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TRANSCRIPT
Removal of Exogenous DNA From Tooth Samples
Zury Phillips, MS, F-ABC
June 29th, 2012
Association of Forensic DNA Analysts and Administrators
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 decomposition, water immersion, burial, or fires (up to 1100°C)
http://forensicodontology.net/wp-content/uploads/2011/12/DSC00073.png
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 handled the tooth
From webspace.ship.edu From thevisualmd.com
Decontamination Methods
Physical Means Chemical Means
• 5% Bleach
• DNA Exitus
• Tergazyme
• (95%) Ethanol
• DI H2O
• Scrubbing (Hard bristle toothbrush)
• Soaking
• UV irradiation
• Application • Concentration • Exposure time
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 DuoTM on ABI 7500
Amplification with Identifiler PlusTM on ABI 9700
CE on ABI 3130xl Genetic Analyzer
Data Analysis Using GeneMapper ID
Treatments A-C & P
• Treatment A – Scrub with brush plus DNA Exitus, rinse with DiH20
• Treatment B – Scrub with brush plus 5% bleach, rinse with DiH20
• Treatment C – Scrub with brush plus Tergazyme, rinse with DiH20
• Treatment P – UV irradiation for 20 min, rotating half-way
through
Results
Treatment A (scrub with DNA Exitus)
Treatment B (scrub w/Bleach)
Treatment C (scrub w/Tergazyme)
Treatment P (UV only-20 minutes)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.00
5.00
10.00
15.00
20.00
25.00
Treatment A Treatment B Treatment C Treatment P
Physical Cleaning and UV Target DNA Amount 5ng
Average % Profile Detected Average Total DNA Recovered (ng)
Treatments Studied Treatments DNA Exitus diH20 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 diH20 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
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
0.000.100.200.300.400.500.600.700.80
0.0010.0020.0030.0040.0050.0060.0070.0080.00
Tota
l DN
A (
ng)
% P
rofi
le
DNA Exitus Treatments Target DNA Amount 5ng
Average % Profile Detected Average Total DNA Recovered (ng)
Results
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
0.000.020.040.060.080.100.12
0.00
2.00
4.00
6.00
8.00
10.00
Tota
l D
NA
(n
g)
% P
rofi
le
Bleach Treatments Target DNA Amount 5 ng
Average % Profile Detected Average Total DNA Recovered (ng)
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
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Sensitivity Study Target DNA Amount 10 ng
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
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)
0.0020.0040.0060.0080.00
100.00
% P
rofi
le D
ete
cte
d
Sensitivity Study Target DNA Amount 10ng
Average % Profile Detected
Results
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)
0.00
20.00
40.00
60.00
80.00
100.00
Sensitivity Study Target DNA Amount 25ng
Average % Profile Detected
0.00
1.00
2.00
3.00
4.00
Trea
tmen
t B
Trea
tmen
t C
Trea
tmen
t D
Trea
tmen
t J
Trea
tmen
t L
Trea
tmen
t M
Trea
tmen
t N
Trea
tmen
t P
Tota
l DN
A (
ng)
Sensitivity Study Target DNA Amount 25ng
Average Total DNA Recovered (ng)
Results
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
0.00
20.00
40.00
60.00
80.00
100.00
Sensitivity Study Target DNA Amount 50ng
Average % Profile Detected
0.00
1.00
2.00
3.00
4.00
Tota
l DN
A (
ng)
Sensitivity Study Target DNA Amount 50ng
Average Total DNA Recovered (ng)
Results
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
0.00
20.00
40.00
60.00
80.00
100.00
Sensitivity Study Target DNA Amount 100ng
Average % Profile Detected
0.00
0.50
1.00
1.50
2.00
2.50
Tota
l DN
A (
ng)
Sensitivity Study Target DNA Amount 100ng
Average Total DNA Recovered (ng)
Current Method vs. Treatment M
0.00
50.00
100.00
5 ng 10 ng 25 ng 50 ng 100 ng
Average % Profile Detected
In house Treatment Treatment M
0.00
5.00
10.00
15.00
5 ng 10 ng 25 ng 50 ng 100 ng
Tota
l DN
A (
ng)
Average Total DNA Recovered
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 μL 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
-10.000
-5.000
0.000
5.000
10.000
15.000
20.000
Tota
l DN
A (
ng)
Average Total DNA Recovered
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
Non-Probative Samples
• Teeth were crushed, extracted and typed using our current procedures
• All resulting DNA profiles were single source consistent with the original source
• No drop-in alleles were observed from the contaminating donor samples
Conclusions and Future Studies
• Bleach outperformed DNA Exitus in removing exogenous DNA from teeth
• Treatment M reliably removes significantly more exogenous DNA from teeth than any other methods tested
• Treatment M does not interfere with downstream DNA analysis
• Treatment M was selected as the optimal method • Future Studies:
– Establishing the optimal bleach concentration – Comparing bleach against other cleaning agents
• DNA Away (Molecular Bioproducts) • DNA Erase (Sigma-Aldrich)
Acknowledgements
• Daniel Corona, BS
• Rebecca Mikulasovich, MS
• Michael Donley, MS, F-ABC
• Roger Kahn, PhD, F-ABC
References
Cone, R. W. & Farifax, M. R. (1993). Protocol for ultraviolet irradiation of surfaces to reduce PCR contamination. Genome Research, 3, S15-S17. Davoren, J., Crews, J., Huffine, E., Konjhodzić, Parsons, T. J., & Vaneck, D. (2007). Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves. Croation Medical Journal, 48 (4), 478-485. Gaytmenn, R., & Sweet, D. (2003). Quantification of forensic DNA from various regions of human teeth. Journal of Forensic Sciences, 48 (3), 1-4. Kemp, B. M. & Smith, D. G. (2005). Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. Forensic Science International, 154, 53-61 Sweet, D. & Hildebrand, D. (1998). Recovery of DNA from human teeth by cryogenic grinding. Jounal of Forensic Sciences, 43 (6), 1199-1202. Sweet, D., Hildebrand, D., & Phillips, D. (1999). Identification of a skeleton using DNA from teeth and a PAP smear. Journal of Forensic Sciences, 44 (3), 630-633.
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