DNA APPLICATIONS IN FORENSIC DENTISTRY

DRG. OKKY MARITA ARDY, M.SI

 DENTAL (TOOTH)

• The unique structure and location of teeth within the jawbones protects the DNA within
tooth tissues from the degrading effects of environmental factors such as heat and water,
but also from other insults such as fire (1,100°C).
• The very low porosity of teeth also helps protect the DNA from within and from
environmental contamination and exogenous DNA.
 TOOTH SELECTION

• It is important to select the most appropriate tooth and to sample it in a manner that
targets the DNA‐rich tissues while avoiding complicating compounds such as calcium
and collagen, without causing further destruction to the DNA and while avoiding
contamination.
 TOOTH SELECTION

• Anterior or posterior, condition of the tooth (eroded, carious, or previously restored teeth,
age of the tooth, periodontal status, as well as post‐mortem conditions).
• Teeth with the greatest amount of dental pulp, cementum and dentine  the richest
sources of DNA.
• DNA can be extracted from the crown body, root body, and root tip, although the root body
is the region that yields the highest quantities of DNA.
• The use of molar teeth for DNA sampling is recommended by INTERPOL and the DNA
Commission of the International Society of Forensic Genetics (ISFG).
• Canines are often recommended because they offer a balance between ease of extraction
(due to the single root) and size.
 TOOTH COLLECTION

• Contamination of tooth samples with exogenous DNA can occur during initial collection
and storage of teeth, so special precautions should be taken to minimise this risk.
• All personnel working on or near samples that will be used for DNA analysis should wear
clean disposable gowns that fully cover the torso, neck and arms, facemasks, gloves and
hair covers.
 TOOTH COLLECTION

• Teeth being simply cleaned of gross debris by washing with ‘DNA‐free’ water.
• Teeth taken for DNA analysis should be placed in a sterile container that can be sealed
(e.g. a specimen container) and labelled with the DVI number. Details of what tooth has
been removed must be included on the post‐mortem forms.
• Careful sampling can also retain sections of the tooth, for example the enamel, which has
no DNA but may be useful for chemical analysis or morphometric comparison.
• If pulp tissue is sampled then direct polymerase chain reaction (PCR) or an extraction
protocol with a short lysis time will be sufficient to liberate the DNA from the cellular
components of the pulp.
• If, however, dentine and/or cementum are sampled an extraction protocol with a much
longer lysis time will be required to liberate sufficient DNA from the tissues. DNA
contained within the dentine and cementum can be intimately associated with the
hydroxyapatite mineral so removal of this mineral via a demineralisation step may also be
required and involve an overnight incubation period, which then greatly increases the
processing time.
• How to obtain dental DNA:
1. Determine if any soft tissue or blood is adherent to the tooth that should be sampled.
2. Debride that tooth of any plaque or calculus with a curette, and wash thoroughly with hydrogen
peroxide followed by ethanol.
3. If the tooth is intact and is supposed to have been removed from the alveolus recently, a
conventional endodontic access and instrumentation can be performed.
4. Sectioning the tooth provides greater access to pulp.
5. Once the tooth is opened, the walls of the pulp chamber can be curetted or instrumented with a
slow rotary burr. Then pulp tissue can be collected in a wide open sterile tube.
6. Finally, crushing the tooth may be required
COMMON METHOD OF DNA EXTRACTION FROM TEETH

• In an adapted horizontal sectioning procedure, the tooth is circumferentially scored 1 mm

below the cementoenamel junction with a long-shanked round burr, leaving a 3- to 4-mm
wide isthmus of intact tooth structure on the buccal surface. The crown is then physically
separated from the root. A large round burr is subsequently used to remove as much
coronal and root dentin as possible.
• Teeth and bone samples must be cleaned and pulverized prior to DNA extraction.
Skeletal remains are prone to surface DNA contamination from handling, and this must
be removed. Bleach is a common decontaminant and is used routinely for soaking intact
teeth and also for bone fragments by some laboratories. Alternatively, bone surfaces can
be sanded to remove the top layer prior to subsectioning for further grinding. Intact teeth
or bone fragments are often cryogenically ground in a freezer mill to produce a powdered
sample amenable to extraction. Some protocols will incorporate a demineralization
process using high levels of EDTA to completely dissolve the bone powder in order to
maximize recovery of DNA
DNA SAMPLE FROM THE BITE MARK SITE

• The currently recommended swabbing technique is Sweet’s Double Swabbing Technique,

which is also recommended by the Australian Society of Forensic Odontology.
• Materials required
• Two sterile cotton swabs (no preservatives).
• 3 ml of sterile distilled water.
• Fitzpak swab box or similar (holds two swabs)
• The technique is as follows:
1. Systematically moisten the head of a cotton swab in sterile, distilled water.
2. Roll the head of this swab over the area of the saliva stain while using moderate pressure
and a constant circular motion.
3. Permit this first swab to air-dry in a contamination-free environment for at least 30 min.
4. Within 10 s of completing the first swab, roll the tip of the second, dry swab crossways
through the now moist area of the stain.
5. Use a circular motion and light pressure to absorb the moisture from the skin into the
second swab.
6. Allow the second swab to air-dry in a contamination-free environment for at least 30
min.
7. After drying, both swabs are packaged together, sealed, and marked with unique
sample and case numbers.
8. The chain-of-document is completed and samples are presented to the laboratory.
• FTA™ paper (a chemically treated card designed to safely store biological samples like
blood and saliva at room temperature).
PRINSIP PENGAMBILAN SAMPEL DNA

• Spesimen bisa diambil dari barang bukti biologis, barang bukti

yang diambil secara langsung dari orang hidup
• Dalam pengambilan specimen dari orang hidup baik itu korban
atau tersangka, hrs memperhatikan aspek ETIKA.
• Surat permintaan tertulis dari pihak kepolisian
• Inform consent
 PRINSIP PENGAMBILAN SAMPEL DNA

• Ambil jaringan yang kaya akan sel.

• Yg utama adalah darah, bisa darah vena atau darah perifer/tepi. Jika tidak
memungkinkan baru ambil kerokan epitel mukosa mulut, saliva, rambut.
• Pengambilan dari epitel mukosa :
Kumur-kumur terlebih dahulu, mukosa dikerok dgn cotton bud steril. Jika
lgsg diperiksa, taruh cotton bud dalam tabung steril, beri label. Jika
diperiksa di laboratorium lain, cotton bud dikeringkan dahulu dgn suhu
ruang agar tidak timbul jamur.
• Pengambilan dari saliva :
Kumur-kumur dahulu, pakai kertas saring taruh setengahnya diantara lidah dan palatum,
kemudian tekan perlahan. Tulis identitas pada bagian kertas yg kering, kasih selotip,
kemudian keringkan. Setelah kering, masukkan dalam amplop kertas dan beri label.
• LABEL : keterangan nomor kasus, tgl dan waktu pengambilan, nama org yg diperiksa,
jenis kelamin, umur, jenis spesimen, nama petugas yg mengambil spesimen.
• Hindari kontaminasi :
1. Selalu pakai alat-alat yg steril, sekali pakai
2. Jgn menyentuh bagian yg kemungkinan mengandung DNA
3. Jgn berbicara, bersin, batuk di atas barang bukti
4. Jgn menyentuh wajah, bibir, hidung ketika mengumpulkan barang bukti
PRINSIP PENYIMPANAN SPESIMEN

• Any sample (soft tissue, bone, or tooth) that is wet should be stored in a leakproof
container and stored frozen (−20°C) prior to testing. Skeletal elements that are completely
dry can be stored in breathable packaging (paper, box, etc.) and stored at room
temperature.