Purpose: To obtain quantitative value of human (higher primate) nuclear DNA isolated from samples. Materials: Previously extracted DNA 96 well optical reaction plate Optical adhesive cover Compression pad Splash free support base DNA Quantification Standards Dilution Series (50 ng/µl to 0.023 ng/µl) Sterile TE Quantifiler Human Primer Mix Quantifiler PCR Reaction Mix 15 ml polypropylene tube or 1.5 ml snap-cap tube Equipment: Micropipettors and appropriate aerosol resistant tips Microcentrifuge Vortex ABI Prism 7000 Sequence Detection System ABI Prism 7000 SDS Software v1.0 Procedure: 1. Turn on the computer and monitor and log on. 2. Press the power button on the lower left front of the 7000 SDS instrument. 3. Launch the software by double clicking the ABI Prism 7000 SDS Software shortcut on the desktop. 4. To create a new plate document, select File > New. When the New Document window appears, select Assay: Absolute Quantitation, Container: 96-Well Clear, Template: Quantifiler Human. Sdt or Quantifiler Y. Sdt. 5. To add/modify the sample description in a well, click on the desired well and select View > Well Inspector. When the Well Inspector window appears, type in the sample description (i.e., case number and item number) across from sample name. For unknown samples, check to insure unknown is selected from the task menu in the Well Inspector. For unused wells on the 96 well reaction plate, click omit wells in the well inspector window. AN DNA 40-1 of 5
6. Save the plate document by selecting File > Save. In the save as type menu, select SDS Document (*.sds). The file can be used to run the quantification protocol. 7. Place 96 well reaction plate in splash free support base. Do not touch wells or allow wells to come in contact with the counter. 8. Calculate the volume of Quantifiler Human or Y Human Male Primer Mix and Quantifiler PCR Reaction Mix needed to prepare the reactions: 10.5µL Quantifiler Human or Y Human Male Primer Mix Per Well, 12.5 µl Quantifiler PCR Reaction Mix Per Well. *Include additional reactions in calculations (i.e., 1 additional reaction for every 24 wells) to provide excess volume for loss that occurs during reagent transfers. 9. Thaw Quantifiler Human or Y Human Male Primer Mix completely, then vortex 3 to 5 seconds and centrifuge briefly before opening tube. Swirl Quantifiler PCR Reaction Mix gently before using (do not vortex). 10. Pipette required volumes of components into appropriately sized tube. Vortex PCR master mix 3 to 5 seconds then centrifuge briefly. 11. Dispense 23 µl of the PCR master mix into each reaction well. 12. Add 2 µl of sample/standard to the appropriate wells. Seal the reaction plate with an optical adhesive cover. 13. Centrifuge the plate at 3000 rpm for 20 seconds in a table-top centrifuge to remove any bubbles. Place compression pad over the optical adhesive cover with gray side down and brown side up with the holes positioned directly over the reaction wells. 14. Lift the handle at the bottom of the door on the front of the 7000 SDS instrument until the door is raised completely. Gently push the carriage back until it stops and locks into place. 15. Position the plate in the instrument thermal block so that well A1 is in the upper left hand corner. 16. Gently push then release the carriage to unlatch it. The carriage automatically slides forward into position over the sample plate. After the door moves to the front, gently pull the door handle down into place to close the cover. 17. In the 7000 SDS software, open the plate document set up for the run. Select the instrument tab and then click Start. Results: 1. Upon completion of the run, highlight the wells containing data you are interested in analyzing. Review analysis settings by selecting Analysis > Analysis Settings. In AN DNA 40-2 of 5
the Analysis Settings dialog box, verify the settings are as follows: Detector All, Threshold 0.20, Baseline Start (cycle) 6, Baseline End (cycle) 15. Click Analyze. 2. View the standard curve to insure the R 2 value is 0.98. The standard curve slope value should range between -2.9 to -3.3 for Human and -3.0 to -3.6 for Y Human Male. If the standard curve R 2 value is less than 0.98 or the slope value varies beyond the typical range, up to two outlier standard curve wells may be omitted in analysis. If omission of up to two outlier wells fails to increase the R 2 value ( 0.98) and affect the slope value (should range between -2.9 to -3.3 for Human and -3.0 to -3.6 for Y Human Male), the standard curve should not be used, and samples should be re-quantified. 3. View Human DNA and Internal Positive Control quantification results for samples by selecting the Results Tab > Amplification Plot Tab. Then select the desired well(s) and use the data and detector menus to choose a data type or detector. Data may also be reviewed by selecting the Results Tab > Report Tab. In the lower pane, select the desired wells and review results. 4. Use the Quantifiler Human results to determine the volume of isolated DNA to use in STR amplification procedures. Use the Quantifiler Y Human Male DNA results to report. Conclusions/Reporting: Note: As routine practice, DNA quantification yields < 0.025 ng/µl (or <0.0125 ng/µl for sperm fraction) will be reported using the following: PCR amplification procedures were not performed on item(s) ### due to an insufficient amount of human DNA. Note: The results from this procedure for Human quantitation are used in subsequent procedures. Note: The following reporting statements are for reporting results of screening sexual assault collection kits using Y Human Male DNA. If Y DNA is detected in any amount report using the following: No-suspect kits: Examination of swabs from item reveals the presence of human male DNA. Samples will be submitted for DNA analysis. AN DNA 40-3 of 5
Suspect kits where both victim and suspect knowns are present: Examination of swabs from item reveals the presence of human male DNA. Samples will be submitted for DNA analysis. Suspect kits where either the victim s or suspect s knowns are not present: Examination of swabs from item reveals the presence of human male DNA. Upon receipt of adequate known samples from victim and/or subject, samples will be submitted for DNA analysis. If Y DNA is not detected, report the following: from item (number). Examination of swabs from item fails to reveal the presence of human male DNA. Measurement Uncertainty: Measurement to be made Quantity of DNA in a well by analyzing cycle to cycle changes in fluorescence signal as a result of amplification. Accuracy/Precision The R 2 value of the standard curve is 0.98. The standard curve slope value ranges between -2.9 to -3.3 for Human and -3.0 to -3.6 for Y Human Male. The Ct value should not fall below 35 in the blank well. AN DNA 40-4 of 5
References: Quantifiler Human DNA Quantification Kit and Quantifiler Y Human Male DNA Quantification Kit User s Manual. Rev C. ABI Prism 7000 Sequence Detection System User Guide. Rev C. Validation on file AN DNA 40-5 of 5