Ancient DNA as a multidisciplinary experience

JOURNAL OF CELLULAR PHYSIOLOGY 202:315–322 (2005)

REVIEW ARTICLES

Ancient DNA as a Multidisciplinary Experience M. CIPOLLARO,* U. GALDERISI, AND G. DI BERNARDO Dipartimento di Medicina Sperimentale, Sezione di Biotecnologie e Biologia Molecolare, 2nd University of Naples, Naples, Italy Investigation into DNA from archeological remains offers an inestimable tool for unraveling the history of humankind. However, a series of basic and technical difficulties renders the analysis of ancient DNA (aDNA) molecules troublesome, depending either on their own peculiar characteristics or on the complexity of processes affecting the bone matrix over time, all compromising the preservation of ancient DNA. This review underlines the contribution of many different disciplines, in particular molecular biology and genetics, to overcome these obstacles. The role of each expertise is illustrated to appropriately address the questions arising in aDNA investigations. J. Cell. Physiol. 202: 315–322, 2005. ß 2004 Wiley-Liss, Inc.

Prodded by his own innate curiosity, present day man strives to answer a number of questions including the origin of humankind, the advent of ancient societies, the construction of social organization, migration patterns, expansions and extinctions, and infectious diseases spreading throughout ancient populations. Exploring the genetic structure of ancient populations through the application of molecular biology techniques can answer a number of these questions. It is noteworthy that the knowledge of DNA nucleotide sequences of ancient animals, plants, and bacteria might also provide a spin-off to many other fields, including phylogenetic relationships of extinct animals, plant breeding, and the spread of infectious diseases. Unfortunately, the study of ancient DNA (aDNA) involves several problems that are illustrated below along with the strategies proposed to overcome these problems. They are the product of mutual interactions among many disciplines, also helping a reciprocal validation of aDNA data. In the last few years, a number of reviews on aDNA have been focused on different and peculiar aspects of this new research field. For example, O’ Rourke et al. (2000) described the role of aDNA studies in physical anthropology, while Hofreiter et al. (2001b) illustrated the possibility to study genetic relationships of extinct organisms with their contemporary relatives. Collins et al. (2002) reviewed highlights on the biomolecular information retrievable from bones; Marota and Rollo (2002) reviewed some of the most important investigations based on molecular paleontology; Pusch et al. (2003) described the molecular phylogenetic approach used to tackle aDNA studies. In July 2002, the 6th International Conference on Ancient DNA & Associated Biomolecules was held in Israel. Many topics were discussed ranging from population genetics, ancient pathogens, animal genomics, plants and wine, to forensic studies. Some of them, such as the reports on Moa taxa phylogenetic relationships (Huynen et al., 2003) and Pompeian horse phylogeny (Di Bernardo et al., 2002), first illustrated at the Conference, gave rise to full text publications. ß 2004 WILEY-LISS, INC.

This review intends to underline the advantages offered by a multidisciplinary mutual approach to guarantee the reliability of aDNA sequence data, along with the most recent acquisitions in the aDNA field. DISCIPLINES INVOLVED IN aDNA RESEARCH Archeology

This support is necessary both to put a historical problem to be solved within a framework, and to identify those archeological sites that better fit with the research hypothesis to be validated. For instance, the colonization of Pithekoussai, a Southern Mediterranean island (today’s Ischia), is reported by two of the major Roman historians Livio (‘‘Ab urbe condita libri’’ VII, 22, 5) and Strabone (‘‘Rerum Geographicarum’’ V, 247) as the first settlement of the Greeks coming from Eubea in the 8th century BC. This hypothesis could be validated, for instance, by an aDNA investigation of human remains found in Pithekoussai, in particular from San Montano, in comparison with data from contemporary Greek remains found in Eubea sites. Some archeologists (Ridgway, 1984) have suggested analyzing remains from the Eretria and Lefkandi necropolis since they hypothesize that the first Greek people coming to Italy were from just these sites. Figure 1 shows the geographic position of the Greek colonies and of Pithekoussai Island. Geology and volcanology

These disciplines provide invaluable contributions toward understanding the characteristics of burial

*Correspondence to: M. Cipollaro, Dipartimento di Medicina Sperimentale, Sezione di Biotecnologie e Biologia Molecolare, 2.a Universita` degli Studi di Napoli, Via Costantinopoli 16, 80138 Naples, Italy. E-mail: [email protected] Received 17 March 2004; Accepted 23 March 2004 DOI: 10.1002/jcp.20116

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Fig. 1. Map of the Southern Mediterranean area which includes Pithekoussai island (todays Ischia), on the top to the left, and Eubea island. The insert shows the two villages of Lefkandi and Eretria from which the Pithekoussai colonization was supposed to begin.

conditions and the physical history of archeological sites. This kind of information helps by acquiring preliminary data on bone preservation conditions, such as histological evaluation, before performing aDNA analysis. The analysis of bone preservation can highlight archeological sites at risk for aDNA investigations, prone to rapid bone destruction and DNA degradation. Anthropology and paleopathology

Their support is indispensable for establishing sex and age of individuals and for skeleton reconstruction useful in selecting bones for DNA extraction. They are also irreplaceable in the recognition of skeletal traces of genetic (i.e., thalassemia), infectious (i.e., tuberculosis), or acquired diseases (i.e., arthrosis) or of other pathologies that leave evident skull stigmas (i.e., Paget’s disease). Histology

This discipline offers techniques often used in archeological investigations for determination of age and species, when fragmented bones are to be studied. It also offers a diagnostic aid in the case of paleopathology. The determination of bone microstructure preservation (Stout, 1978), evaluating the degree of diagenetic changes, might help in the selection of bones for DNA extraction. Diagenetic alterations, consisting in microscopic focal destructions, presence of inclusive material, presence of microfissures, and intensity of birefringence, seem in fact to correlate with the biochemical preservation of the bone (Hagelberg and Clegg, 1991; Colson et al., 1997; Di Bernardo et al., 2004). A recent report describes three pathways of diagenesis due to the

chemical deterioration either of the organic phase or of the mineral phase, or due to the microbiological attack of the composite (Collins et al., 2002). Chemistry

This is essential for designing and synthesizing new and necessary compounds, for instance to optimize DNA extraction procedures. One of these compounds is Nphenacyl thiazolium bromide (PTB) (Vasan et al., 1996; Poinar et al., 1998; Di Bernardo et al., 2002), able to increase the rescue of DNA from the intricate network of crosslinks between proteins and sugars. Chemistry also plays a role in determining the amino acid racemization level, a parameter assumed to be a predictor of aDNA preservation (Poinar et al., 1996). The transformation of L- into D-enantiomers, such as the depurination of DNA, is affected by environmental conditions, and the kinetics of aspartic acid racemization is approximately equal to that of DNA depurination. Physics

It gives an important contribution to dating remains by Accelerated Mass Spectrometry (AMS) radiocarbon based on 14C decay (Conard and Bolus, 2003) and by acquiring paleodietary information through stable carbon and nitrogen isotopes. In fact, it is possible to assess the relative proportion of aquatic and terrestrial resources in ancient individuals through analyses of bone collagen carbon d13C and nitrogen d15N. Humans who consume significant amounts of aquatic foods will have much higher d15N values than humans who consume only terrestrial plants and herbivores (Schwarcz and Schoeninger, 1991; Van Klinken et al., 2000;

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Richards et al., 2001) used these techniques to date midUpper Paleolithic human remains in Europe. They revealed significant amounts of aquatic foods in some of their diets. In contrast, European Neanderthal collagen carbon and nitrogen stable isotope values do not indicate significant use of inland aquatic foods, showing that the majority of their proteins were from terrestrial herbivores. The limits of this approach consist in the necessity of good collagen preservation, a feature often absent in archeological bones. Molecular biology

Along with genetics, nucleic acid expertise constitutes the core of the aDNA field. The molecular biology techniques used in aDNA investigations span from specific protocols on nucleic acid extraction, assessment of criteria and strategies for PCR amplification, sequencing of the most informative genomic loci (such as mitochondrial D-loop/control region, nuclear STRs of the autosomes and of the Y-chromosome), to quantitization by real-time PCR of ancient molecules and their enzymatic repair. The molecular biology contribution will be extensively expanded below. Genetics

The knowledge and the correct interpretation of ancient genetic loci patterns and, when possible, their analysis compared to modern sequences is the essential prerequisite to guide construction of models of evolution, or historical models of migration thus allowing us to tackle problems related to population genetics, phylogeny, taxonomy, and relationships. Microbiology

Infectious diseases have accompanied humankind from its beginning. The identification of aDNA pathogens in ancient remains provides data on host-pathogen interaction and opens new possibilities to answer questions raised by historians and paleopathologists about the actual incidence and prevalence of infectious diseases. It is possible, indeed, to detect ancient bacterial genomes persisting in ancient bones where stigmas of pathologies are visible. Alternatively, screening can be performed for infectious diseases of the past by developing methods so that a number of disease genomes can be detected in a single sample (multiplex PCR) or with batteries of primers bound to membranes or silica (spoligotyping). The detection of tuberculosis by aDNA has been reported as far back as 3000 BC in humans, while a recent study demonstrated the possibility to detect Mycobacterium tuberculosis complex in a bison dated 17,000 years before present times (BP) (Rothschild et al., 2001). Recently Zink et al. (2003) detected Mycobacterium tuberculosis aDNA by amplifying a 123-bp fragment of IS6110 in bone and tissue samples of 85 Egyptian mummies, using spoligotyping. By comparing data to those in an international database, the authors concluded that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum rather than to M. bovis, as previously believed. In addition to tuberculosis (Spigelman and Lemma, 1993), malaria (Taylor et al., 1996, 1999), and leprosy (Rafi et al., 1994), other disease pathogens such

as Yersinia pestis have been found by aDNA techniques (Drancourt et al., 1998). The technology for the determination of infectious microorganisms is also based on the amplification of 16S rDNA and on comparative analysis of sequences with a database for infectious microorganism sequences. Recently Christner et al. (2003) detected 16S rDNA fragments related to Pseudomonas and acinetobacter gamma-proteobacterial species in ancient glacial ice, 296 m below the surface. By amplifying the 16S rDNA, it is possible to demonstrate the presence of aDNA from bacteria causing periodontal disease in human skulls found in ancient Caudium, (today’s Montesarchio) a Southern Italy archeological site nearby Benevento, in the Campania Region. This information will enable us to understand the responsibility of bacteria in provoking dental pathology in the past and allow us to infer information on the food habits of Caudium inhabitants, also detectable by stable isotopes analysis. Botany

Brown et al. (1994) analyzed seeds collected in three archeological sites in Europe and the Middle East confirming the possibility to amplify aDNA from these samples. More recently Jaenicke-Despres et al. (2003), analyzing genes involved in different functions in archeological maize samples from Mexico and Southwestern United States, revealed that alleles typical of contemporary maize were already present in Mexican maize 4,400 years ago. Studies on vegetal remains are also intended to address the origin of cultivated plants, as for example the domestication of Vitis Vinifera. One way is to analyze genetically modern representatives and their wild relatives, while another is to find archeobotanical artifacts and attempt to extract DNA from them. Both methods have been assayed for European grapevine cultivars. Recently Arroyo-Garcia et al. (2002) published a report on chloroplast microsatellite polymorphisms found in Vitis species, confirming the varied distribution of haplotype frequencies at the two ends of the Mediterranean growth area. This finding suggested the existence of independent domestication events for grapevines. As for the grapevine in antiquity, DNA has been recovered in a pithos from a Hellenic archeological site in Macedonia revealing a chloroplast microsatellite profile found to be dominant in Western European wild grapes and also present in Greece (Lefort and ArroyoGarcia, 2002). Zoology

Huge collections of animal remains are found in museums all over the world. The aDNA approach gives archeologists a new window into the past, creating a vivid picture of animal diversity over time. The mutual exchanges among this and other disciplines are illustrated in the animal aDNA section. Bioinformatics

The determination of the sequence of many genes or entire genomes gave rise to the development of much software dedicated to the comparison of nucleotide sequences. This software is also widely used in aDNA investigation: examples are the phylogenetic trees

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constructed on the basis of sequence divergences among individuals analyzed. This part of the review will illustrate the contribution of the different disciplines responding to specific questions and the time course of the investigations on bone remains. When an archeological site becomes the object of aDNA research, the first consideration is the burial environment of the remains, since it is well known that chemical, physical, and biological stress can affect the degree of DNA preservation (Lindahl, 1993; Burger et al., 1999). One example that aptly illustrates the importance of thermal history on DNA preservation is provided by the pitfalls of some aDNA studies from Egyptian papyri and mummies (Marota et al., 2002) presumably due to persistent high temperatures (Hedges, 2002; Schmitz et al., 2002) in Egyptian archeological sites, in addition to the humidity, pH values, and the age of the samples. Elevated temperature is also described for the archeological site of Pompeii, whose situation however is quite different from the Egyptian environment since the increase of temperature was time-limited. Moreover, temperatures did not reach a drastic level, which extensively compromises the DNA preservation. Volcanological data collected from the exact spots where the remains under study (Cipollaro et al., 1998, 1999; Guarino et al., 2000; Di Bernardo et al., 2002) were found, showed that the maximum temperature value was approximately 1008C (Luongo et al., 2001). It is also worth considering that the Pompeian bones were fairly well preserved because the volcano’s ashes virtually pasteurized the bones thus preventing microbial attack, one of the worst problems for DNA preservation. Substantial information on the degree of bone preservation can be obtained by histological analysis (Colson et al., 1997). Generally an archeological bone exhibits altered regions consisting of a network of small pores of 0.1–1.0 mm in diameter, which increases the porosity of the bone. The report of Turner-Walker et al. (2002) describes a method for direct measurement of pore volume and pore size distribution as indicators of the degree of post-mortem modification. The method is based on a mercury intrusion porosimetry and back scattered scanning electron microscope. Previous work (Hagelberg et al., 1991) identified a correlation between spongiform porosity and loss of amplifiable DNA. Moreover, the presence of DNA within the osteocytic lacunae of ancient bones is valuable by histochemical analysis, staining bone sections with DNA-specific fluorochromes, such as 40 -60 -diamino-2-phenylindole (DAPI) and chromomycin A3 (CMA), and with the Feulgen reaction (Guarino et al., 2000). Another parameter to be considered when studying the biological characteristics of archeological remains is the measurement of amino acid racemization level useful in correlating bone tissue preservation with amplifiable DNA. Recent reports, however, have shown exceptions to this simple correlation (Pusch et al., 2003), although this might be a consequence of the technique proposed by the original authors (Poinar et al., 1996). In fact the acid hydrolysis of proteins used determines a a priori a consistent amount of racemization background

(more than 50%) which might obscure the naturally occurring racemization. The result is that only slight differences are appreciable among bone samples of different ages. A modified hydrolysis method is based on the activity of a peptidil-D-amino acid hydrolase, an enzyme that can free all aspartic residues. By decreasing the background, this procedure (D’Aniello et al., 1993) should allow greater accuracy in the determination of D-aspartic acid content in bone samples. Both racemization analysis and microscopic observation should be performed together to offer valuable techniques within archaeological heritage management research. aDNA, MOLECULAR BIOLOGY AND GENETICS DNA extraction and amplification

A brief perusal through the Material and Methods sections of manuscripts on aDNA reveals that many different procedures are aimed at recovering, processing, and amplifying heavily damaged and extremely rare molecules (O’ Rourke et al., 2000; Pusch et al., 2003). Since none of these procedures can be considered a priori the most effective, the extraction protocol generally needs to be modified and adapted to the peculiar characteristics of the archeological site where the remains are found. For instance, in the case of the Taq polymerase inhibitors present in the burial soil, the extraction protocol must remove these substances through extensive phenol-chloroform extraction or through adsorption of DNA molecules on silica particles. Recent reviews give an exhaustive picture of the most important procedures and strategies. However a recent report worth citing (Yang et al., 2003) suggests an interesting experimental behavior in establishing PCR amplification protocols and in the interpretation of results. In particular, the authors focused their attention on strategies aimed at minimizing contamination, adjusting the numbers of cycles, and varying the type of Taq DNA polymerase. Animal aDNA: archeological site authentication and phylogeny

It is worth noting that once an archeological site becomes the object of any aDNA studies, the analysis of aDNA extracted from animal bone remains found there is extremely important for a number of reasons. First of all, data on animal remains allow the authentication of the site itself in terms of suitable conditions for nucleic acid preservation. In fact, modern DNA contamination, one of the most serious problems in aDNA studies, becomes meaningless when analyzing these kinds of remains, since there is no possibility that they could derive from present-day animals, especially in those laboratories where no modern animal DNA is handled. For instance, studies on a young Barbary macaque (Bailey et al., 1999) kept in Pompeii at the ‘‘Terme del Sarno’’ collection, as well as on the five equids (Di Bernardo et al., 2002) found in the ‘‘Casti Amanti’’ house showed the presence of amplifiable aDNA. This finding supported the hypothesis that burial conditions in Pompeii were favorable for aDNA preservation already proposed for human remains (Cipollaro et al., 1998, 1999).

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The low probability of contamination when animal remains are investigated also allows an in-depth study of basic problems strictly related to aDNA characteristics, such as enzymatic repair of damaged molecules. Several research groups have reported data from animal remains to establish the relationship between species or to test the hypotheses on the status of a given taxa. A recent report by Huynen et al. (2003) clarifies the number of Moa species, extinct ratite birds that showed extreme variation in size. This work illustrates the role that nuclear DNA sequences can play in testing previously intractable hypotheses about extinct organisms. Edwards et al. (2003) explored microsatellite markers in ancient cattle bones from a Viking age settlement in Dublin and suggested an Irish origin for these medieval cattles. Vila et al. (2001) favored a widespread origin of domestic horse lineage analyzing mtDNA control region of 191 domestic horses, while Lambert et al. (2002) showed the rate of evolution of hypervariable region I of mtDNA in well-preserved subfossil bones of Adelie penguins dating back 7,000 years BP. Further studies suggested a transition from wild to domestic status in Neolithic goats (Bar-Gal et al., 2002), while Leonard et al. (2002) demonstrated that native American dogs originated from multiple Old World lineages. Poinar et al. (2003) provided information about phylogeny of the Pleistocene ground sloth and the three-toed sloth. mtDNA and nuclear genes

aDNA investigations are largely based on the amplification of mtDNA DNA. This small genome is more prone to give positive amplification, since it has a cell copy number higher than nuclear genes, having only two copies per cell. mtDNA is particularly useful in studying human evolution: first, it is maternally inherited and thus does not undergo recombination; second, the mtDNA evolution rate is much higher than that of nuclear genes. As a consequence, a substantial number of mtDNA mutations have accumulated sequentially along radiating maternal lineages that have diverged as human populations colonized different geographical regions of the world. The sequencing of the mtDNA control region hypervariable segments I and II, together with restriction fragment length polymorphism (RFLP) investigation, have revealed a number of stable polymorphic sites that define related groups of mtDNAs, called haplogroups. Most of the mutations observed both in mtDNA coding and control regions in modern human populations have occurred on these pre-existing haplogroups and they define the individual mtDNA types or haplotypes. The analysis of mtDNA in extant individuals allowed us to understand the phylogeny of Homo sapiens sapiens (Stringer and Andrews, 1988; Pusch et al., 2003) or to infer routes and times for human expansion out of Africa (Dayton, 2003). In particular, Ingman and Gyllensten (2003) presented a report on the mitochondrial genome variation and evolutionary history of Australian and New Guinea Aborigines. They showed that the genetic diversity of the Australian mitochondrial sequences is remarkably high and is similar to that found across Asia, in contrast to the pattern seen in previously described Ychromosome data, where a specific Australian haplotype

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was found at high frequency (Dayton, 2003). The predominance of an unique Y-chromosome haplotype, contrasting with the high mitochondrial diversity would be, according to the Ingman and Gyllenstein hypothesis, the result of a founder effect, since the population expansion started from a few hundred individuals (Kayser et al., 2001). If this is the case, the authors interpreted their data as the results of an inappropriate sampling across subpopulations, rather than within a single tribe. They concluded that additional studies of autosomal loci are necessary to obtain a balanced view of the evolutionary history of the peoples in this region. Many reports based on human aDNA are focused also on mtDNA, such as those analyzing Neanderthal mtDNA sequences (Cooper et al., 1997; Krings et al., 1997, 1999, 2000; Ovchinnikov et al., 2000; Gutierrez et al., 2002). The report of Schmitz et al. (2002) described a further study on Neanderthal remains, whose sequences cluster with other Neanderthal sequences already published. The author also sustained the importance of an integrated biological approach for a complete and reliable perspective on fossil material with the collaboration of geneticists, morphologists, archeologists, and dating specialists. Aside from mtDNA, the analysis of nuclear DNA and in particular of autosomal microsatellites (STR) are useful to study the genetic structure of human populations. A recent report (Rosenberg et al., 2002), exploring 377 autosomal microsatellite loci in 1056 extant individuals from 52 populations identified six main genetic clusters, five of which correspond to major geographic regions, and subclusters that often correspond to individual populations. STR are also explored to identify historical families as reported in the Romanov family investigation, one of the first attempts at molecular identification of skeletal remains (Gill et al., 1994). Recently, however, data related to the Romanov family reconstruction have raised doubts about their molecular and forensic consistency. In fact, Knight et al. (2004) claimed the impossibility to amplify a 1,223-bp fragment in aDNA as Gill et al. had reported in their work. Knight et al. found that the mtDNA consensus haplotype of Elisabeth, sister of Empress Alexandra, differs from that reported for her sister Alexandra at four sites thus sustaining that samples analyzed in Gill study had been contaminated with non-degraded high molecular weight ‘‘fresh’’ DNA. A more recent report by Keyser-Tracqui et al. (2003) is focused on the analysis of biparental, paternal, and maternal genetic systems to reconstruct genealogies in a protohistoric necropolis of Mongolia. A particularly effective way to screen samples is to carry out multiplex analyses of autosomal STRs, Ychromosomal STR markers, and sex typing marker like amelogenin locus (Lassen et al., 1996; Faerman et al., 1997; Cipollaro et al., 1998). Recent reports have illustrated that also single-locus nuclear DNA sequences can be consistently recovered from ancient material (Huynen et al., 2003). This possibility is of great interest in aDNA investigation since it paves the way to analyze genes involved in genetic diseases. Filon et al. (1995) detected a betathalassemia mutation in the aDNA of skeletal remains

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from the archeological site of Akhziv, Israel. More recently, Rabino et al. (2002) detected a sickle cell anemia in three Egyptian mummies stored at the Anthropological and Ethnographic Museum of Turin, Italy. However, this latter finding is in contrast with data reported by Marota et al. (2002) in their study on Egyptian papyri varying in age from 1,300 to 3,200 years BP. They showed the complete loss of authentic aDNA also in more recent papyri dating from the 8th century AD. This is in agreement with the level of amino acid racemization found to be higher than 0.08, commonly indicated as the highest value compatible with the presence of aDNA. The authors, claiming that the amino acid racemization level in Egyptian bones and tissues is consistent with that found in papyri, have also raised doubts about the Egyptian aDNA data published so far. Post-mortem aDNA damages and base modifications

aDNA is always represented by heavily damaged and highly fragmented molecules (Lindahl, 1993). Damage includes either base modification (oxidation and deamination) or base loss (apurinic and/or apyrimidinic sites) as well as single-strand DNA breaks, with either nicks, gaps, or protruding ends. Interstrand crosslinks have also been shown to exist at relatively high frequencies (Paabo, 1989). Such damages interfere to different extents with common PCR procedures used in aDNA studies. Among base modifications, accumulating when an organism dies (Hansen et al., 2001; Hofreiter et al., 2001a; Gilbert et al., 2003a,b), the most often represented is the cytosine deamination, a miscoding modification that causes a G/C ! A/T substitution, producing artifacts during PCR procedure. Aside from misincorporations due to the damages present in the aDNA molecules, there is also the possibility of misincorporation by Taq polymerase itself. To exclude the presence of post-mortem base modifications, some authors have sequenced a DNA fragment of the best preserved region in the genome, that is, 16S rDNA (Di Bernardo et al., 2002; Lambert et al., 2002). The rationale was that, since this DNA region exhibits a low rate of mutations, the finding of base substitutions at an increased rate could indicate either post-mortem base modifications or Taq DNA polymerase mistakes (Poinar et al., 1998). The absence of polymorphisms in this genome region infer either the absence of nucleotide base modifications also in other genome regions or that these events are negligible. Another strategy is to set up an asymmetric PCR (Hofreiter et al., 2001). This strategy allows each strand of the target to be preferentially amplified using an unbalanced number of primers. Different nucleotides found at the same position when sequencing the product of the two unbalanced amplifications will confirm this hypothesis. Pre-amplification and aDNA enzymatic repair

Different strategies have been set up to enhance the rate of successful amplifications or to repair aDNA molecules. Pre-amplification procedures, such as Degenerated Oligonucleotide PCR (DOP-PCR) (Pusch et al., 2000) and Primed Extension PCR (PEP-PCR)

(Satoh et al., 1998), have been proposed. The authors suggest performing a preliminary amplification on aDNA extracts using degenerated primers, thus increasing the number of molecules available for a specific PCR. However, no significant report has been produced applying these techniques. Some authors have proposed enzymatically repairing aDNA molecules in the attempt to increase the number of mtDNA molecules to be amplified (Pusch et al., 1998) or to also rescue nuclear gene sequences other than mtDNA molecules (Di Bernardo et al., 2002). aDNA enzymatic repair can be performed successfully only for molecules presenting unmodified 30 OH and/or 50 P termini. It can be terminally elongated by DNA polymerase I, sealed by T4 DNA ligase, or filled in and then sealed by the concerted action of these two enzymes. CONCLUSIONS

Studies on aDNA are primarily useful for the advancement of knowledge in a new field of research. The results acquired so far open a new frontier on problems related to aDNA characteristics as data are collected from different archeological sites. At present, one of the most concrete conclusions to be drawn is that multidisciplinary studies play an essential role in studying an archeological site using biomolecular research techniques. On the other hand, it seems clear that each archeological site exhibits peculiar characteristics. Consequently, bone diagenesis features and aDNA damages may be different or may be present to different extents. Thus aDNA extraction methods and PCR strategies must be adjusted to the particular characteristics of each site. As far as mtDNA is concerned, experience has shown that these molecules are amplifiable most of the time. Nuclear genes, in the past only occasionally amplifiable, today can be successfully rescued even taking advantage of the physical features of the aDNA, since some damages are substrates of DNA enzymes. Moreover, it is well established that animal remains are extremely useful in authenticating aDNA data from a given archeological site. Future studies should be oriented toward understanding in greater detail basic questions related to the characteristics of ancient nucleic acids. They will be aimed toward developing new strategies to by-pass difficulties either in amplification procedures or in DNA repair using, for instance, newly discovered polymerases (Goodman and Tippin, 2000) as appropriate in vitro systems are established. ACKNOWLEDGMENTS

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