 Organic In House 2 090 2 070 29 68 1 0 5 Organic In House 1 370 0 757 32 54 81 0 6 Organic In House 1 350 1 340 29 48 0 7 7 Organic In House 1 840 1 720 29 69 7 0 8 Organic In House 1 780 1 820 29 47 2 2 1 Chelex In House 0 506 0 505 28 91 0 2 2 Chelex In House 14 660 13 280 29 00 10 4 3 Chelex In House 0 307 0 267 30 79 15 0 4 Chelex In House 0 425 0 332 30 88 28 0 1 Silica Membrane QIAGEN Blood kit 0 457 0 443 29 90 3 2 2 Silica Membrane QIAGEN Blood kit 4 020 3 880 29 80 3 6 3 Silica Membrane QIAGEN Blood kit 0 306 0 312 29 76 1 9 4 Silica Membrane QIAGEN Blood kit 0 522 0 517 29 73 1 0 1 Magnetic Beads Promega DNA IQ 0 732 1 840 29 59 60 2 2 Magnetic Beads Promega DNA IQ 2 620 9 760 29 80 73 2 3 Magnetic Beads Promega DNA IQ 0 645 0 715 29 60 9 8 4 Magnetic Beads Promega DNA IQ 1 140 1 460 29 42 21 9 Quantifiler Duo DNA Quantification Kit User s Manual 6 37 Chapter 6 Experiments and Results 6 38 For all sample
20. 10 female 12 11 0 486 8 94 29 87 0 007 0 04 E 1 female 1 14 0 049 6 55 29 77 0 018 0 12 E 0 10 female 0 12 0 016 21 07 29 73 0 089 0 60 E 0 05 female 0 06 0 008 67 71 29 71 0 047 0 32 synsey pue sjueuuedx3j 9 sajdeyD Developmental Validation The 95 confidence interval shows the approximate range expected for results when using the Quantifiler Duo DNA Quantification Kit The average 95 confidence interval is 24 2 Yo and 21 4 for the human and the male target respectively The reproducibility results for the samples containing ng uL are shown graphically in Figure 6 20 At this concentration the range of standard deviations for each target is Human Target 0 027 to 0 083 Male Target 0 039 to 0 142 E RPPH1 Plate 1 El RPPH1 Plate 2 ORPPH Plate 3 B RPPH1 Mean Reproducibility 1 8 SRY Plate 1 16 m SRY Plate 2 m SRY Plate 3 B SRY Mean Quantity ng uL Male A Male B Male C Male D Female Figure 6 20 Reproducibility at 1ng uL using the Quantifiler Duo DNA Quantification Kit Quantifiler Duo DNA Quantification Kit User s Manual 6 35 Chapter 6 Experiments and Results 6 36 Case Type Samples Std 2 6 This experiment was performed to evaluate different sample types that are commonly processed in a forensic laboratory Experiment A variety of forensic type samples Table 6 8 were prepared using semen saliva and blood obtai
21. 100 0 118 0 100 18 000 0 121 0 145 16 552 D 0 050 0 059 0 066 10 606 0 073 0 074 1 351 E 20 000 24 910 27 880 10 653 female female female E 10 000 12 107 13 270 8 764 female female female E 1 000 1 137 1 300 12 538 female female female E 0 100 0 116 0 162 28 395 female female female E 0 050 0 056 0 060 6 667 female female female synseu pue sjuewEdxXZ 9 sajdeyD 04 2008 Part Number 4391294 Rev B 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 Alonso A Martin P Albarran C Garcia P et al 2004 Real time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies Forensic Sci Intl 139 141 149 Andresson H Nilsson M Budowle B Lundberg H and Allen M 2006 Nuclear and mitochondrial DNA quantification of various forensic materials Forensic Sci Intl 164 56 65 Forster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Ann of Phys Leipzig 2 55 75 Green R L Roinestad I C Boland C and Hennessy L K 2005 Developmental validation of the Quantifiler real time PCR kits for the quantification of human nuclear samples J Forensic Sci 50 809 825 Horsman K M Hickey J A Cotton R W landers J P and Maddox L O 2006 Development of a human specific real time PCR assay for the
22. 15 on the 7500 Real Time PCR instrument 1 The software generates a baseline subtracted amplification plot of AR versus cycle number 2 An algorithm defines the cycle where the AR value crosses the threshold setting as the threshold cycle C The following equation describes the exponential amplification of the PCR m n X Xm 1 By where X number of target molecules at cycle n so that n gt m X number of target molecules at cycle m Ey efficiency of target amplification between 0 and 1 n m number of cycles elapsed between cycle m and cycle n Quantifiler Duo DNA Quantification Kit User s Manual 1 13 Chapter 1 1 14 Overview Amplicons designed and optimized according to Applied Biosystems guidelines amplicon size lt 150 bp have amplification efficiencies that approach 100 Therefore Ey 1 so that m X n Na KG To define the significance in amplified product of one thermal cycle setn m 1 so that X Xm 2 2X4 Therefore each cycle in the PCR reaction corresponds to a two fold increase in product Likewise a difference in Cy values of 1 equates to a two fold difference in initial template amount Quantifiler Duo DNA Quantification Kit User s Manual Quantifiler Duo Kit Workflow Quantifiler Duo Kit Workflow Use of the Quantifiler Duo Kit involves the following workflow Software Setup PCR Amplification Data Analysis Interpretation of Re
23. Amplification Delta Rn vs Cycle Plateau IT Phase Linear Phase Geometric Phase 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Cycle Number Figure 1 8 Phases of PCR amplification Initially R appears as a flat line because the fluorescent signal is below the detection limit of the sequence detector Phase 1 Geometric Exponential Upon detection the signal 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 1s characterized by a high and constant efficiency Amplification occurs between the first detectable rise in fluorescence and 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 real time PCR system is sufficiently sensitive to detect at least 3 cycles in the geometric phase assuming reasonably optimized PCR conditions Quantifiler Duo DNA Quantification Kit User s Manual Real Time Data Analysis 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
24. Assessing Quantity i xua kk aa ak lal eee eens 5 19 Calculating Male Female DNA Ratio WR KK RR KK KK 5 20 Improving Assay Performance Wk kk kk KK KK eee eee KK 5 20 Assessing Sensitivity and Results 5 20 Assessing and Troubleshooting False Positive Results 5 22 Preventing PCR Contamination aa 5 23 Experiments and Results i MC c 6 2 Developmental Validation llle 6 3 Quantifiler Duo DNA Quantification Kit User s Manual Preface This preface contains How to Use This Guide 0 Wu KK KK KK eee eee V Salelys ciu cs Sues rette moteur ok PE a ptos xe pere dS oe vii How to Obtain More Information a xi How to Obtain Support xi How to Use This Guide Purpose of This The Quantifiler Duo DNA Quantification Kit User s Manual Guide provides information about and instructions for using the Quantifiler Duo DNA Quantification Kit Audience This manual is intended for scientists who use the Quantifiler Duo DNA Quantification Kit for the quantification of human DNA extracted from a variety of sample types It requires familiarity with the 7500 Real Time PCR System and 7500 SDS software 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
25. Before analyzing always prepare fresh matrix A right arrow bracket 7 separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set Right click the sample row then select View Filter gt View All Runs Quantifiler Duo DNA Quantification Kit User s Manual V Preface User Attention Words Safety Alert Words Pull out Chapters vi Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical Examples of the user attention words appear below Note The size of the column affects the run time Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password IMPORTANT You must create a separate Sample Entry Spreadsheet for each 96 well plate Safety alert words also appear in user documentation For more information see Safety Alert Words on page vii This User Manual contains individually bound chapters Chapter 2 through Chapter 6 that the user can pull out of the binder to use in the lab Qu
26. Chapter 2 Software Setup Creating a Plate After you create a template you can use it to create a plate document Document from a Template To create a plate document from a template 1 If the SDS software is not already started select Start gt Programs gt 7500 System gt 7500 System Software 2 Select File gt New to open the New Document Wizard Define Document window and make the following selections For Assay select Absolute Quantitation For Container select 96 Well Clear For Template select an applicable template from the list Note Ifthe template is not available in the list click Browse to locate and select an applicable template 3 Click Finish and go to the Plate Setup tab 4 If the template doesn t contain all the information you need complete the plate document as follows Add detectors to the plate document using the open plate alternative method page 2 19 Apply detectors and assign sample name task and quantity to standards unknown samples and NTC wells page 2 20 and page 2 22 Setthermal cycler conditions page 2 24 Note The tasks that you perform at this stage vary according to which settings were defined in the template 5 Save the plate document page 2 25 Note For Save as type select SDS Documents sds 2 28 Quantifiler Duo DNA Quantification Kit User s Manual 04 2008 Part Number 4391294 Rev B Chapter 3 PCR A
27. For the AmpF STR MiniFiler PCR Amplification Kit reactions varying quantities of input DNA including 0 1 0 25 and 0 5 ng were used The electropherograms displayed in Figure 6 12 and Figure 6 14 represent the optimal input DNA amounts at the corresponding inhibitor level For simplicity in the STR panels Figure 6 9 through Figure 6 14 we refer to the inhibited samples using the inhibitor concentration indicated in Table 6 4 in the Sample Name column final concentration in the quantification reaction or 2 uL in a 25 uL reaction volume However the actual final inhibitor concentration in the STR reactions will vary and is determined by the volume of sample used for amplification Results 2 The presence of hematin and humic acid adversely affected the quantification of DNA in a sample due to the inhibition of PCR The quantification results obtained from each sample are summarized in Table 6 4 and indicate that the quantity of DNA was underestimated when the concentrations of hematin and humic acid were increased Hematin and humic acid at concentrations higher than 12 5 uM and 7 5 ng uL respectively completely inhibited the PCR and no DNA was detectable in these samples Quantifiler Duo DNA Quantification Kit User s Manual 6 17 Chapter 6 Experiments and Results Table 6 4 Quantification of inhibited samples using the Quantifiler Duo DNA Quantification Kit Samp
28. NA 40 40 Neisseria gonorrheae NA 40 40 Staphylococcus aureus NA 40 40 Saccharomyces cerevisiae NA 40 40 Candida albicans NA 40 40 Sensitivity Studies Std 2 3 6 6 The Quantifiler Duo DNA Quantification Kit Detected DNA from a human male individual with a Cz value of 27 RPPH1 and 27 9 SRY Detected DNA from a human female individual with a C value of 27 5 RPPH1 Detected DNA from two chimpanzees with relatively lower efficiency producing Cy values of 32 3 and 31 1 for the SRY assay only Did not detect DNA from the remaining non human species Thus the Quantifiler Duo DNA Quantification Kit detected DNA from only one non human species at a significantly reduced efficiency This degree of cross reactivity of primers and probes for higher primates is well documented Sensitivity studies were performed to determine the range of DNA concentrations that can be reliably quantified and detected using the Quantifiler Duo assay Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Experiment Two human male DNA samples obtained from commercial sources Table 6 2 were each diluted to obtain concentrations of 20 0 5 0 1 0 0 1 0 05 0 04 0 03 0 023 0 0115 0 00575 0 002875 and 0 00144 ng uL inT 6E buffer Each dilution was quantified in triplicate using the Quantifiler Duo DNA Quantification Kit For each 25 LL reaction 2 0 uL of DNA sample was used
29. Optical 8 Cap Strip 8 tubes strip Applied Biosystems 125 strips PN 4323032 Table 1 5 Documents Applied Biosystems Document Part Number Real Time PCR Systems Applied Biosystems 4348358E 7900HT Fast Real Time PCR System and 7300 7500 Real Time PCR Systems Rev E 1 18 Quantifiler Duo DNA Quantification Kit User s Manual Chapter 2 software Setup Quantifiler Duo DNA Quantification Kit User s Manual Software Setup This chapter covers OVERVIEW Ld SRM EV RUEDA RR RR pas a Sete and 2 2 Starting the 7500 Real Time PCR System 2 3 About Plate Documents 0 0 cece KK KK KK KK IK 2 6 Setting Up a Plate Document 20 2 0 RR KK KK ee eee 2 8 Setting Up a Plate Document Template 2 26 Quantifiler Duo DNA Quantification Kit User s Manual 2 1 Chapter 2 Software Setup Overview Purpose During software setup you start up the 7500 Real Time PCR System and set up a plate document for DNA quantification using the Quantifiler Duo DNA Quantification Kit Configuration The Quantifiler Duo DNA Quantification Kit is supported using the 7500 Real Time PCR System with SDS Software v1 2 3 for real time data collection and analysis 2 2 Quantifiler Duo DNA Quantification Kit User s Manual Starting the 7500 Real Time PCR System Starting the 7500 Real Time PCR System Overview Starting the 7500 Real Time PCR System involves 1 Starting th
30. each sample the Cy values obtained for the RPPHI target were lower than those obtained for the SRY target because there are two copies of the autosomal human target locus and only one copy of the Y chromosome target locus per genome equivalent The C values did not vary significantly from run to run or from instrument to instrument The data showed that at lower DNA concentrations the standard deviations increased most likely due to stochastic effects Systematic differences between instruments are not expected to affect final sample quantification results when samples and quantification standards are run on the same plate and instrument the C values are affected proportionately 6 52 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Optimization of The quantities of critical reagents in the PCR mix such as primers PCR Reaction probes and IPC template were optimized using the following thermal Conditions cycling conditions Std 2 10 1 amp Hold 50 C 2 min Como GLO Hold 95 C 10 min in Multiplex PCR Cycle 95 C 15 sec 60 C 1 min for 40 cycles Std 2 10 3 The primers and probes for amplification and detection of RPPHI SRY and IPC targets were investigated at concentrations ranging from 50 to 725 nM In a separate experiment copies of the IPC varied from 1 000 to 40 000 copies per assay Contamination Analysis of non template control samples was performed to Std 3 6 demon
31. extremely sensitive and detection of Cy values 735 may indicate the presence of exceedingly low quantities of DNA 3 copies Some user laboratories have reported the detection of Cy values 40 for extraction blank and negative control samples while performing a real time PCR reaction with the previously released Quantifiler Kit assays Detection of such a low quantity of DNA can vary from amplification to amplification based on stochastic effects Such levels may be considered background and may not produce detectable product when the AmpF STR Kits are used The same observation applies to the Quantifiler Duo DNA Quantification Kit Quantifiler Duo DNA Quantification Kit User s Manual Improving Assay Performance The Quantifiler Duo DNA Quantification Kit reagents undergo rigorous quality control to ensure that the reagents are free of extraneous DNA However due to the extreme sensitivity of the test background DNA from the environment can be detected on rare occasions Each laboratory should take standard precautions to minimize contamination in its own facility Each laboratory should also establish a Cy value above which a positive result represents background DNA only In this way samples that are successfully amplified using the AmpF STR Kits can be distinguished from those samples lacking sufficient target to generate an interpretable result Establishing the limits of the test is common practice in forensic labo
32. for amplification of the sample in panel A 0 1 ng of human DNA is used for amplification of samples in panels B C and D 10 uL of the extract is used for amplification of samples in panels E through J Label corresponds to inhibitor concentration in the Quantifiler Duo reaction Figure 6 11 MiniFiler Kit analysis of hematin inhibited samples before dilution of highly inhibited samples Quantifiler Duo DNA Quantification Kit User s Manual 6 23 Chapter 6 Experiments and Results Ta ma PT Pa TE Ca zl Wee ee ee ee AN ST YY oe TE NE ee E 0 uM 0 25 n n H g in A E d ca NAA a E a YY YYULAA sa 2 5 uM 0 1 n ja gt UM U T ng i B 4 8400 initia jn Hp Jatin Sud 6 ng F4 ntin SUNO Ing Mile 65800 vt mm EE TT TE E TE TET ET TET TE TUE YE ETO Na 5 uM 0 1 n n W HM U 1 ng aa C a E i a 7 5 uM 0 1 Ni o UM U T ng M D i st inita n di NG Gn Kai JO I iria 6380 v i a ee ee ee ee ee DE DE ee ear sd 10 uM 1 2 a 0 uM 1 20 PA E ii lu E al 1 07 jai nan NONIE ain ALANO FS GEI WW ES sa 12 5 uM 1 20 imo a Nag f F i Fea AAA NT EUT DD ee TEMERE a 15 uM 1 20 imo QU d AE a E a TT TC TE Ty cc Z977 sad 17 5 uM 1 50 s H QU p m ea aD li E EE TET TE TET ET TE EE ee TET YE TT TE DET EE TY pa 20 uM 1 20 jn pM 1 00707 nie jnhib ip hematin dtu Hes GOB nain AGWN Test mira G5500 31 Tm TE d 40 uM 1 50 n J 4200
33. genetic markers that are used as Std 2 1 2 target regions for quantification of human and male DNA are described in this section Human DNA The quantification of human DNA using the Quantifiler Duo DNA Quantification Kit is based on the amplification of a region from the ribonuclease P RNA component H1 RPPH1 gene Official Symbol RPPHI Name ribonuclease P RNA component H1 Homo sapiens Other Aliases HI RNA HIRNA Chromosome 14 Location 14q11 2 GeneID 85495 Insert the GeneID number into the Search For box on the following linked page http www ncbi nlm nih gov entrez query fcgi CMD search amp D B gene Male DNA The quantification of human male DNA using the Quantifiler Duo DNA Quantification Kit is based on the amplification of a region from the sex determining region Y SRY gene Official Symbol SRY Name sex determining region Y Homo sapiens Other Aliases TDF TDY Other Designations essential protein for sex determination in human males sex determining region protein sex determining region on Y testis determining factor Quantifiler Duo DNA Quantification Kit User s Manual 6 3 Chapter 6 Experiments and Results Detection Std 2 1 3 Species Specificity Std 2 2 Chromosome Y Location Yp11 3 GeneID 6736 The assay maps upstream of the reference sequence mRNA gi 4507224 ref NM 003140 1 Insert the GeneID number into the Search For box o
34. guidelines viii chemical waste hazards ix safety guidelines ix cleavage in 5 nuclease assay 1 5 computer starting for the 7500 Real time PCR System 2 3 contents of kit 1 16 conventions bold text v for describing menu commands v IMPORTANTS vi in this guide v italic text v Notes vi user attention words vi Cy See threshold cycle customer feedback on Applied Biosystems documents xi Index 1 D DANGER description vii data collection 1 8 detectors 7500 SDS adding to plate document 2 16 analysis settings for 5 2 creating 2 10 DNA quantification standards dilution series guidelines for 3 2 guidelines for preparing 3 4 materials required to prepare 3 2 omitting Std 8 5 4 preparing 3 4 reaction recommendation 3 6 See also standards DNA standard curve 1 6 documentation related xi E equipment not included with Quantifiler kits 1 17 exponential phase See geometric phase exporting data analysis results 4 8 F fluorescence detection of 1 7 G geometric phase amplification plot 1 10 guidelines chemical safety viii chemical waste safety ix waste disposal x H hazards biological x chemical waste ix human DNA standard alternate standard curve 1 6 DNA standard curve 1 6 human male genomic DNA 1 6 Index 2 IMPORTANT description vii Information Development department contacting xi instrument powering on for the 7500 Real time PCR System 2 4 Internal PCR Control system See IPC
35. human genomic DNA RPPHI target and male DNA SRY target Based on the results from the SRY male target approximately 1 0 ng of human genomic DNA from each sample was added to a Y filer Kit reaction Results 2 The male DNA was detected and quantified in all mixture samples with as high as a 1 1000 M F ratio using the Quantifiler Duo DNA Quantification Kit Table 6 14 and Figure 6 26 Detection of male DNA as low as 25 pg uL in the presence of 1000 fold excess female DNA demonstrates the robustness and specificity of the Quantifiler Duo DNA Quantification Kit The observed ratio of the male and female DNA varied between 10 to 40 from the expected ratio most likely because of stochastic variations in the PCR Table 6 14 Mixture study 2 ratio and quantification results Male Female Quantity Quantity Male Female ng uL ng uL DNA Ratio 1 0 0 027 0 026 1 0 04 1 50 0 029 1 260 1 42 45 1 100 0 029 2 460 1 83 25 1 200 0 022 6 405 1 288 16 1 500 0 025 13 770 1 545 43 1 800 0 027 24 410 1 896 43 1 1000 0 020 28 210 1 1388 66 0 1 Female 0 016 Quantifiler Duo DNA Quantification Kit User s Manual 6 47 Chapter 6 Experiments and Results Mixture Study Il 100 000 El SRY FAM m RPPH1 VIC 1 0 1 50 1 100 1 200 1 500 1 800 1 1000 0 1 Ratio male female 10 000 1 000 Quantity ng ul 0 100 0 010 Figure 6 26 Mixture study 2 quantifi
36. ng of human DNA is used for amplification of the sample in panel D 0 03 ng of human DNA is used for amplification of the sample in panel E 10 uL of the extract is used for amplification of the samples in panels F G H and I Label corresponds to inhibitor concentration in the Quantifiler Duo reaction Figure 6 13 MiniFiler Kit analysis of humic acid inhibited samples before dilution of highly inhibited samples 6 25 Quantifiler Duo DNA Quantification Kit User s Manual Chapter 6 Experiments and Results CU UN HE E 3000 A O ng uL 0 25 ng N al ae TEN RE ENE on 000707 minitier nhi ip Jummic Ingul 0 Ing DOGS humic Ingul O Ing MiniBle G5500 v1 mm B 1 ng uL 0 1 ng s i 090707 nini inhib flip Jumic 2ngul 01g CU f humic 2ngul 0 ing iniBie G556 v mm C 2 ng LL 0 1 ng s i _ Ll l d _Li 160707_minif ler_inhi _flip_humic_3ngul_l20_D05 f huic 2ngul He20 MiniF ler_G 5500_v1 mm 3000 D 3 ng LL 1 20 i 0707 uita ah fip Poe 3 7Engul N20 Ct lumi 375ngul Jo Mile G5500 vi m m 3 75 ng uL 1 20 7 E 9 NQ UL 1 Ha a FA 7 uL 1 20 ae 5 ng L 1 ka F L tt it 080707 _minifiler inhib fip humic 11 25ngul 1020 G hmc 11 25ngul Ho20 MiniPiler G 500 vl 11 2 uL 1 50 ka 25 ng UL 1 5 2 G a NN KEREMA UN Jf 15 ng LL 1 20 T H a Alul ii ar man EA 30 ng L 1 50 sa n m v Li z 4 l l Note 0 25 ng of hum
37. of The SDS software displays cycle by cycle changes in normalized Reporter Signals reporter signal R The SDS software normalizes each reporter signal by dividing it by the fluorescent signal of the passive reference dye Because the passive reference is one component of the PCR master mix 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 the reporter signal Real Time Data Analysis The 7500 Real Time PCR instrument 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 Figure 1 8 Amplification Plot When using TaqMan probes with the 7500 Real Time PCR Example instrument the fluorescent signal or normalized reporter R increases as the amount of specific amplified product increases Figure 1 8 shows the amplification of PCR product in a plot of R vs cycle number during PCR This amplification plot contains three distinct phases that characterize the progression of the PCR Quantifiler Duo DNA Quantification Kit User s Manual 1 9 Chapter 1 Overview Delta Rn 1 000e 001 1 000e 000 1 000e 001 1 000e 002 1 000e 003 paaa 1 000e 004 1 0006 005 1 000e 006 Phases of
38. on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com www appliedbiosystems com Applied Biosystems 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 2008 Part Number 4391294 Rev B an Applera business
39. phase is termed linear because amplification approximates an arithmetic progression rather than a geometric increase Because amplification efficiency is continually decreasing during the linear phase the amplification curves exhibit low precision Phase 3 Plateau The amplification plot achieves the plateau phase when the PCR stops the R signal remains relatively constant and the template concentration reaches a plateau at about 1077 M Martens and Naes 1989 Quantifiler Duo DNA Quantification Kit User s Manual 1 11 Chapter1 Overview Relationship of Amplified PCR Product to Initial Template Concentration 1 12 Because of the progressive cleavage of TaqMan fluorescent probes during the PCR as the concentration of amplified product increases in a sample so does the R value The following equation describes the relationship of amplified PCR product to initial template during the geometric phase No 5 NG E where N is the concentration of amplified product at any cycle N is the initial concentration of target template E is the efficiency of the system and c is the cycle number For example with the dilutions of RNase P target in the TaqMan RNase P Instrument Verification Plate the ratio of template concentration to detectable signal is preserved in the geometric phase for all dilutions Figure 1 9 As the rate of amplification approaches a plateau the amount of product is no longer proportional to the ini
40. 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 Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 http www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html Additional information about biohazard guidelines is available at http www cdc gov Quantifiler Duo DNA Quantification Kit User s Manual How to Obtain More Information How to Obtain More Information Related Documentation User Bulletin ABI PRISM 7000 Sequence Detection System and Applied Biosystems 7500 Real Time PCR System Instrument Calibration and Maintenance Procedures for Use with Quantifiler DNA Quantification Kits PN 4374416 Rev A User Bulletin Quantifiler Kits Validation Using SDS Software v1 2 3 on the Applied Biosystems 7500 Real Time PCR System and the ABI PRISM 7000 Sequence Detection System PN 4374659 Rev B Real Time PCR Systems Applied Biosystems 7900HT Fast Real Time PCR System and 7300 7500 Real Time PCR Systems Chemistry Guide PN 4348358 Rev E Note For additional documentation see How to Obtain Support below Send Us You
41. product Nonfluorescent quencher 68 Minor groove binder R Reporter P AmpliTaq Gold DNA Polymerase Figure 1 1 Legend for 5 nuclease assay process figures During PCR the TaqMan MGB probe anneals specifically to a complementary sequence between the forward and reverse primer sites Figure 1 2 When the probe is intact Figures 1 2 and 1 3 the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence primarily by F rster type energy transfer Forster 1948 Lakowicz 1983 Forward 42 TaqMan i y Primer MGB probe z a 3 5 5 3 P 5 Reverse Primer Figure 1 2 Polymerization Quantifiler Duo DNA Quantification Kit User s Manual Chemistry Overview Forward z TaqMan Primer MGB probe 5 3 3 5 5 3 apo 5 Reverse Primer Figure 1 3 Strand displacement AmpliTaq Gold DNA polymerase cleaves only probes that are hybridized to the target Figure 1 4 Cleavage separates the reporter dye from the quencher dye resulting in increased fluorescence by the reporter This increase in fluorescence signal occurs only if the target sequence is complementary to the probe and is amplified during PCR Because of these requirements nonspecific amplification is not detected Forward d TaqMan Primer MGB probe 5 3 3 5 5 3 Pc 5 Reverse Primer Figure 1 4 Cleavage Polymerizati
42. properly or the compression pad was not used during the run 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 than unaffected wells and they should correspond with the inconsistent results For subsequent runs make sure that the Optical Adhesive Cover is sealed to the reaction plate properly Delta Rn replicates 4 4 2 3 4 8 67 8 9 1014121244 1516 17 18 19 20 21 22 23 24 25 25 27 28 29 20 31 3232 31 25 36 37 38 3040 Cycle Number AR and C values inconsistent with Incorrect volume of Quantifiler Duo PCR Reaction Mix added to some reactions 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 Duo PCR Reaction Mix should generate significantly different amounts of fluorescence compared to unaffected wells S O q uogeoyiduuy Burjoouse qnoj
43. st St JE 5 S6e 000 E 555e 000 m m mU ju m m m m I 5 56e 000 B 5566 000 m m m 185e00 Biese E sta 5 Std E 6 20 E 620 m mn u m li m m mn E 6 20e Ei 520 m m m With the well s selected select View gt Well Inspector or Ctrl double click to open the Well Inspector dialog box The Well Inspector displays the detectors that were added to the plate document m Well Inspector Well s A3 Sample Name neporer auencner Task Quantity Color v Duo Human none men Duo IPC none Unknown M Duc le none Unknown p Omit Well Passive Reference Add Detector Remove Close Rox E Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document To assign parameters to unknown samples and non template control NTC wells continued 3 Enter the parameters a In the Task column keep the default Unknown for all three detectors b Enter the Sample Name for example Unknown 1 for unknown samples and NTC for NTC wells Note For the Passive Reference select ROX Example Unknown samples m Well Inspector Wells A3 Sample Name Unknown 1 V Duo Human Unknown v Duo IPC Unknown v Duo Male none Xo Unknown A Task for all 3 detectors set to Unknown r Omit Well A Passive Reference Add Detector Remove Close Y Example NTC Wells m Well Inspector Wells A4 Sample Name NTC V Duo Human D
44. target approximately 1 0 ng of human male DNA from each sample was profiled using the Yfiler Kit Figure 6 25 The Yfiler Kit amplifies STR targets on the human Y chromosome only Therefore one would expect a good correlation between the quantification values obtained from the SRY target in the Quantifiler Duo DNA Quantification Kit and the performance of the Yfiler Kit Complete conclusive and consistent male profiles were obtained from all mixture samples investigated 02 stare run mixture 7 ANG fox man Km mm r 400 ao 120 am 140 150 160 m 480 aso E 210 220 230 240 250 ano 310 320 SERT La 1a dla 072407 pictures rerum ixture 06a id bai dh ETR TAW WR TAN 072407 _pistures rerun pnixture 10 D06 fsa tela Top panel Male contributor DNA profile Remaining panels Male female DNA mixtures Figure 6 25 Mixture Study 1 STR analysis using the Yfiler Kit Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Experiment 2 The limit of detection of male DNA in the presence of large excesses of female DNA was studied Mixture samples were prepared containing 25 pg uL of male DNA and increasing quantities of female DNA resulting in M F ratios of 1 0 1 50 1 100 1 200 1 500 1 800 1 1000 and 0 1 The mixture samples were processed in triplicate using the Quantifiler Duo DNA Quantification Kit to determine the concentration of total
45. the Online Help for more information about the Report Settings dialog box Report Settings Report Orientation Portrait Landscape Landscape recommended with 9 or more Data Columns Data Columns r Braph s to Print in the Report Well Number IV Tm v Raw Spectra Amplification Plot Sample Name D Portrait Portrait elector c c Task Landscape Landscape Ct IV Standard Curve Portrait Landscape IV Dissociation Quantity Portrait Mean Qty and StdDev Qty C Landscape Filtered StdDev Ct Show detector results in detector color Additional Data to Print in the Report Show gray white rows IV Document Comments v Analysis Methods tt of White rows F IV Thermal Profile IV Detector Setup HofGrayrows 4 cae Quantifiler Duo DNA Quantification Kit User s Manual 4 7 Chapter 4 Data Analysis and Results Exporting the You can export numeric data into text files which can then be Results imported into spreadsheet applications such as Microsoft Excel To export the results 1 In the analyzed plate document select File gt Export then select the data type to export Sample Setup txt Calibration Data csv Spectra csv Component csv Delta R csv Cr csv Dissociation csv Results 317500 System SDS Software 062007 validation reproducibility 4 sds Absolute Quantification E Tek Insturrent Anaysis W
46. uL when 0 2 units of DNase I was used Similar values were obtained from the SRY target assay 6 28 Quantifiler Duo DNA Quantification Kit User s Manual Table 6 5 Quantifiler Duo DNA Quantification Kit results Developmental Validation Sample DNase pis StdDev Papang StdDev C value StdDev Name Units ng uL SRY ng uL RPPH1 IPC IPC 1 0 7 07 0 08 7 69 0 64 29 61 0 24 2 0 002 5 92 0 20 6 51 0 39 29 60 0 13 3 0 01 4 77 0 06 5 11 0 13 29 67 0 11 4 0 02 3 23 0 10 3 43 0 33 29 75 0 08 5 0 05 0 50 0 04 0 57 0 13 29 77 0 07 6 0 1 0 10 0 01 0 08 0 01 29 90 0 11 7 0 2 0 02 0 01 0 03 0 01 29 83 0 05 Delta Rn Delt ys Cycl Sample 3 Sample 4 a LL Zar J 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 In reasing d g Sample 5 Sample 6 radation Sample 7 Figure 6 16 Degraded DNA Quantifiler Duo RPPH1 amplification plot Quantifiler Duo DNA Quantification Kit User s Manual Samples 1 and 2 6 29 Chapter 6 Experiments and Results Delta Rn vs Cycle Samples 1 and 2 Sample 3 Sample 4 Sample 5 r Sample 6 74 amp Sample 7 Increasing de ira 1 12 3 4 5 6 7 8 9 10 11 12 13 14
47. 0 an 20 20 cm 20 2s0 ano ao xn aao woo sm 2000 lug dq do nG TD MU iid Au 070607 Rainbow validation degraded 3 CO6fs2 degraded 3 Tamiitla VI mm 3 ani 10420 180 ao aso ao 170480 180 2o 20 zo 9 2w 250 200 zo 20 2s0 300 ao s20 330 340350 2009 D 070607 Raimbow validation degraded 4 HOGfsa degraded 4 Heute vt m m 4 agio E AA 20 2 00 2m 29 28 es __ ze 20 20o 280 doo ato ss so su aso 2000 Ha te aay Cees TES ES TER L 4 l a a aasa a Ahaa 070607 Rainbow viden degraded 5_C02 fsa degraded 5 niii v1 m m 5 40 10 120 480 1w 459 460 470 189 490 200 210 220 280 2 28o 20 20 230 290 soo 300 320 39 3w 3s po 2606 a i il i 5 070607 Rainbow yalidatien degraded 6 D02 fsa degraded 6 Tener Vi mm 6 400 no 120 180 1n 150 16o 170 180 1o 2o 20 20 2o 2w 250 280 270 20 2s0 300 30320 330 s a50 1200 eT U pup qme GS 070667 Raimbow validaiien degraded 7_E02 fsa degraded 7 Tdendifiler vi m m T 400 oba ah Aii A a al i 1 Legend The sample numbers to the left of the figure refer to the sample numbers in Table 6 5 Figure 6 18 STR analysis of samples from the degraded DNA series using the Identifiler Kit Based on the DNA quantification results from the RPPH1 human target of the Quantifiler Duo DNA Quantification Kit 0 25 ng of each DNA sample was added to MiniFiler Kit reactions The data is presented in Figure 6 19 Conclusive and comple
48. 1 In the Results tab select the Amplification Plot tab 2 In the Detector drop down list select a detector Duo Human Duo Male PC 3 Select the applicable 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 that reanalysis is needed After reanalysis it changes from red back to green The report displays data for selected wells in tabular form and summarizes the quantity of DNA present in the samples For information about the quantities reported see Assessing Quantity on page 5 19 To view the report 1 In the analyzed plate document select the Results tab then select the Report tab 2 Select the reactions in the 96 well plate representation below the report to display the results in the report Quantifiler Duo DNA Quantification Kit User s Manual Viewing Results To view the report continued 3 View the Qty column to determine the quantity of DNA in each sample Note The values in the Qty column are calculated by interpolation from the standard curve for a given sample Quantities are calculated only if quantification standards were run and set up correctly in the software Otherwise only C values are shown Note Go to Tools gt Report Settings to format the report for printing Refer to
49. 15 16 17 18 18 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Figure 6 17 Degraded DNA Quantifiler Duo SRY amplification plot 1 0 ng of each sample in the degraded DNA series was added to Identifiler Kit reactions based on DNA quantification results obtained using the Quantifiler Duo DNA Quantification Kit RPPHI human target As the extent of degradation increased the concentration of amplifiable DNA decreased resulting in the need to add a higher volume of sample to the subsequent PCR reaction 10 uL of sample was added to the Identifiler Kit reactions for those samples containing 0 1 ng uL DNA or less The peak heights rfu values of the alleles for STR loci with longer amplicons decreased for those degraded samples generated with 0 01 and higher units of DNase I samples 3 to 7 However complete interpretable STR profiles were obtained for samples generated with up to 0 02 units of DNase I samples 1 to 4 Partial STR profiles were obtained for those samples generated with 0 05 and higher units of DNase I samples 5 to 7 Figure 6 18 6 30 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation 070607 Rainbow validation degraded 1 E06 fsx degraded 1 Tdenfif la_v1 mm 1x 1 pa 110 10 m0 tan 150 60 ao de aso 2o 2o mo 2o 2m 250 260 2m mo 28o 300 ao sm EJ a aso 2000 0 070607 Rainbow validam degraded 2 FU6sa degraded deni vi mm 2 p a 0 1o 40 150 am LANE mo 2o 0 2
50. 2526 27 28 2930 31 3233 34 35 3637 38 3 Abnormal AR values or negative AR values Incorrect passive reference was selected when setting up the plate document In the examples Cy5 Cy3 or none was selected as passive reference respectively 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 S O q uoneoduy Burjoouse qnoj Table 5 4 Troubleshooting amplification plots continued Co Observation Possible Cause Recommended Action Incorrect PCR Reaction Mix From the plate document select the standard Delta Rn vs Cycle enue s Jesf NA uoneoynueno YN ONG elalynUeNd 1234598798 9101112191415 1617 1919 20 21 2223 24 2526 27 28 2930 31 3233 34 35 3637 38 39 40 Abnormally low AR values straight amplification plots and low sensitivity across the whole plate em ET TT 1234567 8 9 1011121314151617 1919 2021 2223 24 2526 27 28 2930 31 32 33 34 35 3637 38 39 40 was used to set up the reactions e g Quantifiler Human or Y PCR Reaction Mix instead of Quantifiler Duo PCR Reaction Mix The first example shows the amplification plots of the standard curve wells when the Quantifiler Human or Y PCR Reaction Mix is used The second example shows the amplification plots of the standard curve wells with the expected AR
51. 60 170 den 130 200 210 EJ 230 240 250 280 270 200 3 lul 0 25 Ad Ldu al d bs 71707 minitilr degraded 4 6 25ng_C08 fra a E MiniTile C550 v1 200 210 220 230 240 250 260 270 280 2am A 8 z mrn annam 4 0 25 ng 071707 initi degraded 5 0 25ng_ M03 fsa degraded 5 0 25ng MiniFiler GS500 vi 200 210 220 230 240 250 260 270 200 2400 5 2 0 25 ng 071707_minif ler_degraded_6_0 25ng_GL1 fra E graded anane nine GSS0 v mm den 130 200 210 20 230 240 250 280 270 200 so 240 6 m 0 25 ng e IM L zl u PY Aa 071707 minifiler degraded 7 0ng HO fsa MiniF e G5500 vi 7 Wa 0 1 ng E ahha la Ada acd Legend The sample numbers to the left of the figure refer to the sample numbers in Table 6 5 6 32 Figure 6 19 STR analysis of samples from the degraded DNA Series using the MiniFiler Kit In general these results demonstrate that the Quantifiler Duo DNA Quantification Kit can be used to assess the amount of amplifiable DNA in a degraded sample At extreme levels of degradation it may not be possible to obtain results from the Quantifiler Duo DNA Quantification Kit or the STR kits Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Reproducibility Replicate analysis of human DNA samples was performed using the Study Std 2 5 Quantifiler Duo Kit to assess the reproducibility of the
52. 62 0 28 27 22 0 15 29 56 0 17 1 85 28 26 0 23 28 88 0 18 29 57 0 19 0 62 29 79 0 29 30 44 0 19 29 64 0 19 0 21 31 32 0 34 32 01 0 28 29 66 0 21 0 068 32 83 0 32 33 61 0 40 29 62 0 19 0 023 34 48 0 58 35 33 0 63 29 55 0 18 Figure 6 28 Figure 6 29 and Figure 6 30 provide the human human male and IPC C4 mean values obtained using the Quantifiler Duo DNA Quantification Kit 6 50 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Precision of Human Assay El Instrument 1 E Instrument 2 O Instrument 3 n sen TT TT TE lll 50 16 7 5 56 1 85 0 62 0 21 0 068 0 023 DNA quantity ng ul Figure 6 28 Precision using the Quantifiler Duo DNA Quantification Kit RPPH1 human target at the standard curve concentrations Precision of Male Assay El Instrument 1 m Instrument 2 swe ETT Instrument 3 50 15 7 5 56 1 85 0 62 0 21 0 068 0 023 DNA quantity ng ul Figure 6 29 Precision using the Quantifiler Duo DNA Quantification Kit SRY male target at the standard curve concentrations Quantifiler Duo DNA Quantification Kit User s Manual 6 51 Chapter 6 Experiments and Results Precision of IPC Assay O Instrument 1 E Instrument 2 O Instrument 3 B Mean 50 16 7 5 56 1 85 0 62 0 21 0 068 0 023 DNA quantity ng ul Figure 6 30 Precision using the Quantifiler Duo DNA Quantification Kit IPC target at the standard curve concentrations For
53. A Quantification Kit User s Manual Chapter 4 Data Analysis and Results Analyzing the Plate Document 4 2 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 l To open the plate document for analysis Navigate to the folder where the run file is stored and double click the run file or Launch the software from the shortcut on your desktop Double click the 7500 System Software icon Click File gt Open Then select the run file and click Open or double click the run file Duo samples sds My Recent Documents My Network File name Duo samples sds Places Files of type All SDS Files sds sdm sdt Quantifiler Duo DNA Quantification Kit User s Manual Analyzing the Plate Document To analyze a plate document continued 2 Verify the analysis settings a On the menu bar select Analysis gt Analysis Settings to open the Analysis Settings dialog box b Verify that the settings are as shown below then click OK Analysis Settings Absolute Quantific 3 Ct Analysis Detector Auto Ct Manual Ct Threshold 02000000 a C Automalic Baseline ManualBaseline Start cycle 3 End cycle 15 T Use System OK amp Reanalyze Cancel Apply IMPORTANT If the analysis settings differ from those show
54. C system template DNA is added to the reaction at a fixed concentration therefore the NED C4 should range between 28 and 31 with a variation of 1 C across the standard curve samples 5 13 Chapter 5 Interpretation of Results Negative Results Invalid IPC Results IPC Results Inconclusive PCR Inhibition Determining the Normal Range for IPC Evaluating PCR Inhibition 5 14 No human DNA is detected when No VIC or FAM dye signal is detected indicating that the human and or male specific targets did not amplify NED dye signal Cr NED between 28 and 31 indicates that the IPC target was amplified the PCR was not inhibited If the human and or male specific targets and the IPC target failed to amplify then it is not possible to distinguish between the absence of DNA PCR reaction failure and PCR inhibition With extremely high concentrations of human genomic DNA gt 10 ng uL competition between the human and or male specific and IPC PCR reactions may suppress IPC amplification for that sample If the target amplifies with low C4 and high AR results it is unlikely that PCR inhibitors are present In these cases appearance of suppression or failure of IPC amplification render the IPC result inconclusive Weak amplification high C4 value and low AR value of the human and or male specific targets and no or weak amplification of the IPC may indicate PCR inhibition partial or complete in the sampl
55. Instrument Control Temperature Sect Estimated Time Remaining hh mm Sample Heat Sink Cover Block Cycle ct Status Stage Rep Time mm ss Step State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Reps 1 Reps 1 Reps 40 950 950 10 00 0 15 60 0 50 0 100 2 00 Add Cycle Add Hold Add Step Add Dissociation Stage Delete Help Settings Sample Volume uL IV 9600 Emulatig Data Collection St ge 3 Step 2 60 0 e 00 Select this box Set the volume to 25 uL Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document When you are finished save the plate document and start the run as described in the following section Saving the Plate Before running the reaction plate save the plate document as an SDS Document and Document sds file Starting the Run Note To save the plate document as a template see Setting Up a Plate Document Template on page 2 26 To save the plate document and start the run 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 then Start to start the run Quantifiler Duo DNA Quantification Kit User s Manual 2 25 Chapter 2 Software Setup Setting Up a Plate D
56. Quantifiler Duo DNA Quantification Kit User s Manual Applied Biosystems Copyright 2008 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 U S Patent Appl No 60 890 817 is filed for the Quantifiler Duo DNA Quantification Kit 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 solely in forensic and p
57. Quantifiler Duo 3 0 to 3 6 3 3 The slope values listed in Table 5 1 represent the typical range of slope values observed during the development and validation of the Quantifiler Duo kit Some deviations from this range may be observed due to instrument performance If the slope varies beyond the typical range indicated in Table 5 1 check the following Assay setup Software setup Reagents Instrument Quantifiler Duo DNA Quantification Kit User s Manual Troubleshooting the Standard Curve Troubleshooting the Standard Curve The following table and corresponding sections provide examples of errors that can result by applying incorrect detectors to standards during setup of the plate document For instructions on how to apply detectors for standards correctly see Creating Detectors on page 2 10 The standard curves shown in the following examples represent plots that result from incorrect detector setup and should not be used Table 5 2 Troubleshooting the standard curve Observation Possible Cause Recommended Action Slope for the standard curve is outside the typical range or R value is significantly less than 0 98 The PCR reaction exhibits stochastic effects at the lowest concentration point Omit Standard 8 of the DNA quantification standard 23 pg uL from analysis see the procedure To omit Standard 8 from analysis on page 5 4 When applying detec
58. Quantifiler Duo Kit 95 Mean 95 95 Sample Mean Sampe panon ameny Sigpev Gom Oy Sape Coi pco Sipev Gont ng uL ng yL 96 ng pL A 20 20 10 1 051 10 46 21 15 0 804 7 60 29 70 0 037 0 25 A 10 8 98 0 400 13 36 9 11 0 341 11 49 29 73 0 034 0 23 A 1 0 85 0 109 17 84 0 87 0 051 8 67 29 92 0 034 0 23 A 0 10 0 08 0 007 25 83 0 09 0 015 17 86 29 97 0 039 0 26 A 0 05 0 05 0 028 34 00 0 04 0 002 63 43 30 00 0 032 0 21 B 20 23 09 2 219 19 23 24 36 1 656 13 59 29 79 0 038 0 25 B 10 11 22 0 485 1 45 11 49 0 529 15 11 29 77 0 046 0 31 B 1 1 15 0 142 20 76 1 14 0 083 29 23 29 89 0 030 0 20 B 0 10 0 11 0 013 42 93 0 10 0 008 42 32 29 98 0 040 0 27 B 0 05 0 05 0 015 16 92 0 06 0 007 43 10 29 98 0 012 0 08 C 20 23 11 0 821 710 22 51 0 294 2 61 29 62 0 055 0 37 C 10 9 25 0 601 35 03 8 72 0 562 32 37 29 67 0 053 0 36 C 1 0 89 0 039 10 40 0 82 0 027 5 40 29 81 0 057 0 38 C 0 10 0 11 0 027 10 00 0 10 0 008 23 96 29 89 0 053 0 36 C 0 05 0 04 0 020 68 07 0 04 0 008 90 52 29 81 0 047 0 32 D 20 26 49 2 116 15 98 27 28 1 835 13 45 29 90 0 106 0 71 D 10 13 09 0 596 12 20 13 26 0 261 9 44 29 87 0 028 0 19 D 1 1 26 0 136 8 54 1 22 0 081 16 18 29 76 0 055 0 37 D 0 10 0 12 0 032 43 59 0 12 0 008 28 35 30 02 0 034 0 22 D 0 05 0 07 0 021 14 12 0 06 0 006 22 12 29 97 0 042 0 28 E 20 female 24 91 0 586 4 70 29 97 0 023 0 16 E
59. Real Time PCR instrument on the 7500 Real Time PCR Instrument l Press the tray door to open it 2 Load the plate into the plate holder in the instrument Ensure that the plate is correctly aligned in the holder Quantifiler Duo DNA Quantification Kit User s Manual 3 7 Chapter 3 PCR Amplification To run the plate on the 7500 Real Time PCR instrument continued 3 Load standard 96 well plates with the notched A12 position Well A1 4 Close the tray door 3 8 Quantifiler Duo DNA Quantification Kit User s Manual Running the Reactions To run the plate on the 7500 Real Time PCR instrument continued 5 Apply pressure to the right side of the tray and at an angle to close the tray door Press forward here at slight right angle 6 In the 7500 SDS software open the plate document that you set up for the run 7 Select the Instrument tab then click Start Quantifiler Duo DNA Quantification Kit User s Manual 3 9 Chapter 3 PCR Amplification 3 10 Quantifiler Duo DNA Quantification Kit User s Manual 04 2008 Part Number 4391294 Rev B Chapter 4 Data Analysis and Results Quantifiler Duo DNA Quantification Kit User s Manual Data Analysis and Results 4 This chapter covers Analyzing the Plate Document 00 00 00 eee Viewing Results kk KK KK KK KK KK KK KK KK KK eens Quantifiler Duo DN
60. Standard Curve Duo IPC selected as Standard instead of Duo Human Duo Male Disconnected Legend The Standard Task and Quantity were applied to the Duo IPC detector instead of the Duo Human detector resulting in an incorrect representation of the Duo Human standard curve as a horizontal line Figure 5 1 Example 1 Quantifiler Duo DNA Quantification Kit User s Manual 5 7 Chapter 5 5 8 Interpretation of Results Plate Y Spectra Y Component Y Amplification Plot Y Standard Curve Y Dissociation Y Report Detectr DuolPC N ae emane PAGAN uz R2 0 300013 Duo IPC selected as Standard instead of Duo Human Disconnected Legend The Standard Task and Quantity were applied to the Duo IPC detector instead of the Duo Human detector resulting in an incorrect representation of the Duo Human standard curve AND only the Duo IPC Detector is displayed in the drop down list Note the adjustment of the Y axis scale based on Detector selection Figure 5 2 Example 1 Possible Cause When applying detectors for the standards the Task and Quantity were applied to the Duo IPC detector instead of to the Duo Human detector as shown in Figure 5 3 below m Well Inspector Well s A1 Sample Name Std 1 ssa v Duo Human IC none Unknown Quantity V Duo Ipc NED none Standard V Duo Male FAM none Stanaara EE applied to wrong d
61. To create a plate document template continued Setting Up a Plate Document Template 3 Apply the applicable template settings to the plate document Add detectors to the plate document page 2 15 Apply detectors and assign sample name task and quantity to standards unknown samples and NTC wells page 2 20 and page 2 22 Set thermal cycler conditions page 2 24 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 7500 System gt Templates where X is the hard drive on which the 7500 System 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 Wizard Define Document window see step 2 in Creating a Plate Document from a Template on page 2 28 c For File name enter a name for the template For example enter Duo Template ave the template file in the Templates folder Save in Templates B AQ RNase P Install sdt My Recent Documents 3 Desktop My Documents My Computer My Network File name Places Save as type d Click Save Duo Template SDS Template sat _ Save as sdt template type Quantifiler Duo DNA Quantification Kit User s Manual 2 27
62. aid in determining Ifthe sample contains sufficient human DNA and or human male DNA to proceed with short tandem repeat STR analysis The amount of sample to use in STR analysis applications The relative quantities of human male and female DNA in a sample that can assist in the selection of the applicable STR chemistry If PCR inhibitors are present in a sample that may require additional purification before proceeding to STR analysis Product The Quantifiler Duo DNA Quantification Kit contains all the Description necessary reagents for the amplification detection and quantification of a human specific DNA target and a human male specific DNA target The reagents are designed and optimized for use with the Applied Biosystems 7500 Real Time PCR System and SDS Software v1 2 3 1 2 Quantifiler Duo DNA Quantification Kit User s Manual Chemistry Overview Chemistry Overview Assay Overview The DNA quantification assay combines three 5 nuclease assays A target specific human DNA assay A target specific human male DNA assay An internal PCR control IPC assay Target Specific The target specific assays consist of Assay Components Two primers for amplifying human DNA One TaqMan MGB probe labeled with VIC dye for detecting the amplified human target sequence Two primers for amplifying human male DNA One TagMan MGB probe labeled with FAM dye for detecting the human male amplified target se
63. ale FAM reporter dye detectors increase exponentially and the curves quickly cross the threshold and reach AR values gt 1 as shown clean the work area according to the guidelines described in Preventing PCR Contamination on page 5 23 and repeat the experiment with a new set of reagents G Jej deu2 synsey Jo uoiejeJdieju Improving Assay Performance Preventing PCR Contamination Laboratory PCR assays require special laboratory practices to avoid false Practices to positive amplifications as detailed in Table 5 5 The high sensitivity Minimize False ofthese assays may result in the amplification of a single DNA Positives molecule To minimize false positives due to the presence of amplified material in your work area follow these recommended laboratory practices When possible maintain separate work areas dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Wear a clean lab coat not previously worn while handling amplified PCR products or during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect they are contaminated and before leaving the work area Use positive displacement pipettes or aerosol resistant pipette tips Never bring amplified PCR products into the PCR setup area Open and close all sample tubes and reaction plates carefully Try not to splash o
64. an DNA is used for amplification of the sample in panel A 0 1 ng of human DNA is used for amplification of samples in panels B and C 10 uL of the diluted extract as indicated is used for amplification of samples in panels D through I Label corresponds to inhibitor concentration in the Quantifiler Duo reaction Figure 6 14 MiniFiler Kit analysis of humic acid inhibited samples after dilution of highly inhibited samples 6 26 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Stability Studies Forensic samples may be exposed to environmental conditions that Degraded DNA degrade DNA molecules and reduce amplification efficiency in PCR Studies Std 2 4 reactions Exposure to environmental conditions may cause fragmentation of full length DNA molecules and can reduce the overall concentration of amplifiable DNA Because of such potential occurrences the validation of forensic DNA methods involves studies of the effects of degradation on the amplification and detection of DNA Degraded DNA samples were tested with the Quantifiler Duo DNA Quantification Kit to determine the quantity of amplifiable DNA at increasing levels of degradation Results obtained using the RPPH1 human target of the Quantifiler Duo DNA Quantification Kit were used to calculate DNA input for subsequent STR analysis Experiment A sample of high molecular weight human genomic DNA was used to generate a series of samples with varyin
65. andard 23 pg uL particularly for the male specific standard curve Quantifiler Duo DNA Quantification Kit User s Manual 5 8 Chapter 5 R Value lt 0 98 Slope Interpretation of Results If the R value is lt 0 98 you may choose to omit Std 8 of the DNA quantification standard 23 pg uL from analysis The Quantifiler Duo DNA Quantification Kit assay can quantify 23 pg uL of human genomic DNA in a sample When 2 0 uL of a sample at this concentration is loaded in a reaction the well contains approximately 7 diploid human genome equivalents These equivalents correspond to approximately 14 copies of the Duo Human target locus and approximately 7 copies of the Duo Male target locus Y chromosome loci are haploid Because of stochastic effects when using the lowest concentration point the Cy values are more variable and may affect the closeness of fit between the standard curve regression line and the individual data points of the quantification To omit Standard 8 from analysis 1 Select the wells in the plate document that correspond to Standard 8 and open the Well Inspector 2 Change the Task assignment for the applicable detector from Standard to Unknown 3 Reanalyze the plate to incorporate the change A slope close to 3 3 indicates optimal 100 PCR amplification efficiency Table 5 1 Range and average of standard curve slope values Kit Typical Slope range Average Slope
66. antifiler Duo DNA Quantification Kit User s Manual Safety Safety Alert Words Chemical Hazard Warning Safety Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicatesinformation that is necessary for proper instrument operation accurate chemistry kit use or safe use ofa chemical N iz Ye Indicates a potentially hazardous situation that 1f not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices P WAGNINe Indicates a potentially hazardous situation that f not avoided could result in death or serious injury bied Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations pa T Zan CHEMICAL HAZARD Some of the chemicals used with Applied Biosystems instruments and protocols are potentially hazardous and can cause injury illness or death Quantifiler Duo DNA Quantification Kit User s Manual vii Preface Chemical Safety To minimize the hazards of chemicals Guidelines Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work wi
67. antifiler Duo DNA Quantification Kit User s Manual Bibliography 3 Bibliography 4 Quantifiler Duo DNA Quantification Kit User s Manual Index Numerics 5 nuclease assay 1 3 1 4 7500 Real time PCR System fluorescence detection on 1 7 1 8 PCR instrument 1 9 reactions running on 3 7 starting 2 3 supported configuration 2 2 target nucleic acid relative quantity of 1 9 7500 SDS analysis settings checking 5 2 baseline settings 4 3 detectors creating 2 10 detectors settings 5 2 fluorescence emission data 1 7 New Document dialog box 2 9 plate document analyzing 4 2 product registration 2 5 results viewing 4 4 template setting up 2 26 threshold settings 4 3 9600 Emulation box selection on the 7500 SDS 2 24 A amplification plot about 1 9 example 1 9 inconsistent replicates example of 5 15 jagged plot example of 5 16 phases of 1 10 troubleshooting 5 15 undefined plots example of 5 16 viewing 4 6 analysis settings checking on the 7500 SDS 5 2 verifying on the 7500 SDS 4 3 Quantifiler Duo DNA Quantification Kit User s Manual Applied Biosystems contacting xi customer feedback on documentation xi Information Development department xi Services and Support xi Technical Support xi B baseline about 1 13 settings for the 7500 SDS 4 3 biohazard warning x biohazardous waste handling x biological hazard safety See biohazard warning bold text whentouse v C CAUTION description vii chemical safety
68. aternity 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 Applera Applied Biosystems AB Design ABI PRISM AmpFLSTR Blood Prep GeneMapper Identifiler MicroAmp Quantifiler VIC and Yfiler are registered trademarks and FAM JOE MiniFiler NED ROX and TAMRA 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 4391294 Rev B 04 2008 Contents Preface Chapter 1 Chapter 2 Chapter 3 How to Use This Guide kk kk kK kK KK KK KI KI KK KK KI KK KK KI KIR eens V oafely ce et SCR eet enini uiia rese t a bane deyek e bA hand HANG vii How to Obtain More Information kk kk KK KK RR RR ees Xi How to Obtain Support kk kk KK KK KK KK KK KK esl xi Overview Pfrod ct OVervi W lt ax k sk BI bee daka WEN KE Re NAG 1 2 Chemistry Overview
69. ation Kit User s Manual Starting the 7500 Real Time PCR System Starting the 7500 Select Start gt 7500 System gt 7500 System Software SDS Software The first time you use the software the Product Registration window displays and you are prompted to register the product Product Registration Relative Quantification Study Your Name Organization Registration Code OK Cancel Ok Enter your name organization registration code then click OK On subsequent start ups if the Product Registration window displays click Cancel The software starts and displays the word Disconnected in the status bar on the bottom right corner The status changes to Connected only after the New Document Wizard is completed the software is initialized and the software is connected to the 7500 instrument If the connection is successful the software displays was in the status bar on the bottom right corner Quantifiler Duo DNA Quantification Kit User s Manual 2 5 Chapter 2 Software Setup About Plate Documents How Plate Running a reaction plate on the 7500 system requires creating and Documents Are setting up a plate document using the 7500 SDS software A plate Used document is a representation of the arrangement of samples standards and unknowns and reagents on the reaction plate The 7500 SDS software uses the plate document to Coordinate the instrument operation such as thermal cycling and data co
70. c acid were diluted at 1 20 or 1 50 conclusive interpretable profiles were provided when amplified using the MiniFiler Kit Figure 6 12 and Figure 6 14 As previously described these concentrations correspond to the final inhibitor concentration in the quantification reaction and not to the actual final concentration in the STR reaction 6 22 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation 00070 sinite hib nhie conirl 0 25mg AGL T inhibir cenirol 0 25mg Mia 65500 vl BI 1 E a E am ao dm am 10 Ll Lj m Ll Ll 200 20 zm zm za 20 Ej Ed 2m a40 0 uM 0 25 ng gt A 0707 minister nhi i Juni 2 Sul 01g Ge nanatin 2 SUMLO Ing MiniBle G5500 91 mm 2 5 uM 0 1 ng B a sm D 070 iit jb t natn LO Ing 064 hematin Su t ng Mimina GS v mm n E ano ao am Lj tao Lj Lj m 190 200 20 zm 20 240 280 aw 20 200 2a 5 uM 0 1 ng k C g am 0 00070 inia inhib hematin 7 SUNO Ing D01 fsa hematin_7 5uM_0 lng_ MiniBla C5600 vi mm E 7 5 uM 0 1 ng x 3 10 WM 10pL E AA a E j 12 5 uM 10 uL e F z li i SST 15 uM 10 uL es G 2009 N A HE Til 17 5 uM 10 uL H a GS E 20 uM 10 uL 2000 A kal ENS 071207 Minifsle validation hematin 4buM C05fsa hemalin 4buM a 0H 40 uM 10 uL J zaa Note 0 25 ng of human DNA is used
71. cation graph Based on the results from the SRY target the maximum allowed volume 10 uL of each mixture sample was profiled using the Yfiler Kit Figure 6 27 Complete conclusive and consistent male profiles were obtained from all mixture samples investigated The results demonstrate the utility of the Quantifiler Duo DNA Quantification Kit in the analysis of mixture samples 6 48 Quantifiler Duo DNA Quantification Kit User s Manual 1 0 1 50 1 100 1 200 1 500 1 800 1 1000 Developmental Validation 072407 mistures rerum Mixture 13 He0 CO3fsa Mixture 13 He0 Yes 2 mm E 8 poh Hl 4x hog 4 Nf j 072407 sisctures xerun J14_He50_B03 fsa Mixture 14 Mes TT waa v2 E a UI e ee li NUN 2 W 072407 mixtures erum Mixture 15 Holt Afjfse Mixture 16 t0100 wman m m CEN IA EM SL SE TEL NE TEN S NN 072407 mixtures rerum M iure l6 lie200 H04fsa M xiure 16 Hie200 Yav a u a B BERE Oe ET pi L i Pa M BE a SNN SEB SE SE GER ki ma EE a la da AL i i sak 5 a 5 Hai o WA B xl Top panel Male contributor DNA profile Remaining panels Male female DNA mixtures Precision and Quantifiler Duo DNA Quantification Kit User s Manual Accuracy Std 2 9 Figure 6 27 Mixture study 2 STR analysis using the Yfiler Kit The precision and accuracy of the Quantifiler Duo Kit was assessed by replicate standard curve analysis
72. d The concentrations described here are final concentrations of respective inhibitor in 25 uL PCR when 2 uL of sample is added 2 uL of each sample containing approximately 1 0 ng of DNA was quantified in triplicate using the Quantifiler Duo DNA Quantification Kit The same amount of each replicate sample was subsequently added to AmpF STR Identifiler PCR Amplification Kit reactions in order to have the same final concentration of inhibitor in both the quantification and the STR reaction The STR reactions were analyzed on an Applied Biosystems 3130x Genetic Analyzer instrument Data were analyzed using GeneMapper JD Software v3 2 1 Results 1 The histograms Figure 6 3 and Figure 6 4 illustrate higher C values as the concentrations of hematin and humic acid increased C4 values were relatively stable up to 7 5 uM hematin and 3 0 ng uL humic acid with results displaying more pronounced C shifts at higher concentrations As the concentrations of hematin and humic acid increased the PCR efficiency in the Quantifiler Duo DNA Quantification Kit decreased Complete inhibition of the amplification occurred at 15 0 uM hematin and 11 25 ng uL humic acid In general the C values for all targets human human male and IPC were affected similarly using comparable concentrations of inhibitors see Figure 6 3 and Figure 6 4 Quantifiler Duo DNA Quantification Kit User s Manual 6 11 Chapter 6 Experiments and Results Inhibitor He
73. d to demonstrate the performance of the Quantifiler Duo kit when used to quantify samples subjected to environmental and chemical insults such as those samples containing PCR inhibitors and degraded DNA Forensic DNA extracts commonly contain compounds that inhibit the amplification of nucleic acids These PCR inhibitors can interfere with the reaction and cause varying levels of reduced PCR efficiency including complete inhibition of PCR A wide variety of compounds that may inhibit PCR have been reported 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 hematin is co extracted and purified with the DNA The presence of hematin in DNA samples may interfere with PCR by inhibiting DNA polymerase activity Humic acid is yet another example of a PCR inhibitor that has been found in forensic type samples contaminated with soil Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Experiment 1 Human genomic DNA was mixed with varying concentrations of hematin and humic acid to assess the impact of inhibitors on both the Quantifiler Duo reactions and the subsequent STR reactions performed using the AmpF STR Identifiler Kit Hematin concentrations of 0 2 5 5 0 7 5 10 12 5 15 17 5 20 and 40 uM and humic acid concentrations of 0 1 0 2 0 3 0 3 75 7 5 11 25 15 and 30 ng uLwere evaluate
74. dd Ingul 07060 Juinbew validtien mi dd 2ngul A04 ss humic add ngu 070607 Ruinbew validation Jum scd 3 qul BOA unie acd 2 qul ul aut Had uL Luh aeo Hoo f se wo se M mo wo o om 20 29 29 AA E 2000 eme D aeo HO ta no te 180 16o tO o go 20 0 22 2w 20 280 ze 20 za zw 2o a0 ao ao M0 ao EA 2000 aoo D avo HO PU ore wo ts ts AE EE 300 3o X G0 0 e 0 09 D AA 070607 Rainbow validation humic add 3 7Engul C04 humic acid 3 T ngul 070607 Rainbow validation namic add 75 ngul D04 humi add 7 5 mgl 07060 Juinbew validtin Tum add 25 qul mc acd 25 ul 070607 Rainbow validation Tum dd I5ngul F4 fs humi add L ngut 070607 Rainbow validation Inanit add 30 nqul A10 humi add 20 ngul axo SO GP te te ts Hb Roo te doo zm z ze 29 20b es 29 c6 o 280 Er E pr 000 D a e E E A E ts te tr te t E E E E E E 300 Go za sese se sese E E 2000 4000 D aeo MO t0 1a t0 4s teo tO teo too 20 20 22o 2w ze 280 28o 20 za 28o soo M0 so so sa so a0 amo 4000 D aeo MO e no ta 180 too AO wo oo om 20 ze 29 2 280 x0 2o ch 28 soo 20 sao sao sw aso 000 2000 4000 D aeo HD ta te te do go moo do doo om z ze 29 20 207 28 Z0 Ze ze __ se 900 X e eo E 2000 4006 D a4 Figure 6 10 Identifiler Kit profiles obtained for sampl
75. e To determine the normal range of Cy values for the IPC view the NED dye signal in the amplification plots for the quantification standards If the assays were set up correctly the reactions should show normal IPC amplification across a broad range of input DNA that is NED C4 which falls between 28 and 31 with a variation of approximately 1 C4 across the standard curve 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 analysis of the sample Repeat the DNA extraction from the sample Perform additional cleanup of the sample to remove potential inhibitors Quantifiler Duo DNA Quantification Kit User s Manual jenueyy s Jesf 4y uoneoygueno YN ong g e llnueno G1 G Troubleshooting Amplification Plots Table 5 4 Troubleshooting amplification plots Observation Possible Cause Recommended Action Deta Re E 1011121314 replicates 5 161718 192021 Cycle Number AR and C values inconsistent with 28283091 32 33 34 35 36 37 38 39 40 Evaporation of reaction mixture from some wells because the Optical Adhesive Cover was not sealed to the reaction plate
76. e Computer page 2 3 2 Powering On the Instrument page 2 3 3 Starting the 7500 SDS Software page 2 5 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 if applicable 4 If required type your password in the Password field Powering On the Note Wait for the computer to finish starting up before powering on Instrument the 7500 instrument YN anG PHYSICAL INJURY HAZARD Moving parts can crush and cut Keep hands clear of moving parts while operating Disconnect power before servicing the instrument Quantifiler Duo DNA Quantification Kit User s Manual 2 3 Chapter 2 Software Setup Press the power button on the lower right front of the 7500 instrument The indicator lights on the lower left of the front panel cycle through a power on sequence When the green power indicator is lit not flashing communication is established between the computer and the instrument If the green power on indicator is flashing or the red error indicator is lit see the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Installation and Maintenance Guide PN 4347828 Power button Indicator lights Quantifiler Duo DNA Quantific
77. e Plate window select wells and detectors to apply e Ctrl click all wells containing quantification standards unknown samples and Non Template Control shown in color Check all three detectors listed in the Use column Duo Human Duo Male and Duo IPC IMPORTANT Apply all three detectors to the plate document for all the wells that contain Quantifiler Duo DNA Quantification Kit assay reagents New Document Wizard Set Up Sample Plate Setup the sample plate with tasks quantities and detectors v Detector Reporter Queneher Task Quantity M Duo Human VIC none Unknown Duo Pc NED none Unknown M Duo Male FAM none Unknown Check all 3 detectors listed All wells are selected 5 Click Finish in the New Document Wizard Set Up Sample Plate window to complete the New Document Wizard and to be automatically directed to the Plate Setup tab The 7500 System Software initializes and connects to the 7500 instrument If the connection is successful Bennesied displays in the bottom right corner status bar Quantifiler Duo DNA Quantification Kit User s Manual 2 17 Chapter 2 Software Setup When you are finished in the 7500 System Software Plate Setup tab Figure 2 1 assign sample names tasks and quantities to standards as necessary see page 2 20 for procedure 00 System SDS Software Plate1 Absolute Quantification
78. e Quantifiler Duo DNA Quantification Kit Measured Measured ng pL ng uL 1 20 18 500 19 540 1 5 4 000 4 330 1 1 0 832 0 909 1 0 1 0 099 0 111 1 0 05 0 050 0 048 1 0 04 0 039 0 053 1 0 03 0 026 0 033 1 0 023 0 020 0 022 1 0 01150 0 014 0 009 1 0 00575 0 010 0 007 1 0 00288 0 000 1 0 00144 0 006 2 20 20 910 20 383 2 5 4 943 4 800 2 1 0 802 0 751 2 0 1 0 096 0 108 2 0 05 0 056 0 058 2 0 04 0 038 0 039 2 0 03 0 038 0 031 2 0 023 0 022 0 033 2 0 01150 0 015 0 016 2 0 00575 0 010 2 0 00288 0 006 0 007 2 0 00144 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Sensitivity SRY Target gt eo ao D Sample 1 lt Sample 2 a eo N a SRY Cr FAM o o 28 0 010 0 100 1 000 10 000 100 000 DNA Quantity ngtul Figure 6 1 Sensitivity for the male target using the Quantifiler Duo DNA Quantification Kit Quantifiler Duo DNA Quantification Kit User s Manual 6 9 Chapter 6 Experiments and Results Stability Studies Inhibited Sample Studies Std 2 4 6 10 Sensitivity RPPH1 Target 36 34 RPPHI C VIC J e 26 24 0 010 Sample 1 Sample 2 0 100 1 000 10 000 100 00 DNA Quantity ng ul Figure 6 2 Sensitivity for the human target using the Quantifiler Duo DNA Quantification Kit The stability studies were conducte
79. e ratios of male and female DNA in the mixture samples derived from the quantification results correlated well with the expected ratios Table 6 13 Mixture Study 1 ratio and quantification results Expected SRY RPPH1 Measured Male Female Quantity Quantity Male Female DNA Ratio ng pL ng pL DNA Ratio 1 0 0 228 0 236 1 0 04 1 1 0 229 0 507 1 1 21 1 5 0 240 1 410 1 4 88 1 10 0 280 3 030 1 9 82 1 20 0 235 4 070 1 16 32 0 1 Female 0 217 Quantifiler Duo DNA Quantification Kit User s Manual 6 43 Chapter 6 Experiments and Results 6 44 Mixture Study 10 000 El SRY FAM m RPPH1 VIC 1 000 Quantity ng ul 0 100 1 0 1 1 1 10 1 20 0 1 1 5 Ratio male female Figure 6 23 Mixture Study 1 Quantification values obtained fora constant amount of male DNA in a background of increasing female DNA Based on the results from the RPPHI target approximately 1 0 ng of human genomic DNA from each sample was analyzed using the Identifiler Kit Figure 6 24 As expected the peak height of male alleles decreased with increases in the ratio of female to male DNA reflecting the reducing amount of male DNA present in each sample Interpretation of the minor male profile in such mixture samples was challenging due to the occurrence of shared alleles minor male alleles at stutter positions of female alleles and dropout of minor alleles Alleles from the minor male contrib
80. e standard thoroughly c Repeat steps 4a and 4b until you complete the dilution series 3 4 Quantifiler Duo DNA Quantification Kit User s Manual Preparing the Reactions Preparing the Reactions Required Materials Quantifiler Duo Primer Mix Quantifiler Duo PCR Reaction Mix 10 mL polypropylene tube 96 well reaction plate Extracted DNA samples DNA quantification standards dilutions series Optical adhesive cover Preparing the While preparing the reactions keep the 96 well reaction plate in its Reactions base and do not place it directly on the bench top to protect it from scratches and particulate matter To prepare the reactions 1 Calculate the volume of each component needed to prepare the reactions using the table below Volume Per Component Reaction pL Quantifiler Duo Primer Mix 10 5 Quantifiler Duo PCR Reaction Mix 12 5 Note Include additional reactions in your calculations to provide excess volume for the loss that occurs during reagent transfers N eue CHEMICAL HAZARD Quantifiler Duo PCR Reaction Mix may cause eye and skin irritation Exposure may cause discomfort if swallowed or inhaled Read the MSDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Quantifiler Duo DNA Quantification Kit User s Manual 3 5 Chapter 3 PCR Amplification To prepare the reactions continued 2 Pre
81. eal Time PCR System Quantifiler Duo DNA Quantification Kit User s Manual 1 7 Chapter 1 Overview SDS Software Overview The Applied Biosystems 7500 Real Time PCR System is calibrated with several dyes including FAM SYBR Green JOE VIC TAMRA NED CY3 ROX TEXAS RED and CYS The 7500 system uses the data obtained from the pure dye calibration to distinguish the individual contribution of each dye in the collective fluorescence as gathered by the instrument during a run After each run the SDS software receives raw spectra signal data for each reading To make sense of the raw data the software determines the contribution of each fluorescent dye used in the sample by comparing the raw spectra data to a set of pure dye standards contained in the pure spectra file When a plate is saved after analysis the software stores the pure spectra information with the collected fluorescent data for that experiment Figure 1 7 shows the emission spectrum for each dye and the filters and wavelengths at which each dye is read sps 7500 7500 Fast System A System 7300 System JIL Filters Spectrum nm Emission Spectra Pure Dyes 520 nm 550 nm 580nm 610nm 670 nm FAM JOE TAMRA ROX CY5 SYBR Green VIC NED TEXAS RED CY3 Figure 1 7 Example of an emission spectrum 1 8 Quantifiler Duo DNA Quantification Kit User s Manual Real Time Data Analysis Normalization
82. either the Quantifiler Human or Quantifiler Y Human Male DNA Quantification Kit The differences in the quantities of DNA obtained in the present experiment may be due to one or more of the following The difference in the amplification targets used for quantification of human DNA RPPHI in the Quantifiler Duo DNA Quantification Kit and hTERT in the Quantifiler Human DNA Quantification Kit The difference in the sizes of the human DNA targets 140 bases in the Quantifiler Duo DNA Quantification Kit and 62 bases in the Quantifiler Human DNA Quantification Kit The difference in the sizes of the male DNA targets 130 bases in the Quantifiler Duo DNA Quantification Kit and 64 bases in the Quantifiler Y Human Male DNA Quantification Kit though in both kits the male target is SRY gene The differences in assay complexity the Quantifiler Duo DNA Quantification Kit is a triplex PCR assay and the Quantifiler Human and Quantifiler Y Human Male DNA Quantification Kits are duplex PCR assays Differences in the quantification standards used in the respective kits human male genomic DNA in the Quantifiler Duo DNA Quantification Kit cell line DNA in the Quantifiler Human and Quantifiler Y Human Male DNA Quantification Kits Differences in the optimized reaction mix each PCR reaction mix is optimized to deliver the expected performance for a given kit Applied Biosystems recommends that laboratories deter
83. ence standard DNA sample Quantification results may vary based on the method used e g UV absorbance binding of intercalating dyes hybridization PCR and so on Among the different PCR based quantification methods the quantity of DNA obtained for any given sample may vary due to different aspects of PCR see Results section below The ultimate goal of the DNA quantification step in the STR analysis workflow is not to obtain an absolute value but to determine the volume of the DNA extract to be used for amplification to produce high quality STR genotyping results Experiment Four male genomic DNA samples A through D and one female genomic DNA sample sample E at 20 0 10 0 1 0 0 1 and 0 05 ng uL were quantified in triplicate using the Quantifiler Duo Quantifiler Human and Quantifiler Y Human Male Quantification Kits 6 54 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Results All male samples provided quantification results for both human and male targets using the Quantifiler Duo Quantifiler Human and Quantifiler Y Human Male DNA Quantification Kits Table 6 16 No detectable male target signal was obtained for the female DNA sample at any concentration using the Quantifiler Duo and Quantifiler Y Human Male DNA Quantification Kits The quantities of human and male DNA obtained from the Quantifiler Duo DNA Quantification Kit were similar to the quantities obtained using
84. ent Both methods are explained below Quantifiler Duo DNA Quantification Kit User s Manual 2 15 Chapter 2 Software Setup To add detectors to the plate document 1 In the New Document Wizard Select Detectors window Ctrl click the Duo Human Duo Male and Duo IPC detectors If the detectors for the Duo DNA Quantification Kit are not listed in the Detector Name field see the Creating Detectors section page 2 10 New Document Wizard x Select Detectors Select the detectors you will be using in the document Find a z Passive Reference ROX x Detectors in Document Duo Human Duo IPC none Remove NED none Remove FAM none v New Detector lt Back Next gt Finish Cancel 2 Select ROX as the Passive Reference and click Add to add the detectors to the Detectors in Document list New Document Wizard Select Detectors Select the detectors you will be using in the document Find aj Passive Reference BOX Select ROX Detectors in Document Duo H Due Pe Add the xs detectors to non the Plate FAM none Document gt New Detector lt Back Nexo Finish Cancel 3 Click Next to continue to the Set Up Sample Plate window 2 16 Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document To add detectors to the plate document continued 4 In the Set Up Sampl
85. ere tested annotated as A and B Most of the reactions utilized 5 0 ng of input DNA For a few reactions 10 ng of input DNA was used For the two human samples 5 0 ng of DNA was used in the amplification reactions Quantifiler Duo DNA Quantification Kit User s Manual Table 6 1 Specificity with a non human DNA panel Results Developmental Validation The two human control samples that were tested produced the expected results as shown in Table 6 1 Organism Sex RPPHI SHY Average C Average C Orangutan Unknown 40 40 Chimpanzee A Unknown 40 32 3 Chimpanzee B Unknown 40 31 1 Gorilla A Unknown 40 40 Gorilla B Unknown 40 40 Macaque Unknown 40 40 Dog Unknown 40 40 Cow Unknown 40 40 Pig Unknown 40 40 Cat Unknown 40 40 Horse Unknown 40 40 Sheep Unknown 40 40 Chicken Unknown 40 40 Fish Unknown 40 40 Rabbit Unknown 40 40 Mouse Unknown 40 40 Rat Unknown 40 40 Hamster Unknown 40 40 Human Male Male 27 27 9 Human Female Female 27 5 40 Dog Male 40 40 Bovine Male 40 40 Pig Male 40 40 Horse Male 40 40 Sheep Male 40 40 Chicken Male 40 40 Quantifiler Duo DNA Quantification Kit User s Manual 6 5 Chapter 6 Experiments and Results Table 6 1 Specificity with a non human DNA panel continued Rabbit Male 40 40 Mouse Male 40 40 Rat Male 40 40 Escherichia coli NA 40 40 Pseudomonas aeruginosa
86. es containing a range of humic acid concentrations Label corresponds to inhibitor concentration in the Quantifiler Duo reaction 6 21 Chapter 6 Experiments and Results The MiniFiler M kit is designed to obtain STR profiles from compromised samples such as those which may be inhibited and or degraded The results of the STR analysis using the MiniFiler Kit with varying DNA input amounts are presented in Figure 6 11 Figure 6 12 Figure 6 13 and Figure 6 14 Control samples that did not contain any inhibitor provided complete profiles when using 0 25 ng of template DNA for amplification Inhibited samples labeled as 2 5 uM 5 0 uM 7 5 uM hematin and 1 ng uL and 2 ng uL humic acid provided complete profiles when using 0 1 ng of DNA template as quantified by the Quantifiler Duo DNA Quantification Kit Using 10 uL of all the other samples because of low or no quantification results resulted in either partial or uninterpretable results see Figure 6 11 panels E J and Figure 6 13 panels F I These results are consistent with observations of an IPC Cy shift during quantification using the Quantifiler Duo kit Every PCR system e g Quantifiler Duo Identifiler and MiniFiler Kits has a unique reagent formulation that provides a varying response to samples containing inhibitors When samples labeled as 10 uM 12 5 uM 15 uM 17 5 uM 20 uM 40 uM hematin and 3 ng L 3 75 uL 7 5 ng uL 11 25 ng uL 15 ng uL 30 ng uL humi
87. etector E Omit Well Passive Reference Add Detector Remove Close ROX X Figure 5 3 Example 1 Possible Cause Quantifiler Duo DNA Quantification Kit User s Manual Troubleshooting the Standard Curve Example 2 Observation One point for one detector either Duo Human or Duo Male lies outside of the standard curve In Figure 5 4 below Duo Human is the affected detector Setup Y Instrument Y Plate Y Spectra Y Component Y Amplification Plot Y Standard Curve Y Dissociation Y Report Standard Curve 36 Detector FIBI Outlier Ct sady Legend One point lies outside the Duo Human standard curve Figure 5 4 Example 2 Possible Cause When applying detectors for the standards the incorrect Quantity was entered As noted in Figure 5 5 below 0 062 was entered for the Quantity instead of 0 62 Quantifiler Duo DNA Quantification Kit User s Manual 5 9 Chapter 5 Interpretation of Results m Well Inspector Well s E2 Sample Name E td5 _Use Detector Reporter Quencher Task Quantity Color Incorrect v Duo Human VIC none Standard 0 062 Quanti ty Duo IPC none Unknown V Duo Male none Standard J entered for standard r mit Well Passive Reference Add Detector Remove Close ROX Figure 5 5 Example 2 Possible Cause Example 3 Observation At each concentration only one standard curve is shown either for the Duo H
88. etup Assigning Sample Name Task and Quantity to Standards 2 20 IMPORTANT Assign Sample Name Task and Quantity parameters for each quantity separately For example assign the parameters for quantification standard 1 and then for quantification standard 2 and so on until you finish assigning the parameters for all wells containing quantification standards To apply parameters to quantification standards 1 In the Plate Setup tab select wells that correspond to a specific quantification standard for the Duo DNA Quantification Kit e g Std 1 ER Isa e Bl E El I 7 setup V instrument Y Resurs ra Plate V Ew de Selected wells m m m m u m m m m u m m m u lu m o lu lu u w u u 2 With the wells selected click View gt Well Inspector or Ctrl double click the wells to open the Well Inspector dialog box Note The Well Inspector displays the detectors that were added to the plate document m Well Inspector Duo Human none Unknown Duo IPC none Unknown Duo Male none Unknown Omit Well Passive Reference Add Detector ROX x Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document To apply parameters to quantification standards continued 3 Complete the fields in the Well Inspector dialog box a For the Duo Human and Duo Male detectors click Unknown in the Task column then select Standard from t
89. exporting 7500 SDS 4 8 printing 7500 SDS 4 8 R R value interpreting 5 3 viewing 7500 SDS 4 4 Quantifiler Duo DNA Quantification Kit User s Manual radioactive waste handling x raw data printing 7500 SDS 4 8 reactions examples of arranging 2 7 real time data analysis 1 9 regression line formula 5 3 report viewing on the 7500 SDS 4 6 reporter signal normalized about 1 9 viewing in amplification plot 7500 SDS 4 5 results viewing 7500 SDS 4 4 R See reporter signal normalized ROX selecting in Well Inspector 7500 SDS 2 21 S safety biological hazards chemical waste ix safety alert words CAUTIONS vii DANGERS vii description vii IMPORTANTS vii WARNINGS vii SDS document See sds file sds file description 7500 SDS 2 6 saving 7500 SDS 2 25 SDS software starting for 7500 Real time PCR System 2 5 SDS template See sdt file sdt file description 7500 SDS 2 6 saving 7500 SDS 2 27 See also template Services and Support obtaining xi slope of standard curve Index 3 about 5 3 interpreting 5 4 outside typical range 5 5 viewing 7500 SDS 4 4 standard curve about results 5 3 differences in Cy values of replicates 5 10 interpreting 5 3 outlier in example of 5 9 print setup 7500 SDS 4 8 replicates example of four 5 10 straight horizontal line 5 6 troubleshooting 5 5 viewing 7500 SDS 4 4 standards applying detectors for 7500 SDS 2 20 See also DNA quantification sta
90. g levels of degradation lug of DNA 100 uL reaction at 10 ng uL concentration was treated for 20 minutes using increasing quantities of the DNase I enzyme 0 002 0 01 0 02 0 05 0 1 and 0 2 units Samples were run on a 4 agarose gel for 25 minutes and visualized by staining with ethidium bromide to estimate the extent of degradation The degraded DNA samples were quantified with the Quantifiler Duo DNA Quantification Kit Results Agarose gel electrophoresis showed that DNase I treatment produced DNA fragments with sizes of 100 basepairs bp or less when 0 1 unit of DNase was used Figure 6 15 Quantifiler Duo DNA Quantification Kit User s Manual 6 27 Chapter 6 Experiments and Results 1 2 34 5 67 8 2652 bp 350 bp 200 bp 150 bp 100 bp 50 bp Legend Lane 1 O units of DNAse l Lane 2 0 002 units of DNAse l Lane 3 0 01 units of DNAse l Lane 4 0 02 units of DNAse Lane 5 0 05 units of DNase Lane 6 0 1 units of DNase Lane 7 0 2 units of DNase Lane 8 Size standards Figure 6 15 DNase degradation of human genomic DNA Lower amounts of amplifiable DNA were detected using the Quantifiler Duo DNA Quantification Kit for those samples treated with higher amounts of DNase I According to results from the Quantifiler Duo DNA Quantification Kit RPPHI target the amount of amplifiable DNA decreased from about 7 69 ng uL to 3 43 ng uL when 0 02 units of DNase I was used and to 0 03 ng
91. h In the Keyword Search field enter the chemical name product name MSDS part number or other information that appears in the MSDS of interest Select the language of your choice then click Search Quantifiler Duo DNA Quantification Kit User s Manual 3 Safety Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Note For the MSDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical Waste n Hazard MSc 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 To minimize the hazards of chemical waste Safety Guidelines 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
92. he drop down list b For the Duo Human and Duo Male detectors select the Quantity field and enter the appropriate quantity of DNA in the well expressed in ng uL for both detectors e g enter 50 for Std 1 for both Duo Human and Duo Male detectors c Enter the Sample Name e g Std 1 Std 2 and so on d For the Duo IPC detector keep the default Task as Unknown Note Make sure that ROX is selected as Passive Reference m Well Inspector Wells A1 A2 Sample Name Btd 1 Sample Name Task and Duo Male AM none Standard 50 Quantity Use Detector Reporter Quencher Task Quantity M Duo Human none Standard 8 M Duo IPC none Unkng Omit well Select Add Detector Remove 5 R OX Click Close to close the dialog box When you are finished assign sample names tasks and quantities to unknown samples and non template controls as necessary Quantifiler Duo DNA Quantification Kit User s Manual 2 21 Chapter 2 Software Setup Assigning Sample Name and Task to Unknown Samples and Non Template Controls 2 22 To assign parameters to unknown samples and non template control NTC wells 1 In the Plate Setup tab select the wells that correspond to an unknown sample or NTC for the Duo DNA Quantification Kit Ko ware Plate1 Absolute Quantification sis s Selected well E 1 87e 001 Blt67e 001 n n m m m m IE 1 57e 001 Bit sresoot m m m c
93. he figure refer to the sample numbers in Table 6 5 Figure 6 22 STR analysis using the MiniFiler Kit and organic extracted case type samples Human DNA samples from three population groups were analyzed to verify the ability to obtain male and human quantification results using the Quantifiler Duo kit Experiment Purified genomic DNA samples from 534 human individual donors from Hispanic Caucasian and African American population groups were analyzed using the Quantifiler Duo DNA Quantification Kit All DNA samples were previously extracted from blood specimens using the Applied Biosystems BloodPrep DNA Chemistry and ABI PRISM 6100 Nucleic Acid PrepStation The samples were previously quantified using the Quantifiler Human DNA Quantification Kit Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation The number of male and female samples in each of the population groups tested is detailed in Table 6 10 Approximately 1 ng of purified genomic DNA was used from the panel for each Quantifiler Duo Kit reaction Table 6 10 Distribution of samples used for population studies Population Male Samples Female Samples Caucasian 130 60 African American 116 24 Hispanic 129 75 Total gender 375 159 Total 534 Results The Quantifiler Duo DNA Quantification Kit detected and quantified DNA in all 534 human DNA samples All 375 male DNA samples exhibited the SRY
94. ies on the right side of the Duo Male standard curve when All Detectors are selected in the Standard Curve tab The gap between the Duo Human and the Duo Male Cy values may vary depending on the relative slopes of the two targets and instrument performance Quantifiler Duo DNA Quantification Kit User s Manual 5 11 Chapter 5 5 12 Interpretation of Results 17500 System SDS Software Duo_samples_troublEx4 sds Absolute Quantification D File View Tools Instrument Analysis Window Help Us 5 Ell E EJ re Setup T instrument Resutts Plate Y Spectra Y Component Y Amplification Plot Y standard Curve Y Dissociation Y Report Y Standard Curve decor TA Duo Human Duo Male Legend The Duo Human and Duo Male standard curves are exchanged Figure 5 8 Example 4 Observation Possible Cause When creating detectors for the standards as shown in Figure 5 9 below FAM was selected as the reporter dye for Duo Human and VIC was selected as the reporter dye for Duo Male instead of the opposite m Well Inspector Well s A2 Sample Name 5td1 V Dua IPC Y Duo Human none Standard v Duo Male none Standard Reporter dyes are flipped x Omit Well Passive Reference Add Detector Remove ROX I Figure 5 9 Example 4 Possible Cause Quantifiler Duo DNA Quantification Kit User s Manual Using the Internal PCR Control System Using the Internal PCR Contro
95. indow 2 Enter a file name for the export file and click Save For more information about exporting data see the Applied Biosystems 7300 7500 7500 Fast Real Time PCR System Absolute Quantification Getting Started Guide PN 4378658 4 8 Quantifiler Duo DNA Quantification Kit User s Manual 04 2008 Part Number 4391294 Rev B Chapter 5 Interoretation of Results Quantifiler Duo DNA Quantification Kit User s Manual Interpretation of Results This chapter covers Checking Analysis Settings a 5 2 Examining the Standard Curve 0 002 KK KK KK aes 5 3 Troubleshooting the Standard Curve a 5 5 Using the Internal PCR Control System 5 13 Troubleshooting Amplification Plots 2 4 5 15 Assessing Quantity 2 0 0 2 5 19 Calculating Male Female DNA Ratio 5 20 Improving Assay Performance 00 0 RR KK KK 5 20 Assessing Sensitivity and Results 5 20 Assessing and Troubleshooting False Positive Results 5 22 Preventing PCR Contamination 5 23 Quantifiler Duo DNA Quantification Kit User s Manual 5 1 Chapter 5 Interpretation of Results Checking Analysis Settings The validity of the results requires correct analysis settings Checking To check analysis settings on the 7500 SDS Analysis Settings on the 7500 SDS
96. l System Purpose Components Interpreting IPC Results Table 5 3 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 The following components of the IPC system are present in the Quantifiler Duo Primer mix Synthetic DNA template Primers that hybridize specifically to the synthetic DNA template Probe labeled with NED dye In the amplification plot window of the SDS software observe amplification of the VIC and FAM dyes Duo Human detector and Duo Male detector and the NED dye Duo IPC detector then use Table 5 3 to interpret the IPC results Interpreting IPC amplification results Duo Human VIC dye and or Duo Male FAM Dye Duo IPC NED Dye Interpretation No amplification Amplification Negative result no human DNA detected No amplification No amplification Invalid result Amplification low C and high AR No amplification or Cz higher than 31 IPC result inconclusive Amplification high Cz and low AR No amplification or Cz higher PCR inhibition than 31 Quantifiler Duo DNA Quantification Kit User s Manual Note Positive amplification occurs when the C value for the detector is 40 Because samples contain unknown amounts of DNA a large range of Cy values is possible The IP
97. le Inhibitor xu StdDev REPRE StdDev C value StdDev Name Name ng uL SRY ng uL RPPH1 IPC IPC 0 uM Hematin 0 411 0 033 0 397 0 045 29 647 0 112 2 5 uM Hematin 0 406 0 099 0 399 0 029 29 833 0 078 5 uM Hematin 0 393 0 041 0 358 0 045 29 677 0 123 7 5 uM Hematin 0 371 0 035 0 253 0 030 30 050 0 122 10 uM Hematin 0 092 0 013 0 024 0 010 31 753 0 377 12 5uM Hematin 0 007 0 004 0 000 35 347 1 048 15 uM Hematin 0 000 0 000 0 000 ses 40 000 sas 17 5 uM Hematin 0 000 0 000 0 000 40 000 20 uM Hematin 0 000 0 000 0 000 sas 40 000 saa 40 uM Hematin 0 000 0 000 0 000 ees 40 000 see 0 ng uL Humic acid 0 411 0 033 0 397 0 045 29 647 0 112 1 ng uL Humic acid 0 381 0 077 0 359 0 034 29 783 0 054 2 ng uL Humic acid 0 370 0 060 0 244 0 029 29 813 0 071 3 ng uL Humic acid 0 275 0 058 0 105 0 023 30 263 0 142 3 75 ng uL Humic acid 0 155 0 041 0 055 0 016 30 807 0 411 7 5 ng uL Humic acid 0 006 0 005 0 000 35 207 1 540 11 25 ng uL Humic acid 0 000 0 000 0 000 40 000 15 ng uL Humic acid 0 000 0 000 0 000 40 000 30 ng uL Humic acid 0 000 0 000 0 000 40 000 6 18 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation The results of the STR analysis with the Identifiler kit using 1 0 ng of template DNA for PCR amplification are presented in Figure 6 9 and Figure 6 10 The volume of the extract used for amplification was based on the quantification results RPPHI
98. llection Organize and store the data gathered during the run Analyze the data from the run Plate Document You can use the SDS software to create two types of plate document Types files Table 2 1 Plate Documents 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 2 6 Quantifiler Duo DNA Quantification Kit User s Manual Example Plate Document Setup About Plate Documents You can arrange the reactions in any well of the reaction plate but 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 Table 2 2 shows one example of arranging reactions Note For each Quantifiler Duo DNA Quantification Kit assay there are eight DNA quantification standards and two reactions for each standard See Preparing the DNA Quantification Standards on page 3 2 for more information about the DNA quantification standards Table 2 2 Example plate setup of reactions UNKN unknown NTC non template control 1 2 3 4 5 6 7 8 9 10 11 12 A Std 1 Std 1 UNKN UNKN UNKN UNKN UNKN
99. lotted 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 C m log Qty b where m is the slope b is the y intercept and Qty is the starting DNA quantity The values associated with the regression analysis can be interpreted as follows e R value Measure of the closeness of fit between the standard curve regression line and the individual C4 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 Cy value for a sample with Qty 1 for example 1 ng uL R Value An R value gt 0 99 indicates a close fit between the standard curve regression line and the individual C data points of quantification standard reactions If the R value is 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 C value for Standard 8 of the DNA quantification st
100. matin 45 B SRY FAM E RPPHI VIC IPC NED DuM 2 5uM 5uM 7 5uM 1 uM 12 5uM 15uM 17 5uM 20uM 40uM Figure 6 3 C values for hematin inhibited samples using the Quantifiler Duo DNA Quantification Kit Inhibitor Humic Acid El SRY FAM E RPPH1 VIC D AE Jo NED ka Ong ul 1ng ul 2ng ul 3ngful 3 75ng ul 7 5ngful 11 25ng ul 15ng ul 30ng ul Figure 6 4 C values for humic acid inhibited samples using the Quantifiler Duo DNA Quantification Kit 6 12 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation STR analysis was performed using the Identifiler Kit The volume of extract used for STR amplification was equal to the volume used for quantification 2 uL The Identifiler Kit results Figure 6 6 and Figure 6 8 were consistent with the results from the Quantifiler Duo DNA Quantification Kit Figure 6 5 and Figure 6 7 As the concentrations of hematin and humic acid increased indicated by the increasing IPC Cy values the overall STR peak heights decreased Complete STR profiles were obtained at 10 uM hematin and 3 75 ng uL humic acid The STR amplification reaction was completely inhibited at 40 uM hematin and 30 ng uL humic acid Thus the results from the Quantifiler Duo DNA Quantification Kit provided reasonable predictions of samples that would produce lower quality STR profiles due to the presence of a PCR inhibitor IPC Cr in hematin inhibited samples
101. mine the optimum amount of input DNA required for each STR genotyping system based on the quantification values obtained using the Quantifiler Duo DNA Quantification Kit Quantifiler Duo DNA Quantification Kit User s Manual 6 55 Oo 9 Human Male Quantification Kit enue s Jesf NA uoneoynueno YN ONG sJelumueno Table 6 16 Correlation of DNA quantification using the Quantifiler Duo Quantifiler Human and Quantifiler Y Sample rem Mean Quantity Human Mean Quantity Male A 20 000 21 153 21 160 0 033 20 103 16 910 18 882 A 10 000 9 110 10 440 12 739 8 983 9 020 0 410 A 1 000 0 869 0 831 4 573 0 854 1 120 23 750 A 0 100 0 091 0 071 28 169 0 083 0 113 26 549 A 0 050 0 043 0 044 2 273 0 046 0 060 23 333 B 20 000 24 363 27 690 12 015 23 087 20 380 13 283 B 10 000 11 493 12 290 6 485 11 220 10 650 5 352 B 1 000 1 143 1 080 5 833 1 147 1 310 12 443 B 0 100 0 104 0 098 6 122 0 110 0 160 31 250 B 0 050 0 061 0 044 38 636 0 049 0 082 40 244 C 20 000 22 513 24 740 9 002 23 112 20 270 14 021 C 10 000 8 720 11 110 21 512 9 250 9 220 0 325 C 1 000 0 822 1 010 18 614 0 894 1 110 19 459 C 0 100 0 098 0 129 24 031 0 108 0 099 9 091 C 0 050 0 044 0 056 21 429 0 044 0 046 4 348 D 20 000 27 283 16 730 63 078 26 487 22 800 16 171 D 10 000 13 263 10 610 25 005 13 090 11 740 11 499 D 1 000 1 217 1 470 17 211 1 263 1 500 15 800 D 0
102. mplification Quantifiler Duo DNA Quantification Kit User s Manual PCR Amplification This chapter covers Preparing the DNA Quantification Standards 3 2 Preparing the Reactions 0 0 KK KK KK KK KK KK KK KK KK 3 5 Running the Reactions 0 0 KK KK KK KK KK KK KK KK KK KK 3 7 Quantifiler Duo DNA Quantification Kit User s Manual 3 1 Chapter 3 PCR Amplification Preparing the DNA Quantification Standards Required The required materials include Materials Pipettors Pipette tips Microfuge tubes Quantifiler Duo DNA Standard Quantifiler Duo DNA Dilution Buffer Note You can store the diluted DNA quantification standards for up to 2 weeks at 2 to 8 C Longer term storage is not recommended Guidelines for The standard dilution series example shown in Table 3 1 on page 3 3 Calculating the is suitable for general use Standards IMPORTANT Applied Biosystems recommends Dilution Series Three fold dilution series with eight concentration points in the standard series Minimum input volume of 10 uL DNA for dilutions to ensure accuracy of pipetting 3 2 Quantifiler Duo DNA Quantification Kit User s Manual Standards Dilution Series Example Preparing the DNA Quantification Standards Table 3 1 shows an example of one standards dilution series with the concentrations ranging from 50 ng uL Std 1 to 0 023 ng uL or 23 pg uL Std 8 When 2 0 uL of a sample at the l
103. n here change them to match the settings then click OK On the menu bar select Analysis 5 Analyze Note For routine analysis that doesn t require any change in the Analysis Settings and to skip all the steps described above click the green arrow on the system software tool bar 47500 System SDS Software Duo samples sds Absolute Quantification 9 File View Tools Instrument apalysis Window Help n ug emus ER Quantifiler Duo DNA Quantification Kit User s Manual 4 3 Chapter 4 Data Analysis and Results 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 6 Viewing the Report page 4 6 Exporting the Results page 4 8 Viewing the For information about interpreting and troubleshooting the standard Standard Curve curve see Examining the Standard Curve on page 5 3 and Troubleshooting the Standard Curve on page 5 5 To view the standard curve 1 In the Results tab select the Standard Curve tab 2 In the Detector drop down list select the applicable detector Duo Human or Duo Male 3 View the C values for the quantification standard reactions and the calculated regression line slope y intercept and R values 4 4 Quantifiler Duo DNA Quantification Kit User s Manual Amplification Plot Results Viewing Results
104. n the following linked page http www ncbi nlm nih gov entrez query fcgi CMD search amp D B gene Quantification of the human and human male DNA and qualitative indication of the presence of PCR inhibitors in a biological sample are determined by the measurement of fluorescent dye released during the amplification process The fluorescent dyes are linked to sequence specific nucleotide probes The principle of detection and details of the assay are described in Chapter 1 Chemistry Overview on page 1 3 in this document The Quantifiler Duo DNA Quantification Kit measures the quantity of human and human male DNA in forensic type samples The quantification results are further used to determine the optimal amount of DNA sample to be used for genotyping assays Since the forensic type samples are often contaminated with non human DNA specificity measurements of primers and probes in the Quantifiler Duo DNA Quantification Kit are crucial Specificity is confirmed through experimentation Experiment Cross reactivity of primers and probes in the Quantifiler Duo DNA Quantification Kit was examined by testing DNA from various non human species Human DNA samples were used as controls see Table 6 1 for details The DNA from non human biological species was either obtained commercially or purified in the laboratory For some of these DNA samples the sex of the donor animal was unknown For some species multiple donor animals w
105. ndards stochastic effects 5 19 storage recommendations for kits 1 16 strand displacement in 5 nuclease assay 1 5 T TaqMan fluorescent probes 1 12 MGB probes 1 4 RNase P Instrument Verification Plate 1 12 targets about 1 3 Technical Support contacting xi template creating plate document from 7500 SDS 2 28 saving 7500 SDS 2 27 setting up for plate documents 7500 SDS 2 26 text conventions v thermal cycler conditions setting 7500 SDS 2 24 threshold settings for the 7500 SDS 4 3 threshold cycle calculation of 1 13 exporting 7500 SDS 4 8 in standard curve 5 3 normal range for IPC system 5 14 Index 4 printing 7500 SDS 4 8 relationship to initial template amount 1 13 viewing in amplification plot 7500 SDS 4 5 viewing in standard curve 7500 SDS 4 4 training information on xi U user attention words described vi V validation importance of 6 2 validation studies 6 3 W WARNING description vii waste disposal guidelines x Y y intercept of standard curve interpreting 5 3 viewing 7500 SDS 4 4 Quantifiler Duo DNA Quantification Kit User s Manual Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries
106. ned from one or two male individual donors a and b The blood samples were spiked with inhibitors spotted onto fabric filter paper and organically extracted The saliva samples were spotted onto swabs and the semen samples were spotted onto fabric A subset of these samples 1 4 were also extracted using other commercially available methods including Chelex resin the QIAGEN QIAamp DNA Blood Mini Kit and the Promega DNA IQ Kit For details on the preparation of the samples see Table 6 8 Extracted DNA was quantified in triplicate using the Quantifiler Duo DNA Quantification Kit Based on the results from the Quantifiler Duo kit RPPH1 human target approximately 1 0 ng of human genomic DNA was added to each Identifiler Kit reaction and approximately 0 1 ng of human genomic DNA was added to each MiniFiler Kit reaction Table 6 8 Preparation of samples Sample Sample Type Volume and Substrate 1 Saliva Swab a 50 uL on cotton swab 2 Saliva Swab b 50 uL on cotton swab 3 Blood Stain a 5 uL on fabric 4 Blood Stain b 5 uL on fabric 5 Blood Stain 5 uL on denim 6 Blood Stain 5 uL on filter paper 7 Blood Stain 5 uL spiked with inhibitor mix on fabric 8 Semen Stain 1 uL on fabric Quantifiler Duo DNA Quantification Kit User s Manual Results Developmental Validation The Quantifiler Duo Kit reactions performed on the case type samples yielded a range of DNA concentrations from
107. oc mw TRINY YY O E TET TE TT DET ET TE TET TET TET TE TT TET 2 e Pill i 23 du nw E i i i A i i i ET mw CIC TENE TELE TAN TERE TET TN TENE TURE AA AYY a 3 p aan titi iit bi ya a dd diui d Day mw uS n Rte ea DEN CHE EDU ee EE TES wann n pli al 070607 Rainbow validation Phenol BSD Tila mal BSD Tamil VI 400 no 120 100 10 150 40 170 1 lar an dn i Lian alia i 4l d 7 000 RAINBOW 2007 05 16 phencl_SSF_DS_ID_DOGfsa phendl SSF D5 ID 40 0 Q0 aw so ao s oo wo 2000 j 0 8 J os AA ae a roe ae ee kai zl Legend The sample numbers to the left of the figure refer to the sample numbers in Table 6 5 Figure 6 21 extracted case type samples STR analysis using the Identifiler Kit and organic Quantifiler Duo DNA Quantification Kit User s Manual 6 39 Chapter 6 Experiments and Results SS as e ae m n J a 2 iod aad i i A iia ka AA Naa SO Me j alla hog A aial dy n a Aa aaa mW b A Aa SO M o dha i Ata 444 071707 _sinifiter_Phencl_BSD_0 Ing_C02 fsa Phencl_BSD_O Ing MiniRler CS500 vl m m a o Muda a a kua m ET a a TT m B i a MR 1 Lak hid ca a um i T T a Ces adi n cz an xn e a KEZWTETEZ Tena nm M al Population Studies Std 2 7 6 40 Legend The sample numbers to the left of t
108. ocument 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 the Quantifiler Duo DNA Quantification 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 reaction volume settings and 9600 Emulation setting Creating a Plate This procedure assumes that you have created the detectors for Document running reactions using the Duo DNA Quantification Kit Template page 2 10 To create a plate document template 1 Ifthe 7500 SDS Software is not already started select Start gt Programs gt 7500 System 7500 System Software 2 Select File gt New to open the New Document Wizard Define Document window then click Next New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and comments Assay Absolute Quantification Standard Curve Container 96 Well Clear Template Blank Document Operator AB Comments SDS v1 23 Plate Name te Finish Cancel 2 26 Quantifiler Duo DNA Quantification Kit User s Manual
109. on of the strand continues but because the 3 end of the probe is blocked there is no extension of the probe during PCR Figure 1 5 Quantifiler Duo DNA Quantification Kit User s Manual 1 5 Chapter 1 Overview TaqMan MGB probe 3 Forward Primer 3 5 5 3 lt 5 Reverse Primer Figure 1 5 Completion of polymerization Human DNA The human DNA used to generate the DNA quantification standards Standard dilution series consists of pooled human male genomic DNA As such the performance of the Quantifiler Duo assay is validated and the results are optimized for use with this DNA standard The use of an alternate DNA standard may result in the reporting of different concentration values for the unknown samples Use of an alternate DNA standard should be supported by appropriate validation studies 1 6 Quantifiler Duo DNA Quantification Kit User s Manual Instrument Overview Instrument Overview Fluorescence Detection on the Applied Biosystems 7500 Real Time PCR Detection System 1 A tungsten halogen lamp directs light to each well on the reaction plate The light excites the fluorescent dyes in each well of the plate 2 During the run the CCD camera detects the fluorescence emission 3 The SDS software obtains the fluorescence emission data from the CCD camera and applies data analysis algorithms AN Attention Physical hazard Figure 1 6 Applied Biosystems 7500 R
110. owest concentration 23 pg uL is loaded in a reaction the well contains approximately 7 diploid human genome equivalents These equivalents correspond to approximately 14 copies of the Duo Human target locus and approximately 7 copies of the Duo Male target locus Y chromosome loci are haploid Table 3 1 Standards dilution series example Concentration NY Dilution Standard ng uL Example Amounts Minimum Amounts Factor Std 1 50 000 50 uL 200 ng L stock 10 uL 200 ng UL stock 4X 150 uL Quantifiler Duo 30 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Std 2 16 700 50 uL Std 1 10 uL Std 1 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Std 3 5 560 50 uL Std 2 10 uL Std 2 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Std 4 1 850 50 uL Std 3 10 uL Std 3 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Std 5 0 620 50 uL Std 4 10 uL Std 4 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Std 6 0 210 50 uL Std 5 10 uL Std 5 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Std 7 0 068 50 uL Std 6 10 uL Std 6 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer S
111. pare the reagents Thaw the Quantifiler Duo Primer Mix completely then vortex 3 to 5 seconds and centrifuge briefly before opening the tube Swirl the Quantifiler Duo PCR Reaction Mix gently before using Do not vortex it Pipette the required volumes of components into an appropriately sized polypropylene tube Vortex the PCR mix 3 to 5 seconds then centrifuge briefly Dispense 23 uL of the PCR mix into each reaction well Add 2 uL of sample standard or control to the applicable wells For a plate setup example see page 2 7 IMPORTANT Applied Biosystems recommends running duplicates of the eight DNA quantification standards for each reaction plate Seal the reaction plate with the Optical Adhesive Cover Centrifuge the plate at 3000 rpm for about 20 seconds in a tabletop centrifuge with plate holders to remove any bubbles Note Ifa tabletop centrifuge with 96 well plate adapters is not available visually inspect the plate for bubbles and lightly tap the plate to remove bubbles in wells Quantifiler Duo DNA Quantification Kit User s Manual Running the Reactions Running the Reactions Before You Run v Before you run the reactions make sure that you have the Reactions Powered on the 7500 Real Time PCR instrument computer and software For setup procedures see page 2 3 Set up a plate document for the run See page 2 7 Running the Plate To run the plate on the 7500
112. 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 Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Quantifiler Duo DNA Quantification Kit User s Manual ix Preface Waste Disposal Biological Hazard Safety 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 ANTITE BIOHAZARD Biological samples such as tissues body fluids and blood of humans and other animals have the
113. quence About the Targets Table 1 1 provides information about the targets of PCR amplification in the Quantifiler Duo DNA Quantification Kit Table 1 1 Quantifiler Duo Kit targets i Amplicon Target Gene Target Location Length Gene ID Ploidy Human Target Ribonuclease P 14q11 2 140 bases 85495 Diploid RNA Component H1 RPPH1 Human Male Target Sex determining Yp11 3 130 bases 6736 Haploid region Y SRY IPC Assay The IPC assay consists of Components IPC template DNA a synthetic sequence not found in nature Two primers for amplifying the 130 base IPC template DNA One TaqMan MGB probe labeled with NED dye for detecting the amplified IPC DNA Quantifiler Duo DNA Quantification Kit User s Manual 1 3 Chapter 1 Overview About the Probes The TagMan MGB probes contain A reporter dye FAM VIC or NED dye linked to the 5 end of the probe A minor groove binder MGB at the 3 end of the probe This modification increases the melting temperature T m without increasing probe length Afonina et al 1997 Kutyavin et al 1997 to allow for the design of shorter probes A nonfluorescent quencher NFQ at the 3 end of the probe 5 Nuclease The 5 nuclease assay process Figures 1 1 through 1 5 takes place Assay Process during PCR amplification This process occurs in every cycle and it does not interfere with the exponential accumulation of
114. r Applied Biosystems welcomes your comments and suggestions for Comments 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 information about customer training Download software updates and patches Quantifiler Duo DNA Quantification Kit User s Manual Xi Preface xii Quantifiler Duo DNA Quantification Kit User s Manual Overview This chapter covers Product Overview KK Chemistry Overview aasa Instrument Overview an SDS Software Overview Real Time Data Analysis Quantifiler Duo Kit Workflow Materials and Equipment Quantifiler Duo DNA Quantification Kit User s Manual 1 1 Chapter1 Overview Product Overview Purpose The Quantifiler Duo DNA Quantification Kit PN 4387746 is designed to simultaneously quantify the total amount of amplifiable human DNA and human male DNA ina sample The results obtained using the kit can
115. r spray PCR samples When pipetting from a kit component tube hold the cap of the tube in your gloved hand or be sure to set it down on a clean decontaminated surface Keep reactions and components sealed when possible Do not open sealed reaction tubes or plates after amplification Clean lab benches and equipment periodically with freshly diluted 10 bleach solution Quantifiler Duo DNA Quantification Kit User s Manual 5 23 Chapter 5 Interpretation of Results 5 24 Quantifiler Duo DNA Quantification Kit User s Manual 04 2008 Part Number 4391294 Rev B Chapter 6 Experiments and Results Quantifiler Duo DNA Quantification Kit User s Manual Experiments and Results 6 This chapter covers OVERVIEW cues bas Be N ai Ue EU coe Vo ee Developmental Validation Quantifiler Duo DNA Quantification Kit User s Manual 6 1 Chapter 6 Experiments and Results Overview About This Chapter Importance of Validation Experiments This chapter provides results of the validation experiments performed by Applied Biosystems using the Quantifiler Duo DNA Quantification Kit Although the Quantifiler Duo DNA Quantification kit is not a DNA genotyping assay it is intended for use before performing genotyping assays such as the AmpF STR PCR Amplification Kits By testing the procedure with samples commonly encountered in forensic and parentage laboratories the validation process establishes attributes and limita
116. ratories when dealing with STR amplification results Applied Biosystems recommends applying a similar approach when interpreting the results generated by the Quantifiler Duo DNA Quantification Kit About False When encountering false positive results positive amplification of Positive Results negative controls note the following The quantities obtained are usually well below the dynamic range of the standard curve Therefore these quantities may produce no STR or variable STR profiles You can set a Cy value threshold to proceed with STR analysis Note Setting a C value threshold requires further internal validation at your facility tis important to distinguish between a real DNA signal due to the contamination of one reagent and a positive result due to spectral overlap between the various dyes Note The 7500 SDS software uses a specialized multicomponenting algorithm that provides precise deconvolution of multiple dye signals in each well This algorithm ensures minimal crosstalk when using multiple fluorophores for multiplex assays However a residual spectral overlap may be observed in the NTC wells especially if the instrument is in need of calibration Quantifiler Duo DNA Quantification Kit User s Manual 5 21 CO N N enue s Jesf NA uoneoynueno YN ONG e linueno Assessing and Troubleshooting False Positive Results Table 5 5 Troubleshooting false positives and or Duo Male
117. s extracted using the organic extraction method human and male DNA quantities were similar except for the blood stain on denim Table 6 8 sample 5 For this sample the quantity of male DNA was 8196 higher than the quantity obtained from the human target Table 6 9 The blood stain on denim is a challenging sample due to inhibitors present on the denim substrate and in the blood The lower quantity of total human DNA may result from inhibition of the amplification of the RPPHI target The presence of PCR inhibitors is suggested by the greater Cy value obtained for the IPC For the other extraction methods the human and male DNA quantities for all samples were similar except for the saliva swabs Table 6 8 samples 1 and 2 extracted with the Promega DNA IQ Kit Table 6 9 Complete and interpretable STR profiles were obtained for all the samples analyzed Peak heights from 500 to 4000 rfu were obtained for samples analyzed using the Identifiler Kit DNA target amount was 1 ng rxn and from 200 to 2000 rfu for samples analyzed using the MiniFiler Kit DNA target amount was 0 1 ng rxn Representataive STR profiles obtained for the phenol chloroform extracted samples are shown in Figure 6 21 and Figure 6 22 Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation 070607 Rainbow validation Phenol_yative di HOS fva Phenol suliva dl 40 d LANE 4 LL i La C pc T
118. signal see Table 6 11 The SRY signal was not detected for any of the female samples tested see Table 6 11 The human male DNA quantity value SRY was within 25 of the total human quantity value RPPH1 obtained for most of the male samples data not shown 5 of the 375 male samples tested Table 6 12 yielded SRY quantity values that deviated from the RPPH1 quantity values by gt 25 This may have resulted from duplication of the SRY gene in these DNA samples Quantifiler Duo DNA Quantification Kit User s Manual 6 41 Chapter 6 Experiments and Results Table 6 11 Quantification of DNA in the samples from different population groups Quantification Results Obtained Sex Duo Human Duo Male Male 375 10096 10096 Female 159 10096 096 Table 6 12 Quantification results for the five samples exhibiting gt 25 variation in the quantities of total human and male DNA Sample Population Duo Human ng uL Duo Male ng pL IPC C Male Human difference 417 Caucasian 0 688 1 300 29 837 88 953 34 African American 0 244 0 475 29 957 94 672 64 African American 0 230 0 458 29 733 99 130 129 African American 0 253 0 444 29 847 75 494 183 African American 0 329 0 542 29 803 64 742 Mixture Study 6 42 The mixture studies were designed to simulate circum
119. simultaneous quantitation of total genomic and male DNA J Forensic Sci 51 758 765 Kontanis E J and Reed EA 2006 J Forensic Sci 51 795 804 Kutyavin I V Lukhtanov E A Gamper H B and Meyer R B 1997 Oligonucleotides with conjugated dihydropyrroloindole tripeptides base composition and backbone effects on hybridization Nucleic Acids Res 25 3718 3723 Lakowicz J R 1983 Energy Transfer In Principles of Fluorescence Spectroscopy New York Plenum Press 303 339 Martens H and Naes T 1989 Multivariate Calibration Chichester John Wiley amp Sons Quantifiler Duo DNA Quantification Kit User s Manual Bibliography 1 Bibliography 2 Nicklas J A and Buel E 2003 Use of real time Alu PCR for quantitation of human DNA in forensic samples J Forensic Sci 48 936 944 Nicklas J A and Buel E 2005 An Alu based Eclipse real time PCR method for quantitation of human DNA in forensic samples J Forensic Sci 50 1081 1090 Nicklas J A and Buel E 2006 Simultaneous determination of total human and male DNA using a duplex real time PCR assay J Forensic Sci 51 1005 1015 Revised Validation Guidelines Scientific Working Group on DNA Analysis Methods SWGDAM approved by SWGDAM July 10 2003 Forensic Science Communications 6 3 July 2004 Available at http www fbi gov hq lab fsc backissu july2004 research 2004_03_ research02 htm Shewale J G Schneida E Wilson J Walker J A
120. stances in Std 2 8 which a small component of male DNA must be discerned from a high background of female DNA When interpreting results consider that evidence samples may contain DNA from more than one individual Experiment 1 Mixture samples containing 0 2 ng uL of human male DNA and varying amounts of female DNA were prepared The ratio of male and female DNA in these samples was 1 0 1 1 1 5 1 10 1 20 and 0 1 Table 6 13 The mixture samples were processed in triplicate using the Quantifiler Duo DNA Quantification Kit to determine the concentration of total human DNA RPPHI target and male DNA SRY target Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation In addition using the results from the RPPH1 human target approximately 1 0 ng of human genomic DNA from each sample was added to an Identifiler Kit reaction Using the results from the SRY male target approximately 1 0 ng of human genomic DNA from each sample was added to a Yfiler kit reaction Results 1 The quantification results obtained using the Quantifiler Duo DNA Quantification Kit are summarized in Table 6 13 and shown graphically in Figure 6 23 For all samples the male DNA produced consistent quantification values regardless of the amount of female DNA present Thus the ability to quantify the male DNA was not adversely affected by the presence of the quantities of female DNA investigated In addition th
121. strate that the Quantifiler Duo assay did not generate results due to the presence of contaminating DNA Experiment To test for contamination all standard assay parameters were used except that the number of cycles was extended from 40 to 50 in the PCR amplification cycling step A 96 well plate was set up with 95 non template controls NTCs and one positive control sample at a concentration of approximately 20 ng L The number of PCR cycles was increased from 40 to 50 in this experiment to study the performance at a higher stringency Results None of the 95 NTCs exhibited any detectable signal for the human and male targets in the VIC and FAM channels respectively Figure 6 31 The C4 values for the IPC signal NED fell within a range of variation of 1 Cp which is within the normal variation of the TaqMan assay The positive control sample provided the expected C4 values for human and male targets Quantifiler Duo DNA Quantification Kit User s Manual 6 53 Chapter 6 Experiments and Results 1 0e 001 Positive Control 1 0e 000 Human amp Male IPC 1 0e 001 NTC Wells 1 0e 002 Human amp Male 1 0e 003 1 0e 004 1 3 5 7 8 11 13 15 17 19 21 23 26 27 29 31 33 35 37 38 41 43 45 47 49 Figure 6 31 NTC and positive control Correlation Quantification of DNA is a step in the STR analysis workflow that Studies provides a value obtained by measuring the DNA content of a given sample against a refer
122. sults Quantifiler Duo DNA Quantification Kit User s Manual 1 15 Chapter 1 Overview Materials and Equipment Kit Contents and Each Quantifiler Duo DNA Quantification Kit contains materials Storage sufficient to perform 400 reactions at a 25 uL reaction volume Store the entire kit at 15 C to 25 C upon receipt Store the kit at 2 C to 8 C after first thaw as described in Table 1 2 Table 1 2 Quantifiler Duo DNA Quantification Kit contents Reagent Contents Quantity Storage Quantifiler Duo Primer Mix Primer pairs for 3 tubes 2to8 C amplification of 1 4 mL keep RPPH1 SRY and IPC each protected from TagMan probes for exposure to RPPH1 SRY and IPC light which are labeled with VIC FAM and NED dyes respectively IPC template Quantifiler Duo PCR Reaction Mix e MgCl dNTPs bovine 1 Tube 2to8 C serum albumin and 5 0 mL keep AmpliTaq Gold DNA protected from Polymerase in buffer exposure to and salts light e Sodium azide 0 02 w v is incorporated as preservative Quantifiler Duo DNA Standard Human male genomic 1 tube 2to8 C DNA 120 uL Quantifiler Duo DNA Dilution Buffer 10 mM Tris HCI buffer pH 2 Tubes 2108 C 8 0 containing 0 1 mM 1 8 mL EDTA see each Quantifiler Duo DNA Quantification Kit User s Manual Additional Storage Guideline For Primer Mix and PCR Reaction Mix Equipment and Materials Not Included Keep Primer Mix and PCR Reac
123. system IPC system about assay 1 3 components 5 13 interpreting results of 5 13 invalid results from 5 14 italic text when to use v K kit contents Quantifiler 1 16 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 v minor groove binder description 1 4 MSDSs description viii obtaining viii xi referring to viii ix N negative results 5 14 New Document dialog box 7500 SDS 2 9 nonfluorescent quencher description 1 4 O Optical Adhesive Cover sealing plate with 3 6 Quantifiler Duo DNA Quantification Kit User s Manual P passive reference normalization using 1 9 selecting in Well Inspector 7500 SDS 2 21 PCR kinetic analysis of 1 9 partial inhibition 5 14 phases of 1 10 process in 5 nuclease assay 1 4 reactions preparing 3 5 standard preparing 3 2 plate document 7500 SDS analyzing 4 2 creating blank 2 9 creating from a template 2 28 detectors adding 2 16 detectors creating 2 10 howused 2 6 saving 2 25 setting up 2 8 setup examples 2 7 template creating 2 26 template setting up 2 26 thermal cycler conditions setting 2 24 types 2 6 plateau phase amplification plot 1 11 polymerization in 5 nuclease assay completion of 1 6 process 1 4 probes about 1 4 pure dye calibration 1 8 Q Quantifiler Kit contents 1 16 quantity assessing 5 19
124. target summarized in Table 6 4 A full and interpretable profile was obtained for samples labeled as 0 uM 2 5 uM and 5 uM hematin as well as O ng uL 1 ng uL and 2 ng uL humic acid Samples labeled as 7 5 uM hematin and 3 ng uL humic acid exhibited partial profiles All other samples did not provide any STR profile As previously described these concentrations correspond to the final inhibitor concentration in the quantification reaction 2 uL in a 25 uL reaction volume and not to the actual final inhibitor concentration in the STR reaction The results indicate that an upward shift in the IPC C4 value with the Quantifiler Duo DNA Quantification Kit can be used to signal that STR analysis may result in a partial profile or in the inability to obtain an STR profile Quantifiler Duo DNA Quantification Kit User s Manual 6 19 Chapter 6 Experiments and Results OpM al Hi L AA E L A Ah EUREN Ja Lad P ONERE NE RE RN RA RR tg amano 25uM 7 060 Juinbew validation hematin Su CHfa henatin Sun Menit vi mm aeo HO ta no e so nao 10 seo ago 20 2o 22 2w 2a 280 ze 20 2 2 ao 20 ao ao M0 ao 5 jiM 7 2000 eo D 07060 Rainbow validation nati 7 Sul Asa hanatin 7 Sut Ment vi mm aeo M0 0 ore o wo o Ho Soo do oo ze 07 90 9 2a 28o 30 0 00 za 28o ae aw ao ao sa ao 75y9M 7 2000 Lal Wa laha Ek ENKA W aq A ah 070605 Rainbow validation hematin TOWN BO sa hematin_10
125. td 8 0 023 50 uL Std 7 10 uL Std 7 3x 100 uL Quantifiler Duo 20 uL Quantifiler Duo DNA dilution buffer DNA dilution buffer Quantifiler Duo DNA Quantification Kit User s Manual 3 3 Chapter 3 PCR Amplification Preparation While preparing the standards keep in mind that Guidelines DNA quantification standards are critical for accurate analysis of run data 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 When using Quantifiler Duo DNA Dilution Buffer you can store DNA _ the prepared DNA quantification standards for up to 2 weeks at Quantification 2 to 8 C Standards To prepare the DNA quantification standards dilution series 1 Label eight microcentrifuge tubes Std 1 Std 2 Std 3 and so on 2 Dispense the required amount of diluent Quantifiler Duo DNA Dilution Buffer to each tube refer to Table 3 1 for volumes 3 Prepare Std 1 a Vortex the Quantifiler Duo DNA Standard 3 to 5 seconds b Using a new pipette tip add the calculated amount of Quantifiler Duo 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 refer to Table 3 1 for volumes b Mix th
126. te Document Setting Up a Plate Document 1 Ifthe 7500 SDS software is not already started select Start 5 Programs 5 7500 System 5 7500 System Software You can also launch the software from the shortcut on your desktop by double clicking the icon comments Template Default Assay Container Operator Comments Plate Name 2 In the 7500 SDS software select File gt New to open the New Document Wizard Define Document window The default settings are shown New Document Wizard Define Document Select the assay container and template for the document and enter the operator name and Absolute Quantification Standard Curve 96 Well Clear El Blank Document Browse AB SDS v1 23 Platel Next gt Finish Cancel Quantifiler Duo DNA Quantification Kit User s Manual 2 9 Chapter 2 Software Setup To create a blank plate document continued 3 Use the default settings shown then click Next to display the Select Detectors window Note If detector names are not listed first time use add new detectors as described in the following section New Document Wizard Select Detectors Select the detectors you will be using in the document Find a Passive Reference ROX zi EZ tame Description Reportar KECE DES Duo Human none none none Add gt gt enone Remove none Remove none Ne
127. te STR profiles were obtained for samples that were generated using up to 0 02 units of DNase I samples 1 to 4 The samples that were generated using up to 0 05 and 0 1 units of DNase I samples 5 and 6 provided interpretable profiles however the amplitude rfus of the alleles for STR loci with longer amplicons decreased Samples that were generated using 0 2 units of DNase I sample 7 when amplified using 0 1 ng of template DNA provided interpretable profiles with low amplitude rfus for all loci The results indicate that interpretable profiles can be recovered from all the degraded samples generated in this study when using the Minifiler Kit Quantifiler Duo DNA Quantification Kit User s Manual 6 31 Chapter 6 Experiments and Results The MiniFiler Kit is specifically designed to obtain STR profiles from compromised samples The size range of the amplicons generated using the MiniFiler Kit is relatively smaller 70 to 283 bases than those generated using the Identifiler Kit 102 to 359 bases and therefore the success rate is higher as demonstrated by the results 130 ta aso 460 am aso 430 200 210 220 230 240 250 260 270 280 gi T d 0 25 ng Aird 071707 mninitter degraded 2 0 26ng A08 fsa MiniFila G5500 v1 m m 140 aso 160 aro aso 130 200 210 220 230 240 250 260 270 200 2 pa i 0 25 ng 071707 minitilr degraded 3 25ng I6 fsa degraded 3 0 25ng nila GS500 v1 m m 140 150 1
128. th 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 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 viii Go to www appliedbiosystems com click Support then click MSDS Searc
129. the Quantifiler Duo DNA Quantification Kit However the amount of DNA present in the sample may be below the working range of certain genotyping methods If the results from Quantifiler Duo DNA Quantification Kit reactions indicate that insufficient DNA 1s present to perform an STR assay the analyst can Re extract the DNA then repeat the test with the Quantifiler Duo DNA Quantification Kit before performing STR analysis Concentrate the sample then repeat the test with the Quantifiler Duo DNA Quantification Kit before performing STR analysis Quantifiler Duo DNA Quantification Kit User s Manual 5 19 Chapter 5 Interpretation of Results Calculating Male Female DNA Ratio The Quantifiler Duo Quantification Kit provides the quantity of human and male DNA in biological samples From these values one can calculate the ratio of male and female DNA using the following equation Male DNA Female DNA Ratio Male DNA Male DNA Human DNA Male DNA Male DNA All quantities in the above equation are ng uL For example assuming Male DNA concentration 2 ng ul Human DNA concentration 8 ng ul then the Male DNA Female DNA ratio is 2 2 8 2 2 1 3 This ratio determines the extent of the mixture and is useful in determining whether to proceed with autosomal STR or Y STR analysis Improving Assay Performance Assessing Sensitivity and Results 5 20 About Assay Sensitivity Real time PCR assays are
130. tial number of template copies Delia Rnvs Cycle Copy Number 1 0006 001 1 000e 000 1 000e 001 4 000 002 Delta Rn 1 000e 003 Delta Rn 1 000e 004 1 0006 006 12345678 940111213 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 20 30 31 32 33 34 35 36 37 38 39 40 Cycle Number at Cycle Number Figure 1 9 Amplification plot from a real time run of an RNase P Instrument Verification Plate Quantifiler Duo DNA Quantification Kit User s Manual About the Threshold About the Threshold Cycle How C4 Values Are Determined Relationship of Threshold Cycles to Initial Template Amount Real Time Data Analysis The SDS software uses a threshold setting to define the level of detectable fluorescence Based on the number of cycles required to reach the threshold the SDS software can compare test samples quantitatively A sample with a higher starting template copy number reaches the threshold earlier than a sample with a lower starting template copy number The threshold cycle C for a specified amplification plot occurs when the fluorescent signal increases beyond the value of the threshold setting The Cy value depends on Starting template copy number Efficiency of DNA amplification by the PCR system To determine the Cy value the SDS software uses the R values collected from a predefined range of PCR cycles called the baseline the default baseline occurs between cycles 3 and
131. tion Mix protected from direct exposure to light Excessive exposure to light may affect the fluorescent probes and or the passive reference dye Tables 1 3 through 1 5 list required and optional equipment and materials not supplied with the Quantifiler Duo DNA Quantification Kit Unless otherwise noted many of the items are available from major laboratory suppliers MLS Table 1 3 Equipment not included Materials and Equipment Equipment Source Applied Biosystems 7500 Real Time PCR Instrument Contact your local Applied Biosystems sales representative Tabletop centrifuge with 96 well plate MLS adapters optional Table 1 4 User supplied materials Material Source Quantifiler Duo DNA Quantification Kit Applied Biosystems PN 4387746 High Throughput Setup MicroAmp Optical 96 Well Reaction Plate with Barcode Applied Biosystems PN 4306737 MicroAmp Optical Adhesive Film Applied Biosystems PN 4311971 MicroAmp Splash Free 96 Well Base Applied Biosystems PN 4312063 Quantifiler Duo DNA Quantification Kit User s Manual 1 17 Chapter 1 Overview Table 1 4 User supplied materials continued Material Source Mid to Low Throughput Setup MicroAmp Optical 8 Tube Strip 8 Applied Biosystems tubes strip 125 strips PN 4316567 MicroAmp 96 Well Tray Retainer Set Applied Biosystems PN 403081 MicroAmp
132. tions that are critical for sound data interpretation Experiments to evaluate the performance of the Quantifiler Duo DNA Quantification Kit were performed at Applied Biosystems according to the Revised Validation Guidelines issued by the Scientific Working Group on DNA Analysis Methods SWGDAM published in Forensic Science Communications Vol 6 No 3 July 2004 http www fbi gov hq lab fsc backissu july2004 standards 2004_03 _standards02 htm perfcheck These guidelines 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 experiments focus 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 Duo DNA Quantification Kit should perform appropriate internal validation studies Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation Developmental Validation The validation studies are described with reference to the standard numbers noted in the Scientific Working Group on DNA Analysis Methods SWGDAM Guidelines The stock DNA samples used for the validation study were quantified using the validation lot of the Quantifiler Duo kit to establish baseline DNA concentrations from which the dilutions were made Mapping The chromosomal location of the
133. tors for standards the Task and Quantity were applied to the wrong detector see Example 1 on page 5 6 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 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 Quantity was entered and reanalyze Quantifiler Duo DNA Quantification Kit User s Manual 5 5 Chapter 5 Interpretation of Results Table 5 2 Troubleshooting the standard curve continued Observation Possible Cause Recommended Action At each concentration only one standard curve is shown either for the Duo Human or for the Duo Male detector Only one detector was applied either Duo Human or Duo Male to each concentration of the standard curve see Example 3 on page 5 10 From the plate document double click a well containing a DNA quantification standard to view the Well Inspector Verify that both detectors Duo Human and Duo Male are applied select the correct Task and Quantity and reanalyze At each concentration the C values for the Duo Human detector are higher than the Cz
134. uM parang mm axo HD PU Ho wo Poo 19 m do doo GA 5 se 0 09 100M gt pr eb E E al i Dil a erai IS pl mmm al AA mmm 12 5uM gt 2000 m D LTD ioi i mm a fo SO te Hei oo Poo d m wo ts c9 cM o ze 9 280 28 e Gb coo ao 390 300 Sb sese so 15 uM x 200 4000 D 070607 Juinbew validation rati 17 suht E03 fsa hematin _17 Sul Meister vi mm aeo Moo t0 te t0 180 teo 10 teo t90 20 20 22o 2w 2a 280 28o 20 zu 28 soo m0 sao sao so oso 17 5 jM 7 2000 4000 D 70607 Rainbow vatidatian hanain 20uM Pos a hemal n_20uM Paang mm aeo Moo e Ho ta s wo SO wo go ze 20 ze 2 2a 280 20o 20 Gh 29o soo 20 sao so sw so 20uM 7 2000 4006 D 070607 Rainbew validation hematin 4buM G03fsa hematin 4uM Menifee v mm aeo SO fe te Po Gb mmo do oo 2m 20 20 29 20 20 z Z0 Ze E se 90 o X G9 seo 09 40 jM 2000 4000 a Jl 1 i Figure 6 9 Identifiler Kit profiles obtained for samples containing a range of hematin concentrations Label corresponds to inhibitor concentration in the Quantifiler Duo reaction 6 20 Quantifiler Duo DNA Quantification Kit User s Manual 0 ng uL 1 ng uL 2 ng uL 3 ng uL 3 75 ng uL 7 5 ng uL 11 25 ng uL 15 ng uL 30 ng uL Quantifiler Duo DNA Quantification Kit User s Manual Developmental Validation 070607 Rin validation umi acd Ingul HI f humic a
135. uman or for the Duo Male detector and only one detector either Duo Human or Duo Male is available in the Standard Curve tab In Figure 5 6 below Duo Human is the only detector available 17500 System SDS Software Duo samples troubIEx3 sds Absolute Quantification TEE B File View Tools Instrument Analysis Window Help Us 462 El E EJ Aa Plate Y Spectra Y Component Y Amplification Plot Y Standard Curve p B8x Standard Curve Detector Duo Human x Slope Binan 1 Intercept 28551771 R2 0 997201 Ct Log CO Ready Disconnected NUM Legend Only the Duo Human curve is available for display in the Standard Curve plot Figure 5 6 Example 3 5 10 Quantifiler Duo DNA Quantification Kit User s Manual Troubleshooting the Standard Curve Possible Cause When applying detectors for the standards Duo Male was not selected as shown in Figure 5 7 below m Well Inspector Well s A2 Sample Name Etdi al v Duo Human VIC none Standard Task and M Duoc NED none Quantity m ra nene Em not selected Detector not applied a Omit Well r Passive Reference Add Detector Remove Close ROX BA Figure 5 7 Example 3 Possible Cause Example 4 Observation At each concentration the Cy values for the Duo Human detector are higher than the Cy values for the Duo Male detector As shown in Figure 5 8 below the whole standard curve for the Duo Human detector l
136. uo Pc v Duo Male Task for all 3 detectors set to Unknown a Omit Well Passive Reference Add Detector Remove Close ROX gt Note Samples with identical sample names are treated as replicates by the 7500 System Software Results for replicate reactions are grouped together automatically for data analysis 4 Click Close to close the Well Inspector window Quantifiler Duo DNA Quantification Kit User s Manual 2 23 Chapter 2 Software Setup Setting Thermal Cycler Conditions 2 24 When you are finished assigning all parameters set the thermal cycler conditions as described in the following section Before running a Duo DNA Quantification Kit assay set the thermal cycler conditions by changing the default thermal cycler Sample Volume To set thermal cycler conditions 1 In the plate document select the Instrument tab 2 Change the Sample Volume to 25 uL and select the 9600 Emulation box Note Selecting the 9600 Emulation box reduces the ramp rate Note The thermal cycler protocol validated for use with the Quantifiler Duo kit includes a hold step at 50 C for 2 minutes This step was deleted from the Quantifiler Human and Quantifiler Y Human Male Kit Thermal Cycler Protocol 47500 System SDS Software Plate1 Absolute Quantification B File View Tools Instrument Analysis Window Help Sh Bi Er Ez gt Ela Bi Setup Yinstrument Y
137. utor were interpretable in the mixture samples having 1 1 1 5 and 1 10 ratios of male female DNA as indicated by the arrows in Figure 6 24 Quantifiler Duo DNA Quantification Kit User s Manual 1 0 1 1 1 5 1 20 0 1 RAINBOW 2007 05 21 mixture 7 ID A02 fsa aoo Developmental Validation 2000 0 so so azo sao aw c aso 250 90 810 0 sao 3D 350 360 lail RAINBOW 2007 05 21 mixture 10 D FU fsa 30 4000 2000 D LANE NE RAINBOW 2007 05 21 mixture 11 ID E0lfra 250 30 o 20 sao sao 350 360 lau ETTE eee l d 40o RAINBOW 2007 05 21 mixture 12 ID D0lfsa mixture_ _IB laevis v mm sn 159 a6 Jiro ato ao ao 10 ture 10 Im deter vl mm tao 190 160 piro aso 1o 20o 10 misture 11 1D amit vl mm 10 180 490 470 ato 190 200 200 mixture 12 1D aene vi mm a9 wo 20 220 20 so 300 20 330 sao 350 360 2000 0 LANE NET 10 so 460 40 9 di iahh dh 1 A W i Top panel Male contributor DNA profile Bottom panel Female contributor DNA profile Middle z panels Male female DNA mixtures Arrow denotes male contributor alleles at one locus Figure 6 24 Mixture study 1 STR analysis using the Identifiler Kit Quantifiler Duo DNA Quantification Kit User s Manual 6 45 Chapter 6 Experiments and Results 1 0 1 1 1 5 1 20 6 46 Based on the results from the SRY
138. values when the Quantifiler Duo PCR Reaction Mix is used curve wells click on the Amplification Plot tab then change the AR scale to linear If the AR values look like the ones in the first example set up the reactions again with the correct PCR Reaction Mix G Jej deu2 sjjnseg Jo uoiejeJdieju Assessing Quantity Assessing Quantity Purpose Assay Sensitivity Stochastic Effects Validity If Insufficient DNA Is Present After viewing the results and assessing the quality of the results the analyst can determine whether sufficient DNA is present to proceed with a short tandem repeat STR assay The Quantifiler Duo DNA Quantification Kit can quantify 23 pg uL of human genomic DNA in a sample When 2 0 uL of a sample at this concentration 1s loaded in a reaction the well contains approximately 7 diploid human genome equivalents These equivalents correspond to approximately 14 copies of the Duo Human target locus and approximately 7 copies of the Duo Male target locus Y chromosome loci are haploid In the 23 pg uL concentration range stochastic effects or the statistical effect of sampling alleles present at a very low copy number can produce significant variability in assay results When using samples containing DNA in this concentration range perform replicate analysis to confirm true absence of DNA Detection and quantification of very low concentration 100 pg DNA samples is valid using
139. w Detector Creating The first time you run the Quantifiler Duo DNA Quantification Kit Detectors assay you must create three detectors in the 7500 SDS Software There are two methods for creating these detectors Youcan create the detectors upon first run from the New Document Wizard Select Detector Window or You can create the detectors from an open plate document Both methods are explained below After you create these detectors you do not need to create any others for subsequent runs of the Quantifiler Duo DNA Quantification Kit assays Upon completion proceed to Adding Detectors to the Plate Document on page 2 15 2 10 Quantifiler Duo DNA Quantification Kit User s Manual Setting Up a Plate Document To create detectors 1 Open the New Detector dialog box From the Document Wizard Select Detectors window click New Detector to open the New Detector dialog box or From an open plate document select Tools gt Detector Manager to open the Detector Manager Detector Manager an od arr NED none 2007 06 28 18 17 49 Duo Male ji FAM none 2007 06 28 18 17 14 Add To Plate Document Help Duplicate Add to Plate Document Import Export 6b I Clear Clear all nun EEE F2 HEE Fy Beez Properties Then in the Detector Manager window select File gt New to open the New Detector dialog box

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Quantifiler® Duo DNA Quantification Kit User`s Manual (PN