Pietro PORTA LA, Fabio Maria GUARINO, Claudio BUCCELLI, Vincenzo GRAZIANO, Marcello MEZZASALMA, Gaetano ODIERNA, Mariano PATERNOSTER, Pierpaolo PETRONE

Recovery and amplification of ancient DNA from Herculaneum victims killed by the 79 AD Vesuvius hot surges

In thermally damaged archaeological bones the quantity, quality, and amplifiability of DNA are very much reliant on both the extent of heating and the environmental conditions of the burial context. In this study we tested the possibility of extracting and amplifying ancient DNA from human bone remains of Herculaneum victims of the 79 AD eruption of Vesuvius using a combination of histochemical and molecular methods. Long bone samples with variable degrees of chromatic and morphological alterations consistent with exposure to temperatures of about 300 °C were taken from four specimens. Using histochemical stains, bone cryostat sections from three individuals revealed DNA within osteocyte lacunae but only for one sample the DNA was suitable for PCR amplification obtained, namely from reactions with the primer pair for X and Y amelogenin (AMEL) loci. The relative sequence differed from the homologous trait of AMELX deposited in GenBank for six bases, probably due to degradation processes following death. Our data are indicative that archaeological bones exposed to high temperatures of about 300 °C should be considered for DNA analysis, given the favorable conditions of corpse burial and skeleton preservation, such as those that occurred for the 79 AD victims in Herculaneum and Pompeii.

PDF

___

Bailey JF, Henneberg M, Colson IB, Ciarallo A, Hedges RE, Sykes B (1999). Monkey business in Pompeii- unique find of a juvenile barbary macaque skeleton in Pompeii identified using osteology and ancient DNA techniques. Mol Biol Evol 16: 1410-1414.
Binladen J, Wiuf C, Gilbert TM, Bunce M, Barnett R, Larson G, Greenwood AD, Haile J, Ho YW, Hansen AJ et al. (2006). Assessing the fidelity of ancient DNA sequences amplified from nuclear genes. Genetics 172: 733-74.
Brown T, Brown K (2011). Biomolecular Archaeology: An Introduction. Oxford, UK: John Wiley and Sons.
Buikstra JE, Ubelaker DH (1994). Standards for Data Collection from Human Skeletal Remains. Research Series No. 44. Fayetteville, AR, USA: Arkansas Archaeological Survey.
Cipollaro M, Di Bernardo G, Forte A, Galano G, De Masi L, Galderisi U, Angelini, F, Cascino A (1999). Histological analysis and ancient DNA amplification results on human bone remains found in Caius Iulius Polybius house in Pompeii. Croatian Med J 40: 392-397.
Cipollaro M, Di Bernardo G, Galano G, Galderisi U, Guarino FM, Angelini F, Cascino A (1998). Ancient DNA in human bone remains from Pompeii archeological site. Biochem Biophys Res Comm 247: 901-904.
Cooper A, Poinar H (2000). Ancient DNA: do it right or not at all. Science 289: 1139.
Di Bernardo G, Del Gaudio S, Galderisi U, Cascino A, Cipollaro M (2009). Ancient DNA and family relationships in a Pompeian house. Ann Hum Gen 73: 429-437.
Di Bernardo G, Del Gaudio S, Galderisi U, Cipollaro M (2004a). 2000 Year-old ancient equids: an ancient-DNA lesson from Pompeii remains. J Exp Zool Part B 302: 550-556.
Di Bernardo G, Galderisi U, Del Gaudio S, DAniello A, Lanave C, De Robertis MT, Cascino A, Cipollaro M (2004b). Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD. J Cell Phys 199: 200-205.
Elsner J, Schibler J, Hofreiter M, Schlumbaum A (2015). Burial condition is the most important factor for mtDNA PCR amplification success in Palaeolithic equid remains from the Alpine foreland. Archaeol Anthropol Sci 7: 505-515.
Ferembach D, Schwidetzky I, Stloukal M (1980). Recommendations for age and sex diagnoses of skeletons. J Hum Evol 9: 517-549.
Gamba C, Fernandez E, Tirado M, Pastor F, Arroyo-Pardo E (2011). Ancient nuclear DNA and kinship analysis: the case of a medieval burial in San Esteban Church in Cuellar (Segovia, Central Spain). Am J Phys Anthropol 144: 485-491.
Geraci G, Del Gaudio R, Di Giaimo R (2002). Le analisi paleogenetiche. In: Petrone PP, Fedele F, editors. Vesuvio 79 A.D. Vita e morte ad Ercolano. Naples, Italy: Fridericiana Napoli, pp. 85-88 (in Italian).
Gilbert MTP, Bandelt HJ, Hofreiter M, Barnes I (2005a). Assessing ancient DNA studies. TRENDS Ecol Evol 20: 541-544.
Gilbert MTP, Hansen AJ, Willerslev E, Rudbeck L, Barnes I, Lynnerup N, Cooper A (2003). Characterization of genetic miscoding lesions caused by postmortem damage. Am J Hum Gen 72: 48-61.
Gilbert MTP, Rudeck L, Willerslev E, Hansen AJ, Smith C, Penkman KEH, Prangerberg K, Nielsen-March CM, Jans ME, Arthur P et al. (2005b). Biochemical and physical correlates of DNA contamination in archaeological human bones and teeth excavated at Matera, Italy. J Archaeol Sci 32: 785-793.
Gotherstrom A, Collins MJ, Angerbjorn A, Liden K (2002). Bone preservation and DNA amplification. Archaeometry 44: 395- 404.
Guarino FM, Angelini F, Odierna G, Bianco MR, Di Bernardo G, Forte A, Cascino A, Cipollaro M (2000). Detection of DNA in ancient bones using histochemical methods. Biotech Histochem 75: 110-117.
Guarino FM, Angelini F, Vollono C, Orefice C (2006). Bone preservation in human remains from the Terme del Sarno at Pompeii using light microscopy and scanning electron microscopy. J Archaeol Sci 33: 513-520.
Hall TA (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp Ser 41: 95-98.
Hansen A, Willerslev E, Wiuf C, Mourier T, Arctander P (2001). Statistical evidence for miscoding lesions in ancient DNA templates. Mol Biol Evol 18: 262-265.
Holden JL, Phakey PP, Clement JG (1995). Scanning electron microscope observations of heat-treated human bone. For Sci Int 74: 29-45.
Hoss M, Jaruga P, Zastawny TH, Dizdaroglu M, Pääbo S (1996). NA damage and DNA sequence retrieval from ancient tissues. Nucleic Acids Res 24: 1304-1307.
Incoronato A, Mastrolorenzo G, Pagano M, Spina G (1998). Determinazione delle temperature di deposizione, mediante procedure paleomagnetiche di piroclastiti del 79 A.D. ad Ercolano. In: Damico C, Livadie A, editors. Le scienze della Terra e larcheometria. Naples, Italy: Pàtron, pp 95-98 (in Italian).
Jans MM, Nielsen-Marsh CM, Smith CI, Collins H, Kars H (2004). Characterization of microbial attack on archaeological bone. J Archaeol Sci 31: 87-95.
Kaestle FA, Horsburgh KA (2002). Ancient DNA in anthropology: methods, applications, and ethics. Am J Phys Anthropol 35 (Suppl.): 92-130.
Kemp BM, Smith DG (2005). Use of bleach to eliminate contaminating DNA from the surface of bones and teeth. For Sci Int 154: 53-61.
Lindahl T (1993). Instability and decay of the primary structure of DNA. Nature 362: 709-715.
Mannucci A, Sullivan K, Ivanov PL, Gill P (1994). Forensic application of a rapid quantitative DNA sex test by amplification of the X-Y homologous gene amelogenni. Int J Legal Med 106: 190-193.
Mastrolorenzo G, Petrone PP, Pagano M, Incoronato A, Baxter PJ, Canzanella A, Fattore L (2001a). Herculaneum victims of Vesuvius in AD 79. Nature 410: 769-770.
Mastrolorenzo G, Petrone PP, Pagano M, Incoronato A, Baxter PJ, Fattore L, Fergola L (2001b). The 79 AD Vesuvius Plinian eruption at Herculaneum and its impact on the people. In: Juvigne E, Raynal JP, editors. Tephras. Les dossiers de lArchéoLogis, n°1. Lausanne, France: CDERAD Goudet, pp. 183-189.
Mastrolorenzo G, Petrone P, Pappalardo L, Guarino FM (2010). Lethal thermal impact at periphery of pyroclastic surges: evidences at Pompeii. PLoS ONE 5 1-12 e11127.
ORourke DH, Hayes MG, Carlyle S (2000). Ancient DNA studies in physical anthropology. Ann Rev Anthropol 29: 217-242.
Ottoni C, Koon HEC, Collins MJ, Penkman KEH, Rickards O, Craig OE (2009). Preservation of ancient DNA in thermally damaged archaeological bone. Naturwissenschaften 96: 267-278.
Pääbo S (1989). Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. P Natl Acad Sci USA 86: 939-1943.
Petrone PP (2011). Human corpses as time capsules: new perspectives in the study of past mass disasters. J Anthropol Sci 89: 1-4.
Pruvost M, Schwartz R, Correia VB, Champlot S, Braguier S, Morel N, Jalvo Y, Grange T, Gelgi EM (2007). Freshly excavated fossil bones are best for amplification of ancient DNA. P Natl Acad Sci USA 104: 739-744.
Quatrehomme G, Bolla M., Muller M, Rocca JP, Grevin G, Bailet P, Ollier A (1998). Experimental single controller study of burned bones: contribution of scanning electron microscopy. J For Sci 43: 417-422.
Rohland N, Hofreiter M (2007). Comparison and optimization of ancient DNA extraction. BioTechniques 42: 343-352.
Salamon M, Tuross N, Arensburg B, Weiner S (2005). Relatively well preserved DNA is present in the crystal aggregates of fossil bones. P Natl Acad Sci USA 102: 13783-13788.
Shipman P, Foster G, Schoeninger M (1984). Burnt bones and teeth: an experimental study of colour, morphology, crystal structure and shrinkage. J Archaeol Sci 11: 307-325.
Sokolov EP (2000). An improved method for DNA isolation from mucopolysaccharide-rich molluscan tissue. J Moll Study 66: 573-575.
Stiner MC, Kuhn S, Bor-Yosef O (1995). Differential burning, recrystallization, and fragmentation of archaeological bone. J Archaeol Sci 22: 223-237.
Tautz D, Renz M (1983). An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 132: 14-19.
Thompson JD, Higgins DG, Gibson TJ (1994). Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acid Res 22: 4673-4680.
White L, Booth TJ (2014). The origin of bacteria responsible for bioerosion to the internal bone microstructure: results from experimentally-deposited pig carcasses. For Sci Int 239: 92- 102.
Winters M, Barta JL, Monroe C, Kemp BM (2011). To clone or not to clone: methods analysis for retrieving consensus sequences in ancient DNA sample. PLoS One 6: e21247.
Zhang L, Wu Q (2005) Single gene retrieval from thermally degraded DNA. J Biosci 30: 599-604.