New uses for old DNA Alan Cooper* and Robert Waynet

Several years have elapsed since the last report of million-year-old DNA, coinciding with increased standards for experimental procedures in ancient DNA research. Whereas many earlier studies are now regarded as erroneous, the recent successful characterisation of Neandertal DNA has set new standards for the field. Researchers continue to find new ways to exploit preserved genetic information in studies of more recent remains, widening the utility of ancient DNA.

Addresses *Department of Biological Anthropology, Oxford University, Oxford OX2 6QS, UK; e-mail: alan.cooper@bioanth.ox.ac.uk #Department of Biology, University of California, Los Angeles, CA 90094, USA; e-mail: rwayne@ucla.edu

Current Opinion in Biotechnology 1998, 9:49-53

http://biomednet.com/elecref/0958166900900049

© Current Biology Ltd ISSN 0958-1669

Introduction Ancient DNA research, or the study of genetic information retrieved from the past, provides the chance to directly measure molecular evolution over large periods of time. Initially of interest to systematists studying extinct taxa, it has become increasingly important in studies covering only tens to hundreds of years, such as conservation biology, population genetics, and forensic science.

Since the initial reports in 1984 [1], the field has evolved from the inefficient and error-prone cloning of multi-copy DNA some hundreds of years old, to sophisticated PCR-based studies of genetic material deposited well before the last Ice Age. During the period since 1984, ancient DNA techniques have offered the first genetic access to what are often high-profile extinct taxa, and the quality of research has sometimes suffered in the rush for spectacular publications. Although the majority of published ancient DNA research appears sound, many of the more spectacular reports have since turned out to be embarrassingly flawed. Fortunately, the recent retrieval of Neandertal DNA [2°'], has set new standards of experimental rigour, especially for studies of ancient human DNA.

Because many new fields are beginning to utilise the potential of ancient DNA, it is important to review what has been learned so far in order to understand the limitations and possible future directions for ancient DNA research.

Death and decay Ancient DNA is any genetic information retrieved from preserved biological remains, ranging from extinct taxa

thousands of years old, to hair or faeces recently deposited by free-ranging animals. The unifying component is that preserved DNA is damaged over time by processes such as oxidation and hydrolysis, leaving only trace amounts of DNA fragments containing cross-links and modified bases [3,4,5°]. Direct biochemical studies of damage are complicated by the small amounts of preserved DNA and authentication requirements, but have the potential to minimise artefacts and improve efficiency during amplification [5°]. Empirical studies of spontaneous DNA decay rates suggest that DNA should not survive beyond 100,000 years, even in favourably cold or anoxic conditions [4,5 ° ] and it is no coincidence that the oldest reliable ancient DNA sequences are from permafrost samples, while many others are from cold environments (Figure 1). Except in the most favourable conditions, the amount of preserved DNA is so small that only multi-copy sequences, such as mitochondrial genes (up to several thousand copies per cell), can be reliably amplified.

The difficulties inherent in using PCR to amplify small amounts of damaged DNA are still being appreciated, particularly the risk of contamination and amplification artefacts [6,7,8"*,9,10]. Early criteria for the authentication of ancient DNA sequences relied on negative controls during extraction and amplification procedures, and the presence of phylogenetically sensible, but novel, ancient sequences [3]. Unfortunately, carrier effects [6] minimise the power of negative controls, and a string of high-profile failures demonstrate that the phylogenetic criterion is inadequate. In fact, the large number of PCR amplification attempts characteristic of ancient DNA studies mean that in the absence of authentic ancient DNA there is a high probability that artefacts, chimeric PCR products, damaged modern templates, or poor sequencing technique will eventually produce a novel, and phylogenetically rea- sonable, sequence [11,12]. Because obvious contaminant sequences are recognised and discarded, a circular logic exists whereby phylogenetically sensible contaminants are first selected for and then accepted as authentic. As a result of these failings, new criteria have been proposed, of which the most important is independent replication by another laboratory [2",13,14]. Quantification of the ancient template using oligonucleotide constructs, cloning of PCR products to determine the homogeneity of the starting template [2",6,8°'], and biochemical analysis of specimens [9] are further important improvements.

Systematics and the million-year plus club Reports of DNA sequences from specimens millions of years old are viewed with some scepticism because they violate the theoretical limit of DNA survival [4] and because none of the results have been independently replicated [15]. Furthermore, support for their authenticity

50 Analytical biotechnology

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Key publications of ancient DNA sequences with estimated sample ages (log scale). Following initial cloning successes with DNA from preserved soft tissues, PCR based amplification of DNA from bone samples became common from 1989 [52]. The length of sequences obtained has increased from around 200 bp [1], to greater than 1000 bp [23",26°]. There have been no independent replications of multi-million year old DNA and it seems likely that they are erroneous [18"°]. The remaining studies are consistent with the theoretical limit of approximately 100,000 years. Samples: *, amber; f, bone; A, cold site; §, compression fossil; #, permafrost site; **, soft tissue; ?, contamination is suggested.

is generally constrained to evidence of phylogenetic uniqueness [15,16]. These concerns were reinforced when putative dinosaur DNA was shown to be a nuclear copy of a human mitochondrial gene [12], supposedly Oligocene bacteria sequences turned out to be those of a modern taxon [17], and comprehensive studies of a variety of amber specimens, including fairly recent material, failed to yield authentic DNA [18°°]. While amino-acid racemi- sation studies have provided circumstantial supporting evidence for amber preserved DNA [19"°], the most revealing testimony is the long silence since the last report of million-year-old DNA.

Doubts also exist about DNA studies of more recent fos- sils, particularly those from human specimens. Ironically, one of the initial ancient DNA studies, the cloning of a 3.4 kilobasc pair piece of phylogenetically uninformative nuclear DNA from a 2,400 year old Egyptian mummy [20], appears likely to be a contaminant in hindsight, both because of its size and nature. Many other studies

report sequences that are too short to be phylogenetically informative, but which are still unique; however, without replication many of these should be regarded with suspi- cion, especially when attempts to replicate results with the same or other specimens fail [21], or where experimental techniques appear particularly contamination prone [22].

When ancient DNA sequences do meet authentication criteria they can provide valuable and novel systematic data from extinct taxa, allowing previously intractable evolutionaw hypotheses to be tested [2"*,23",24,25,26"]. Until now, population-level studies using ancient DNA have generally been constrained to material younger than a thousand years by an insufficient number of DNA-containing specimens [27]. This will improve if the potential of permafrost specimens is fulfilled, as DNA sequences have been obtained from frozen tissue and bone up to 50,000 years old [14,26",28-30], and vast permafrost deposits exist in several parts of the world. If these sites do prove to be genetic museums, then evolutionary change

New uses for old DNA Cooper and Wayne 51

can be studied in populations over hundreds or thousands of generations, finally realising the full potential of ancient DNA.

The problem with people Studies of ancient human DNA can provide a temporal perspective for important issues in human historx; such as changes in population structure and movement pat- terns, language evolution, and the origins of infectious disease [31,32,33",34,35]. However, the difficulty associ- ated with extracting and authenticating ancient human DNA presents unique research problems, and limits the scope of studies. Modern human DNA can contaminate experiments at many stages and phylogenetic criteria are often not appropriate tests of authenticity [6,7,8*',9]. Given these problems, it is unfortunate that research on human remains, like that of million-year-old DNA, has too often ignored the burden of proof. For example, sequences of single copy globin genes from human skeletons up to 12,000 years old [36] were claimed to be authentic because they varied from one another and because one specimen possessed a novel mitochondrial haplotype, although this was soon found in modern Europeans [9,32]. In reply to criticism, the authors stated that in future, more careful analysis will produce % progressive classification of ancient DNA sequences in(to) 'dubious' or 'probably real' categories" [37]. Obviously, more rigour is needed if the field of molecular archaeology is to be taken seriousl'~:

In contrast, the successful sequencing of Neandertal DNA [2"*], probably the most technically difficult ancient DNA study to date, demonstrates how careful attention to authentication critcria can effectively address questions of contamination, although the effort required to obtain and replicate a single short sequence is sobering. The mitochondrial DNA from the Neandertal specimen is ge- netically distant from modern human sequences, strongly supporting replacement rather than genetic assimilation models of human evolution in Europe. Unfortunately; analyses of other Neandertal specimens indicate that the potential for extensive studies of Neandertal population structure may be limited [38].

Recent material While million-year-old DNA appears to belong in the science fiction section with Jurassic Park, ancient DNA studies of material up to just a few thousand years old have been used to address many important evolutionary questions. Material from field studies or museum collec- tions has allowed the analysis of migration patterns, ge- netic admixture, prehistoric population sizes, phylogenetic relationships and pathways of disease transmission.

In conservation genetics, analysis of recent material has provided important information about endangered taxa. For example, subfossil bones of the Laysan duck were identified in lava tubes on the main Hawaiian islands by mitochondrial DNA analysis, justifying reintroduction and suggesting that many endemic island species may be relics

of former cosmopolitan populations [39°]. Hypervariable single copy nuclear loci have also been obtained from young material that is sufficiently well preserved. An analysis of microsatellites in wolf-like canines showed that the red wolf had extensively hybridised with gray wolves and coyotes well before its extinction in the wild [40]. While microsatellite-based studies of ancient populations are a promising area, research on chimpanzee hair collected in the field suggests there is considerable potential for damaged DNA to complicate results [10]. In fact, a systematic study comparing the preservation of nuclear single- and muhi-copy genes to those of mitochondria in ancient specimens is long overdue.

The loss of genetic variation in recently bottlenecked populations has been quantified using museum specimens obtained before the bottleneck. For example, DNA obtained from skin and feather samples of the San Clemete Island Shrike [41] and the Greater Prairie chicken [42] from the beginning of this century has been used to demonstrate marked reductions in the genetic variability of modern populations. Ancient DNA has also been used to track endangered species using their dung [43] and identify violations of wildlife protection treaties, such as the sale of illegally-captured meat from protected whale species in Japanese markets [44°].

Archeological material has been a rich source of DNA. Analysis of human remains has provided information about sex ratios [33°], tribal relationships, and the colonisation of the New World [35,45,46]. In contrast to the difficulty of avoiding contamination during such studies, animal remains in archaeological sites can be analysed with species-specific primers [7,38]. DNA from early cattle remains in Britain show that modern cattle are derived from a diverse ancestral population and have recently expanded in Europe [47]. Similarly, analysis of Bronze age rabbits suggest a recent Holocene population expansion [48].

There are several potentially large areas where ancient DNA research is starting to have an impact. While well established in the field of forensics [49°], studies of the histou of infectious disease using ancient DNA are not yet common. One success story has been the analysis of Lyme disease, where ticks from museum skins where used to demonstrate an early presence in the US and Europe [50]. The potential for similar studies of viral evolution is large, since changes over many thousands of generations can be measured on a known temporal scale. A further promising field is the retrieval of genetic information from prehistoric domestic animals and cultivars, such as maize and wheat [51], which may lead to improvements in disease resistance or yield.

Conclusions and future perspectives While many existing ancient DNA publications require authentication, there have also been many successful stud-

52 Analytical biotechnology

ies which have provided novel information about a variety of evolutionary subjects. As experimental procedures are improved and the quest for DNA sequences older than the theoretical limit of 100,000 years diminishes, the field of ancient DNA is set to become more robust.

In the near future, we can expect analyses of entire prehistoric communities using well-preserved material from permafrost or cave deposits. Vast numbers of large mammals including horses, mammoths, moose, wolves, bears, lions and saber-toothed cats are preserved in Arctic permafrost deposits. Genetic analysis of entire faunal communities will provide a better understanding of the systematics and population genetics of prehistoric species. For example, the effect of climate change on genetic diversity, or immigration events between the Old and New World could be studied directly. Just as the study of DNA from extant mammals has led to a new field of molecular ecology, so might the study of communities that existed in the recent past provide a necessary historical perspective on current levels of genetic diversity. Molecular palcoecology may be the bright hope for those who desire new uses for old DNA.

Acknowledgements Financial support was provided in part by the UK National Fnvironmenta l Research (~ouncil and the l .everhulme Trust to Alan Cooper.

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• of special interest • . of outstanding interest

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