Malaysi*. azlina @ kb. usm. mv lntroduction

POSTER PRESENTATTONS: HUMAN GENETICS
PHO2
Mitochondrial DNA Extraction lsolated from Tooth Dentine - A
Comparison of Two Techniques
Azlina, A.1'2,Zuraitah,B.2, M.R. Sidek.l, and A.R. Samsudin2
Human Genome Centre, Universiti Sains Malaysia Health Campus, 16150
Kubang Kerian, Kelantan, Malaysial and Schootof Dentatsciences,
Universiti Sains Malaysia Health Campus, 16150 Kubang Kerian, Kelantan,
Malaysi*. azlina @ kb. usm. mv
lntroduction
Several studies showed that the tooth is a good source of both nuclear and
mitochondrial DNA. A tooth is made up of 3 major tissues; enamel, dentine and pulp. The
odontoblastic processes (also called Tomes' dentinal fibers) project from the odontoblasts
of the pulp into the dentine (Berkovitz et. al., 1992). These processes contain numerous
mitochondria which can be expected to be well protected in the dentinal canaliculae. The
odontoblastic processes undergo regression in an age dependent manner with the
subsequent occlusion of the canaliculae with calcium phosphate crystals. lt is not known
whether the odontoblastic processes are completely disintegrated, or if there are remains
of mitochondria with mtDNA trapped in the canaliculae. The analysis of teeth as the best
preserved parts of the human body, proved to be the basic and irreplaceable method of
identification, as well as the best source for DNA isolation in the use of difficult
identification cases.
The use of molecular techniques applied to human remains has great potential in
human studies since they can be applied to research concerning forensics, archeology,
population studies and paleoanthropology. However, there is disagreement on the quality
of the results and sometimes on the authenticity. ln the case of extraction of DNA from
dentine of teeth, a few techniques are available (Hernandez et. al., 2OO3; Pfeifer et. al.,
1998; Budowle et al.,2OOO, Rohland et. al.,2OO4).ln this study, we tried to evaluate the
viability of our laboratory set-up to accomplish mitochondrial DNA (mtDNA) isolation and
analysis from teeth by comparing two available methods; Pfeifer et. a1.,1998 and Budowle
et. al., 2000. The aim was to follow the methodology as instructed and compare the
quality of the DNA and evaluate the ability DNA extracted to be utilize as a source of
genetic material by PCR analysis using specific primers for mtDNA.
Materials and Methodology
Samples
We used teeth from six different people to test the efficacy of the two different
techniques to extract DNA from tooth dentin.
DNA extraction and Purification
The extracted teeth were obtained from the dentist after extraction and stored at 20"C until use. Then, the teeth were cleaned mechanically and susequently washed with
25ml of sterile distilled water, 107" commercial bleach and 95% ethanol. Using a burr, the
crowns were separated from the roots. The pulp tissues were extracted out by using the
burr and drilling through the pulp cavity. Roots that consists most of the dentine were then
cut into smaller pieces with the burr. The isolated dentine was washed, dried and crushed
into a powder by using mortar and pestle.
The isolated dentine was then subjected to DNA extraction using methodology
based on an article by Pfeifer et. al. (1998) or Budowle et. al. (2OOO). The main differences
of the techniques are summarised in Table '1.
Proceedings of the dh National Congress on Genetics, 12-14 May 2005, Kuata Lumpur
173
POSTER PRESENTATIONS: HUMAN GENETICS
Pfeifer
Chemical
elements in DNA
Extraction Buffer
m mMTns, pH 8'0; 100 mM
Budowle
10mM TRIS
-
100 mM Sodium
50 Chloride (NaCl) 39 mM
Dithiothreitol (DTT) - 10 mM EDTA
pH
0.5%
8.0;
mM EDTA,
Sodium Dodecyl Sulfate 2% Sodium Dodecyl Sulfate
Sodium Chloride (NaCl);
concentrator
Measurement of DNA Concentration by Spectrophotometry
Spectrophotometer analysis at 260nm and 280nm was carried out to evaluate the
concentration and the purity of the DNA. Readings for DNA concentration were taken at
260nm and for 280nm for protein concentration in the samples. lf there were
contamination with protein or phenol, this ratio wouldl be significantly less than the values
given above, and accurate quantitation of the amount of nucleic acid would not be
possible.
Principles:DNA molecules absorb light of certain wavelength. Light of 260nm passing through
Sgpg/ml DNA solution in 1 cm cuvette will give absorbance reading of 1.0. Therefore, 1.0
ODiOOnm = S0Lrg/ml. Based on the formula, the DNA concentrations were calculated,
The ratio of OD 26}l280nm will give the purity of DNA solution towards protein
contamination; pure DNA = ralio 2601280 of 1.7-2.0.
Results and Discussion
Based on the results in Table 2 and 3, it showed that the concentrations of DNA
extracted using method by Budowle et. a\.,2000 was higher than Pfeifer et. al., 1998.
However, the DNA purity was low in all samples extracted by either method. Both
methods turned out to be reliable techniques of DNA extraction from tooth dentine and
able to produce a good concentration of DNA. However, the Budowle method produced a
higher DNA concentration. The reasons might be because of the usage of dithiothreitol
(DTT) (Amresco, USA), a reducing agent in the DNA extraction buffer that reduce any
disulfide bonds and thus break those covalent bonds of the proteins. Both methods used
centrifugal filter devices to concentrate macromolecules solutions; which was in this study
DNA solution. Budowle method chosed to utilise Centricon YM-100 (Millipore, USA) might
be because of the compatibility of the device's filter membrane to DTT.
Table 2: DNA concentration extracted usinq Pfeifer Method
Samples
G11
G14
Weight (g)
0.18
o.20
0.35
0.10
1.29
1.39
DNA concentration
(p
g/ml)
DNA purity (OD 2697269)
Table 3: DNA concentration extracted usino Budowle Method
Samples
GD
GE
Weight
o.25
0.27
DNA concentration (pg/ml)
1.19
3.36
DNA purity (OD 26s72s9)
1.35
1.48
Proceedings of the dn National Congress on Genetics,
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2-14 May 20o5, Kuala Lumput
174
POSTER PRESENTATTONS: HUMAN GENETTCS
Acknowledgements
This work was supported by short term grants from USM 304/PPSG/6131356.
Authors would also like to thanks for all the supports from Dental Clinic and Human
Genome Centre, USM Health Campus.
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