Institut für Strahlenschutz

Figure1. Egyptian mummy head from a Middle Kingdom tomb in Thebes-West (2050 – 1650 BC).

AFM Imaging and laser based microdissection

Figure 2: Nanoendoscopic analysis of ancient DNA a) AFM image of the collagen network structure (arrow), the three dimensional images shows the preserved collagen network, scale bar 1 µm; b) Isolation of the bone particle, diameter 150 µm, via laser based microdissection; the laser cut is marked with an arrow; c) ejection of the isolated fragment (optical image 10x magnification) and d) isolated bone islet inside the collection device. 

The structural analysis with the AFM revealed a typical three-dimensional collagen distribution and, thereby, shows an excellent state of preservation of the organic matrix. The collagen fibers presented with an average diameter of 400nm, which is within the range of modern bone samples. Therefore, the AFM analysis provided evidence that no severe decomposition processes occurred, which would have had a bad impact on the conservation of the biochemical components of the bone tissue.    

Nested isolation of paraffin embedded blocks was performed and single 150 µm islets were isolated by laser microdissection for subsequent DNA extraction and amplification. The applied laser wavelength of 337 nm is far from the adsorption maximum of DNA of 260 nm and the effective laser energy is limited within the focal area excluding the potential beam damage to DNA.

Ancient DNA extraction

Figure 3: Gel electrophoresis of isolated DNA. a) polyacrylamid electrophoresis of the 202 bp β-actin and 106/112 bp amelogenin gene PCR products: lane 1: amelogenin female control sample 106 bp. lane 2: amelogenin male control sample 106/112 bp, lane 3: molecular weight marker VIII (Roche), lane 4: β-actin gene of the isolated bone material, lane 5: amelogenin gene of the isolated bone material, lane 6: negative control and lane 7: H₂O control. b) β-actin amplification of the pulverized bone sample: lane 1: molecular weight marker VIII (Roche); lane 2-6: different amount of bone powder used for DNA extraction (1, 0.75, 0.5, 0.3 and 0.1g); lane 7, 8: extraction controls; lane 9: PCR control; lane 10: positive control from recent DNA.

We isolated six samples from a histological section of the mummy material and repeated the procedure four times. In each of the four repeated approaches, human ß-actin and amelogenin could be amplified in 1-3 samples showing a successful DNA extraction. Additionally, we were able to amplify mitochondrial DNA in four of six samples of each isolation approach. Extraction and PCR controls were negative in all cases. Amplification products generally showed weak signals in gel analysis, presumably due to low amount of extracted material. Nevertheless, high-resolution polyacrylamide gel electrophoresis demonstrated that the ancient DNA is derived from a female individual, as in all amelogenin PCR products only the X-Chromosome specific 106 bp fragment was visualized (Figure 2a). Sequencing of the mitochondrial DNA revealed no differences in the sequences of the four samples. The DNA extraction of the pulverized material revealed in all 5 samples the presence of ancient DNA as shown by the amplification of a 202bp fragment of the human β-actin gene. Again, all extraction and PCR controls were constantly negative. The band intensity of the amplified DNA revealed slightly differences within the five extractions, but did not depend on the amount of bone powder used. However, the control amplification of a recent human tissue samples showed a much more higher DNA yield compared to the ancient samples. 

Spectrophotometric measurements

Table 1. DNA quantification comparison of a standard technique and the laser based microdissection. The amount of extracted DNA was measured using an UV/Vis spectrophotometer, NanoDrop ND-1000 (NanoDrop, Delaware). Therefore, 1,5 µl of each extracted sample was loaded was onto the instrument in turn and the UV absorbance reading taken at 230, 260, and 280 nm using the “DNA 50” settings. The software automatically calculated the DNA concentrations in ng/µL.

The spectrophotometric measurement of the DNA concentration revealed a higher yield in the samples extracted from the pulverized material compared to the laser microdissection approach. However, the OD 260/280 ratios of the dissected samples are higher and within the range of good quality DNA.  The lower 260/280 ratios of DNA extracted from the pulverized bone specimens indicate a lower DNA quality, probably due to contamination with proteins or other co-extracted material from the soil or the mummification process. This indicates that despite the lower overall amount of extracted DNA, the purity of the laser microdissected material appears to be significantly higher.  

Our new approach has the advantage, that the amount of material used for the subsequent analytic steps as compared to other extraction methods can be reduced significantly. The reduction of the source material could allow decreasing the amount of co-extracted inhibition factors or exogenous DNA. Moreover, the AFM analysis offers the opportunity to evaluate the bone collagen status at the same region from which the material is isolated for the DNA extraction. No additional bone material is necessary to investigate the collagen and only small samples have to be taken from the historic specimens. This situation is also often found in forensic cases, when only minute amounts of biological material are preserved. Here, this new method allows the evaluation of the state of preservation of the specimen combined with a subsequent DNA identification for forensic diagnostics. Moreover, the tissue block can also be used for further histological or immunohistochemical investigations.  

The nanotechnical approach presented here can adapt the preparative and extraction procedures to the low amount of preserved ancient DNA.

References

Thalhammer S, Nerlich AG, Heckl WM, Zink AR. Nanoendoscopic analysis of ancient DNA. J Forensic Sci, in press