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Genomics and Applied Biology 2015, Vol. 6, No. 1, 1-10
http://gab.biopublisher.ca
7
1.8 Evaluation of Y Chromosome Lineage
PCR-RFLP Analysis
Here we present Y chromosome lineage assays based
on a more widely accessible technique Polymerase
Chain Reaction- Restriction Fragment Lenght
Polymorphism (PCR-RFLP). PCR products (male
human bones) are always observed with following
restriction endonuclease digestion protocol. Digested
DNA fragments vary from 201 bp to 12.216 bp, but
no single assay requires this range of DNA fragment
sizes to be discriminated. All Y-chromosome lineages
can be assigned correctly by examining restriction
fragments between 201 bp to 12.216 bp, and these
fragment sizes are easily distinguished following
standart 2% agarose gel electrophoresis. RFLP protocol:
Protocol containing 2 μl 10 x buffer, 0,5 μl Lambda
EcoRI/Hind III , 0.2 μl 100 x BSA, 13 μl water and 2
μl PCR products belonging to female aDNA. 55°C
and 2 hours incubation. These protocols that require
only a minimal molecular biology setup fulfil two
niche roles. PCR-RFLP protocols require little
experimental equipment and limited technical expertise.
1.9 Microsatellite DNA Profiling and Authentication
DNA was extracted from each sample at least twice.
From each extract, at least four PCR amplifications
were made. Only if two of four amplification reactions
resulted in consistent allele determinations, and if this
could be reproduced in another extract, were the
alleles rated reproducible and thus authentic.
1.10 Statistical Analyses
Statistical analyses were performed in Microsoft Excel.
Single factor Analysis of Variance (ANOVA) was
used to examine the effect of weathering stage, sex,
age, and bone type on the DNA quantity and quality
results from the Koranza bone samples. ANOVA was
also used to examine the effect of skeletal weathering
on amplification success of multiple bones from a
single individual, as well as the effect of skeletal
weathering, bone weathering, and bone type on
amplicon size. DNA quantities for sex and age
statistics were averaged when multiple bones
originated from a single individual. Amplification
success DNA quantity was examined using a t-test
with equal variance assumed. In all cases results were
considered significant at p<0.05.
2 Results and Discussion
There has been growing interest in PCR amplification
of DNA from ancient remains and preserved bone
tissues, which is expected to provide important
information in population and evolutionary studies.
The extraction method from fossil bones employed in
this study. In the present article, partial genealogical
reconstruction was obtained using biparental, paternal,
and maternal genetic systems in a sample of 100
human skeletal remains of which is provided by A.
Ahmet TIRPAN and fellowships. The ancient ruins of
Koranza and Necropal area are situated in the region
of modern city Mugla. More than hundred ancient
tombs have been excavated and a lot 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. Sex identification of ancient human
is essential for the exploration of gender differences in
past population. Investigation of gender differences
plays an important role in archeology reconstruction
of the structure of past societies and particularly
demography of historical socities based on skeletal
remains from cemeteries (Faerman M., et. al., 1995).
To the best of our knowledge, no equivalent molecular
analysis has been undertaken so far. Such a study was
possible because the Koranza necropolis was mainly
composed of relatively well-preserved skeletons. In
this study, genomic DNA from 100 individual remains
from Koranza Skeletal Remains in Turkey was
extracted within one month after removal of bone
from cave. Before extraction, cleaned specimens were
stored at room temperature. Under optimal analysis
conditions, they immediately were stored in lab
frezeer. Before DNA extraction from bone,
decalcification procedure was applied to all bone. In
this stage, addition of an aliquot of proteinase K to
bone powder was carried out at 56
o
C in order to
remove proteins such as collagen, osteocalcin and
noncallogen structure from DNA. After removal of
protein, genomic DNA extracted from each sample
was extracted automatically by using EZ1 DNA
isolation system (Qiagen- Germany) with investiagator
kit (Qiagen). They monitored amount and purity of
genomic DNA by UV spectrophotometer (Shimadzu
UV 1700) at 260 nm/280 nm as 1,8 value. Image of
DNA on 1 % agarose gel (W/V) in TAE buffer and
