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Genomics and Applied Biology 2015, Vol. 6, No. 1, 1-10
http://gab.biopublisher.ca
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interpopulation analyses. In conclusion, we have
established that DNA can be recovered from ancient
bone, often less degraded than the DNA recovered
from soft tissue remains, and that aDNA sequences
can be amplified consistently from many
archaeological samples. In addition, archaeologists
and conservators will need to learn of the potential for
genetic information in excavated skeletal remains, and
to develop appropriate methods for the removal and
storage of samples for future study. We are now
beginning to work on a wider range of bone samples,
and although the oldest bones we have studied so far
date to only about 5000 years BP (Hagelberg et al.
1989), the analysis of chloroplast DNA from a 17-20
million-year-old magnolia leaf (Golenberg et al. 1990)
suggests that in exceptional circumstances DNA may
survive indefinitely, which augurs well for the study
of hominid evolution. Although archaeology,
anthropology and forensic science will probably profit
most from PCR bone analysis, these methods will also
be applicable to studies of evolutionary and
population biology, for example in resolving questions
of the phylogenetic relations between extinct and
living taxonomic groups. In the present work, we have
established a sensitive and reliable aDNA extraction
and a PCR reaction based on amplifying part of intron
1 of the AMG- amelogenin encoding gene- is more
suitable. The method facilitates analysis of samples
with degraded DNA, such as frequently encountered
in archaeological specimens, with high confidence.
Furthermore, this study demonstrates the applicability
of the methods for gender determination in skeletal
remains from different periods in Koranza and
Necropal area are situated in the region of modern city
Mugla in Turkey.
1 Material and Method
1.1 Collection of Samples
A subset of 100 bones from the total set obtained from
the ancient ruins of Koranza and Necropal area are
situated in the region of modern city Mugla in Turkey.
More than hundred ancient tombs has been excavated
and a lots of grave gifts and skeletal remains were
found in this graves. According to this finds, the date of
this graves goes back to the 7th century B.C. Upon
recovery of the skeletal remains, the bones were
described in terms of sex, uncontaminated or clear and
pure DNA, estimated age, and skeletal weathering
stages. In order to allow ratings on individual bones, a
new staging system was developed in our laboratory
and assigned to each bone based on visual inspection
for the DNA study (Table 1).
1.2 Contamination Prevention of Fossil Human
Bones
Ancient DNA research has proved a powerful tool for
studying archaeological remains, but technical
problems, as the degradation into small bits (100 to
200 bp on average), the oxidation, the effects of
background radiation and the exogenous
contamination, continue to make these degraded
molecules extracted from fossil and subfossil difficult
to study. The only solution is to adopt strict laboratory
precautions. To prevent any possible risk of
contamination, several precautions were adhered to:
These; with DNA extraction and PCR studies, sample
preparation workings were done dedication of
equipment to the different laboratory rooms.
Furthermore, no-template controls were used in every
PCR run to monitor contamination of reagents.
1.3 Decalcifications of Bone and Genomic DNA
Extraction
Extraction of DNA was carried out using the
laboratory handling and cleaning protocol (Römpler
H., et. al., 2006). After cleaning of bone with
chromatographic water, small piece of ancient bones
were ground to powder with a mixer mill. Aliquots of
the powder were subjected to a calcification method.
150 mg of bone powder was extracted with 0.7 ml of
0.5 M EDTA (pH 8.3) for 48 hours at 56
o
C. After
addition of 1U of proteinase K, solution of bone was
incubated at 37
o
C. Genomic DNA from supernatant
was extracted automatically by using EZ1
Automatic DNA Isolation System (Qiagen,
Germany) with investgator kit (Qiagen, Ilden,
Germany) from ancient bones. Amount and purity
of extracted DNA from ancient bones were
measured by Shimadzu UV 1700 Spectrophotometer.
Extracted DNA was then stored at -20°C until assay
for the amelogenin was performed (Figure 2).
