Removal of Exogenous DNA From Tooth Samples Zury Phillips, MS, - - PowerPoint PPT Presentation

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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

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SLIDE 1

Removal of Exogenous DNA From Tooth Samples

Zury Phillips, MS, F-ABC June 29th, 2012

Association of Forensic DNA Analysts and Administrators

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SLIDE 2

Background

  • DNA identification of

human remains is invaluable when they are significantly decomposed

  • r 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

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SLIDE 3

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

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SLIDE 4

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

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SLIDE 5

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
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SLIDE 6

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

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SLIDE 7

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.

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SLIDE 8

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

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SLIDE 9

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

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SLIDE 10

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)

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SLIDE 11

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

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SLIDE 12

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

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 Total DNA (ng) % Profile

DNA Exitus Treatments Target DNA Amount 5ng

Average % Profile Detected Average Total DNA Recovered (ng)

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SLIDE 13

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

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.00 2.00 4.00 6.00 8.00 10.00 Total DNA (ng) % Profile

Bleach Treatments Target DNA Amount 5 ng

Average % Profile Detected Average Total DNA Recovered (ng)

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SLIDE 14

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

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SLIDE 15

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

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SLIDE 16

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)

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SLIDE 17

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)

0.00 20.00 40.00 60.00 80.00 100.00 % Profile Detected

Sensitivity Study Target DNA Amount 10ng

Average % Profile Detected

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SLIDE 18

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 Treatment B Treatment C Treatment D Treatment J Treatment L Treatment M Treatment N Treatment P Total DNA (ng)

Sensitivity Study Target DNA Amount 25ng

Average Total DNA Recovered (ng)

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SLIDE 19

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 Total DNA (ng)

Sensitivity Study Target DNA Amount 50ng

Average Total DNA Recovered (ng)

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SLIDE 20

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 Total DNA (ng)

Sensitivity Study Target DNA Amount 100ng

Average Total DNA Recovered (ng)

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SLIDE 21

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 Total DNA (ng)

Average Total DNA Recovered

In house Treatment Treatment M

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SLIDE 22

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

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SLIDE 23

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
  • ur current protocols
  • DNA yield and Amplification success were

evaluated

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SLIDE 24

Reproducibility Results

  • 10.000
  • 5.000

0.000 5.000 10.000 15.000 20.000 Total DNA (ng)

Average Total DNA Recovered

Treatment M Current Method

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SLIDE 25

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

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SLIDE 26

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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SLIDE 27

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

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SLIDE 28

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)
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SLIDE 29

Acknowledgements

  • Daniel Corona, BS
  • Rebecca Mikulasovich, MS
  • Michael Donley, MS, F-ABC
  • Roger Kahn, PhD, F-ABC
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SLIDE 30

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

  • f 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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SLIDE 31

Questions??