Association of Forensic DNA Analysts and Administrators Removal of Exogenous DNA From Tooth Samples Zury Phillips, MS, F-ABC June 29 th , 2012
Background • DNA identification of human remains is invaluable when they are significantly decomposed or charred • Teeth are the hardest substances in the human body • Known to survive http://forensicodontology.net/wp- decomposition, water content/uploads/2011/12/DSC00073.png immersion, burial, or fires (up to 1100°C)
Background • Dental pulp is a loose connective tissue of the periodontal ligament through the apex of the root – Cells – Fibers – Ground Substance – Blood Vessels and Nerves • It is the cellular material inside the tooth we are targeting for DNA analysis – Challenge is to collect and type ONLY that DNA http://www.hochendodontics.com/ImagesDR/ toothlabel.jpg
Recent Case • The surface of a tooth was decontaminated using our current protocol • DNA analysis yielded a male profile • Source of tooth was determined to be female • Male profile was consistent with the anthropologist who From webspace.ship.edu From thevisualmd.com handled the tooth
Decontamination Methods • Application • Concentration • Exposure time Chemical Means Physical Means • Scrubbing (Hard • 5% Bleach bristle toothbrush) • DNA Exitus • Soaking • Tergazyme • UV irradiation • (95%) Ethanol • DI H 2 O
Optimization Experiments • Determine the optimal treatment for the removal of exogenous DNA from fragmented tooth samples • 5 ng of saliva added to each treatment set: – 3 Animal experimental teeth – 1 Positive control animal tooth – 1 Negative control animal tooth – 1 Reagent blank
Tooth Extraction • Decontamination treatment applied • Whole tooth extracted using the Bone and Tooth Extraction QIAsymphony Pretreatment procedure – 500uL of master mix – 450uL QIAgen Proteinase K, 450 µL DTT, 3.6 mL ATL buffer • Overnight incubation on a Thermomixer at 900rpm and 56° C • Lysate removed and further analyzed to determine the success of the decontamination method.
DNA Analysis of Lysate Sample Purification on QIAGEN QIAsymphony SP Quant, NORM, and AMP on Tecan Freedom EVO 100 Quantification with Quantifiler Duo TM on ABI 7500 Amplification with Identifiler Plus TM on ABI 9700 CE on ABI 3130 xl Genetic Analyzer Data Analysis Using GeneMapper ID
Treatments A-C & P • Treatment A – Scrub with brush plus DNA Exitus, rinse with DiH 2 0 • Treatment B – Scrub with brush plus 5% bleach, rinse with DiH 2 0 • Treatment C – Scrub with brush plus Tergazyme, rinse with DiH 2 0 • Treatment P – UV irradiation for 20 min, rotating half-way through
Results Physical Cleaning and UV Target DNA Amount 5ng 25.00 0.18 0.16 20.00 0.14 0.12 15.00 0.10 0.08 10.00 0.06 0.04 5.00 0.02 0.00 0.00 Treatment A Treatment B Treatment C Treatment P Average % Profile Detected Average Total DNA Recovered (ng) Treatment A (scrub with DNA Exitus) Treatment B (scrub w/Bleach) Treatment C (scrub w/Tergazyme) Treatment P (UV only-20 minutes)
Treatments Studied Treatments DNA Exitus diH 2 0 Ethanol UV D 20 min 20 min 20 min 20 min E 20 min 20 min 20 min F 20 min 20 min G 10 min 10 min 10 min 10 min H 10 min 10 min 10 min I 10 min 10 min Treatments Bleach diH 2 0 Ethanol UV J 20 min 20 min 20 min 20 min K 20 min 20 min 20 min L 20 min 20 min M 10 min 10 min 10 min 10 min N 10 min 10 min 10 min O 10 min 10 min
Results DNA Exitus Treatments Target DNA Amount 5ng 80.00 0.80 70.00 0.70 Total DNA (ng) 60.00 0.60 % Profile 50.00 0.50 40.00 0.40 30.00 0.30 20.00 0.20 10.00 0.10 0.00 0.00 Average % Profile Detected Average Total DNA Recovered (ng) Treatment D (Exitus, diH2O, EtOH, UV) Treatment E (Exitus, diH2O, EtOH) Treatment F (Exitus, diH2O) Treatment G (Exitus, diH2O, EtOH, UV) 10 min intervals Treatment H (Exitus, diH2O, EtOH) 10 minute intervals Treatment I (Exitus, diH2O) 10 minute intervals
Results Bleach Treatments Target DNA Amount 5 ng 10.00 0.12 Total DNA (ng) 0.10 8.00 % Profile 0.08 6.00 0.06 4.00 0.04 2.00 0.02 0.00 0.00 Average % Profile Detected Average Total DNA Recovered (ng) Treatment J (Bleach, diH2O, EtOH, UV) 20 min intervals Treatment K (Bleach, diH2O, EtOH) 20 min intervals Treatment L (Bleach, diH2O) 20 min intervals Treatment M (Bleach, diH2O, EtOH, UV) 10 min intervals Treatment N (Bleach, diH2O, EtOH) 10 min intervals Treament O (Bleach, diH2O) 10 min intervals
DNA Exitus vs. Bleach • Only the most stringent DNA Exitus treatment removed all detectable amounts of DNA • Bleach far outperformed DNA Exitus – Proprietary – Declared to cause strand breakages towards degradation of DNA molecules • Bleach – Documented to affect DNA through oxidative damage and production of chlorinated base products
Sensitivity Study • Top 8 Decontamination Treatments were tested with increasing amounts of DNA – 10ng, 25 ng, 50 ng, and 100 ng of saliva • Evaluation of the treatment – Successfully remove the maximum amount of DNA and resulting profiles – Determine optimal treatment
Results Sensitivity Study Target DNA Amount 10 ng 1.20 1.00 0.80 0.60 0.40 0.20 0.00 Average Total DNA Recovered (ng) B (scrub w/Bleach) C (scrub w/Tergazyme) D (Exitus, diH2O, EtOH, UV) J (Bleach, diH2O, EtOH, UV) L (Bleach, diH2O) M (Bleach, diH2O, EtOH, UV) 10 min intervals N (Bleach, diH2O, EtOH) 10 min intervals P (UV only-20 minutes)
Results Sensitivity Study Target DNA Amount 10ng 100.00 % Profile Detected 80.00 60.00 40.00 20.00 0.00 Average % Profile Detected B (scrub w/Bleach) C (scrub w/Tergazyme) D (Exitus, diH2O, EtOH, UV) J (Bleach, diH2O, EtOH, UV) L (Bleach, diH2O) M (Bleach, diH2O, EtOH, UV) 10 min intervals N (Bleach, diH2O, EtOH) 10 min intervals P (UV only-20 minutes)
Results Sensitivity Study Sensitivity Study Target DNA Amount 25ng Target DNA Amount 25ng 4.00 100.00 Total DNA (ng) 80.00 3.00 60.00 2.00 40.00 1.00 20.00 0.00 0.00 Treatment B Treatment C Treatment D Treatment J Treatment L Treatment M Treatment N Treatment P Average Total DNA Recovered (ng) Average % Profile Detected Treatment B (scrub w/Bleach) Treatment C (scrub w/Tergazyme) Treatment D (Exitus, diH2O, EtOH, UV) 20 min intervals Treatment J (Bleach, diH2O, EtOH, UV) 20 min intervals Treatment L (Bleach, diH2O) 20 min intervals Treatment M (Bleach, diH2O, EtOH, UV) 10 minute intervals Treatment N (Bleach, diH2O, EtOH) 10 min intervals Treatment P (UV only-20 minutes)
Results Sensitivity Study Sensitivity Study Target DNA Amount 50ng Target DNA Amount 50ng 4.00 100.00 Total DNA (ng) 80.00 3.00 60.00 2.00 40.00 1.00 20.00 0.00 0.00 Average Total DNA Recovered (ng) Average % Profile Detected Treatment D (Exitus, diH2O, EtOH, UV) Treatment J (Bleach, diH2O, EtOH, UV) Treatment L (Bleach, diH2O) Treatment M (Bleach, diH2O, EtOH, UV) 10 minute intervals
Results Sensitivity Study Sensitivity Study Target DNA Amount 100ng Target DNA Amount 100ng 2.50 100.00 Total DNA (ng) 2.00 80.00 1.50 60.00 1.00 40.00 0.50 20.00 0.00 0.00 Average Total DNA Recovered (ng) Average % Profile Detected Treatment D (Exitus, diH2O, EtOH, UV) Treatment J (Bleach, diH2O, EtOH, UV) Treatment L (Bleach, diH2O) Treatment M (Bleach, diH2O, EtOH, UV) 10 minute intervals
Current Method vs. Treatment M Average Total DNA Recovered 15.00 Total DNA (ng) 10.00 5.00 0.00 5 ng 10 ng 25 ng 50 ng 100 ng In house Treatment Treatment M Average % Profile Detected 100.00 50.00 0.00 5 ng 10 ng 25 ng 50 ng 100 ng In house Treatment Treatment M
Optimal Treatment Selected • Treatment M • Selection based on four points – Treatment M removed more of the total DNA than all other treatments in all trials – This method utilizes two wash steps for removal of residual bleach from the sample, reduces toxic interaction with the Qiagen reagents – Method supported by literature
Reproducibility • 1 μ L of ten different donor saliva samples were deposited onto ten tooth samples • Treatment M was conducted in duplicate and the current method was conducted once • All teeth were then extracted and typed with our current protocols • DNA yield and Amplification success were evaluated
Reproducibility Results Average Total DNA Recovered 20.000 15.000 10.000 Total DNA (ng) 5.000 0.000 -5.000 -10.000 Treatment M Current Method
Reproducibility Conclusions • Treatment M reliably removes significantly more exogenous DNA from tooth samples than the existing method. • Some variation from run to run was observed – Uneven tooth surfaces – Varying exposure of enameled portions of teeth • Affects binding of DNA to tooth and therefore removal from the tooth – Saliva itself a highly variable sample
Non-Probative Samples • Nine (9) human tooth samples (including 3 molars, 3 bicuspids, and 3 incisors) were decontaminated using Treatment M – Prior to decontamination, teeth were stored in 50mL conical tubes with multiple other teeth – In addition, 1 µL of saliva from 9 donors were deposited onto teeth • Mimics worst case scenario for addition of exogenous DNA to a single tooth
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