Association of Forensic DNA Analysts and Administrators
Removal of Exogenous DNA
From Tooth Samples
Zury Phillips, MS, F-ABC
June 29th, 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/wpdecomposition, 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
From webspace.ship.edu
• 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 thevisualmd.com
Decontamination Methods
• Application
• Concentration
• Exposure time
Physical Means
Chemical Means
• Scrubbing (Hard
bristle toothbrush)
• Soaking
• UV irradiation
•
•
•
•
•
5% Bleach
DNA Exitus
Tergazyme
(95%) Ethanol
DI H2O
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
Physical Cleaning and UV
Target DNA Amount 5ng
25.00
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
20.00
15.00
10.00
5.00
0.00
Treatment A Treatment B
Average % Profile Detected
Treatment A
Treatment B
Treatment C
Treatment P
Treatment C
Treatment P
Average Total DNA Recovered (ng)
(scrub with DNA Exitus)
(scrub w/Bleach)
(scrub w/Tergazyme)
(UV only-20 minutes)
Treatments Studied
Treatments
DNA Exitus
diH20
Ethanol
UV
D
E
F
20 min
20 min
20 min
20 min
20 min
20 min
20 min
20 min
20 min
G
H
I
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
Treatments
Bleach
diH20
Ethanol
UV
J
K
L
20 min
20 min
20 min
20 min
20 min
20 min
20 min
20 min
20 min
M
N
O
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
10 min
Results
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
Average % Profile Detected
Treatment D
Treatment E
Treatment F
Treatment G
Treatment H
Treatment I
Average Total DNA Recovered (ng)
(Exitus, diH2O, EtOH, UV)
(Exitus, diH2O, EtOH)
(Exitus, diH2O)
(Exitus, diH2O, EtOH, UV) 10 min intervals
(Exitus, diH2O, EtOH) 10 minute intervals
(Exitus, diH2O) 10 minute intervals
Total DNA (ng)
% Profile
DNA Exitus Treatments
Target DNA Amount 5ng
Results
10.00
8.00
6.00
4.00
2.00
0.00
0.12
0.10
0.08
0.06
0.04
0.02
0.00
Average % Profile Detected
Treatment J
Treatment K
Treatment L
Treatment M
Treatment N
Treament O
Average Total DNA Recovered (ng)
(Bleach, diH2O, EtOH, UV) 20 min intervals
(Bleach, diH2O, EtOH) 20 min intervals
(Bleach, diH2O) 20 min intervals
(Bleach, diH2O, EtOH, UV) 10 min intervals
(Bleach, diH2O, EtOH) 10 min intervals
(Bleach, diH2O) 10 min intervals
Total DNA (ng)
% Profile
Bleach Treatments
Target DNA Amount 5 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
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
C
D
J
L
M
N
P
(scrub w/Bleach)
(scrub w/Tergazyme)
(Exitus, diH2O, EtOH, UV)
(Bleach, diH2O, EtOH, UV)
(Bleach, diH2O)
(Bleach, diH2O, EtOH, UV) 10 min intervals
(Bleach, diH2O, EtOH) 10 min intervals
(UV only-20 minutes)
Results
% Profile Detected
Sensitivity Study
Target DNA Amount 10ng
100.00
80.00
60.00
40.00
20.00
0.00
Average % Profile Detected
B
C
D
J
L
M
N
P
(scrub w/Bleach)
(scrub w/Tergazyme)
(Exitus, diH2O, EtOH, UV)
(Bleach, diH2O, EtOH, UV)
(Bleach, diH2O)
(Bleach, diH2O, EtOH, UV) 10 min intervals
(Bleach, diH2O, EtOH) 10 min intervals
(UV only-20 minutes)
Results
Sensitivity Study
Target DNA Amount 25ng
Average Total DNA Recovered (ng)
Treatment B
Treatment C
Treatment D
Treatment J
Treatment L
Treatment M
Treatment N
Treatment P
Treatment P
Treatment N
Treatment M
Treatment L
Treatment J
Treatment D
Treatment C
4.00
3.00
2.00
1.00
0.00
Treatment B
Total DNA (ng)
Sensitivity Study
Target DNA Amount 25ng
100.00
80.00
60.00
40.00
20.00
0.00
Average % Profile Detected
(scrub w/Bleach)
(scrub w/Tergazyme)
(Exitus, diH2O, EtOH, UV) 20 min intervals
(Bleach, diH2O, EtOH, UV) 20 min intervals
(Bleach, diH2O) 20 min intervals
(Bleach, diH2O, EtOH, UV) 10 minute intervals
(Bleach, diH2O, EtOH) 10 min intervals
(UV only-20 minutes)
Results
Total DNA (ng)
Sensitivity Study
Target DNA Amount 50ng
4.00
Sensitivity Study
Target DNA Amount 50ng
100.00
80.00
60.00
40.00
20.00
0.00
3.00
2.00
1.00
0.00
Average Total DNA Recovered (ng)
Average % Profile Detected
Treatment D
Treatment J
Treatment L
(Exitus, diH2O, EtOH, UV)
(Bleach, diH2O, EtOH, UV)
(Bleach, diH2O)
Treatment M
(Bleach, diH2O, EtOH, UV) 10 minute intervals
Results
Total DNA (ng)
Sensitivity Study
Target DNA Amount 100ng
2.50
2.00
1.50
1.00
0.50
0.00
Sensitivity Study
Target DNA Amount 100ng
100.00
80.00
60.00
40.00
20.00
0.00
Average Total DNA Recovered (ng)
Average % Profile Detected
Treatment D
Treatment J
Treatment L
(Exitus, diH2O, EtOH, UV)
(Bleach, diH2O, EtOH, UV)
(Bleach, diH2O)
Treatment M
(Bleach, diH2O, EtOH, UV) 10 minute intervals
Current Method vs. Treatment M
Total DNA (ng)
Average Total DNA Recovered
15.00
10.00
5.00
0.00
5 ng
10 ng
25 ng
In house Treatment
50 ng
100 ng
Treatment M
Average % Profile Detected
100.00
50.00
0.00
5 ng
10 ng
25 ng
In house Treatment
50 ng
Treatment M
100 ng
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
Average Total DNA Recovered
20.000
Total DNA (ng)
15.000
10.000
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
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.
Questions??