Abstract |
The study of ancient genetic material (DNA) and proteins in archaeological human or animal
remains (bones, teeth) is of particular interest, as it provides information about the age and
gender of individuals, the living conditions of individuals or groups of individuals, but also
more broadly, migration and interaction among communities and populations. The analysis of
archaeological samples presents several challenges, which stem from the fact that biological
and bio-organic remains are often found in small or even trace amounts, as well as the presence
of exogenous contamination and the large number of samples available for analysis. The
analysis of ancient genetic material is based on the application of PCR (Polymerase Chain
Reaction, PCR) methods, while paleoproteomics studies rely on the use of liquid
chromatography coupled to high-resolution molecular mass spectrometry (Liquid
Chromatography Mass Spectrometry, LC-MS) techniques.
The aforementioned palaeogenetic or paaleoproteomic methods are of high sensitivity and
specificity, but they are characterized by rather high costs and analysis time, while their
application causes partial or total destruction of samples, which may have significant
archaeological value. These factors make it expensive, time-consuming and often prohibitive
to study a sufficient number of samples and thus draw statistically safe conclusions through the
analysis. Therefore, it becomes apparent the need to develop alternative analytical approaches
that will allow rapid examination of archaeological samples at a preliminary level and then
selection of only those with a higher probability of being rich in information for further analysis
with the specialized methods of palaeogenetics and/or palaeoproteomics. Based on such an
approach it is possible to significantly limit the analysis of samples with negligible content in
ancient organic remains.
Biological dental remains constitute a source of endogenous organic material (protein and
genetic) found mainly in the cementum tissue, which surrounds the part of the tooth that is
inside the jaw. It is understood that the endogenous organic material of a tooth is directly
affected by the burial conditions (grave, soil, etc.) and the environmental ones in the burial area.
In the context of this Master's thesis, a study was carried out on the applicability of Raman
scattering spectroscopy and fluorescence emission spectroscopy in terms of estimating the
amount of endogenous genetic material in samples of ancient teeth, indirectly, through the
determination of their content in protein material. It is noted that the presence of collagen
residues in the teeth reflects the preservation of the tooth and in turn the endogenous genetic
material in it, as has been shown in recently published studies. Raman spectroscopy and
fluorescence emission spectroscopy have certain features that make them suitable for this
application. More specifically, the two techniques provide the possibility and carry out
measurements in a short time (typical analysis time < 5 min), while they can, potentially, be
applied in field measurements, given the development of portable spectrometers. The
techniques are non-destructive and are applied directly onto the sample surface without the need
for any pre-treatment (extraction, fragmentation), only direct optical contact with it is required.
During the Raman study, a total of 16 teeth of archaeological origin were examined and spectra
were recorded in two different spectrometers providing excitation at 1064 nm and 785 nm.
Raman spectra show bands related to the mineral content of the teeth, which consists mainly of
hydroxyapatite, and arise from the characteristic vibrations of the phosphate group.
Accordingly, the organic content, which is mainly proteinaceous in nature, usually collagen
residues, shows bands related to the characteristic vibrations of the peptide bond and the
vibrations of the methyl and methylene side groups of the amino acids.
To evaluate the samples as regards their protein content, Raman spectra were studied, which
were recorded with excitation at 1064 nm, and through them, two spectral indices were
calculated, referred to as Am/P, C/P, representing ratios of values of the integrated intensity of
spectral peaks corresponding to organic and inorganic material in the enamel and cementum
tissues. In particular, the Am factor is associated with the characteristic vibration of the amide
bond of the protein, so it reflects the collagen content of the tooth, while the P factor is related
to the vibration of the phosphate group, so it represents the inorganic matrix of the tooth.
Therefore, the spectral index Am/P expresses the ratio of the organic protein residue in the tooth
to the main inorganic component. The C/P index is used to describe the alteration that
hydroxyapatite crystals undergo due to the addition of carbonate ions (CO32-). The C factor is
calculated from the characteristic peak intensity due to carbonate ions.
It was observed that the values of the Am/P index are significantly different in the cases of
enamel tissue versus cementum tissue, having distinctly higher values in the case of cementum,
which is indeed richer in protein material. Accordingly, the values of the C/P ratio appear higher
in the case of cementum due to the increased porosity of the specific tissue and its low
crystallinity. By taking together the values of the Am/P and C/P ratios, calculated for each tooth,
specific criteria were adopted, the application of which allows the assessment of samples in
terms of their content of endogenous genetic material and, by extension, the distinction of the
well preserved teeth against the rest. In particular, using the C/P index, a 90% rejection of
poorly preserved teeth is obtained, while using the Am/P index, a 40% rejection is achieved.
Additionally, the CH/P spectral ratio obtained from Raman spectra recorded with excitation at
785 nm was also examined. The CH term corresponds to the integrated intensity of the methyl
and methylene vibrational bands, so the CH/P ratio reflects, correspondingly to the Am/P ratio,
the tooth content in organic material. A satisfactory correlation of the CH/P ratio with the
endogenous DNA content of the teeth was found, but due to the reduced sensitivity of the
spectrometer, it was only possible to analyze 6 of the samples.
In the context of the study of ancient teeth by use of fluorescence emission spectroscopy, a
portable spectrometer was employed that provides excitation at 375 nm through the use of an
LED source. UV excitation results in fluorescence emission from the proteinaceous material
which can be indirectly correlated with the endogenous DNA content of the cementum. Spectral
data from the study of enamel (A) and cementum (O) showed that there is a small shift in the
wavelength corresponding to the maximum of the fluorescence emission banmax (O) / λmax (A). The
values of the ratio λmax (O) / λmax (A) showed a rather weak correlation (R2 = 0.42) with the
amount of endogenous genetic material. By setting appropriate criteria, it became possible to
reach a rejection rate of about 85% as regards the poorly preserved samples. It is noteworthy
that applying the criteria for the Am/P and C/P indices in combination with the λmax (O) / λmax
(A) index leads to the rejection of all ancient teeth with endogenous genetic material < 10%,
while the acceptance is achieved of all well-preserved tooth specimens.
The results, mainly those obtained based on Raman spectroscopy, are in agreement with
recently published work using infrared absorption spectroscopy (FT-IR) to estimate the protein
content of archaeological bones after sampling a small amount of bone tissue and grinding it
before analysis. Despite the small number of samples, examined in this preliminary study,
results are very encouraging and suggest that the methodology proposed can offer a reliable
sample screening tool in the context of ancient DNA analysis. As a continuation of the present
study, measurements of a larger number of samples are planned to confirm the preliminary
results that satisfactorily correlate Raman and fluorescence spectral indices with the percentage
of endogenous genetic material in archaeological teeth and bones.
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