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Amplifying DNA from Archeological Remains: A Meeting Report Svante P~abo

Institute for Zoology, University of Munich, D-8000 Munich 2, Germany

During the half-decade of its exis- cloned. This is important because an- numbers of sequences, the original al- tence, the polymerase chain reaction cient DNA contains large amounts of leles of the ancient individual could be (PCR) has transformed most areas of damage that manifests itself in the determined. Since this approach seems molecular biology. In the case of the form of a high frequency of misincor- too laborious to apply to the large study of DNA from archeologica] porated (3) and in the number of samples necessary for work remains, PCR has created an entirely formation of in vitro recombinant at the population level, Hauswirth and new field. Prior to PCR, only two molecules by means of "jumping collaborators intend to use allele- papers appeared in which DNA se- PCR. ''{4) The latter phenomenon is in- specific to address quences from old tissues were present- duced by DNA damage that causes the population genetics questions. ed. (1,2) Since the advent of PCR, at least DNA polymerase to stall or fall off its Endogenous retroviruses are simi- 10 such papers have been published template. The partially extended larly being amplified and cloned from and several more are currently in primers can then "jump" to other al- ancient Egyptian animal remains by review or in the press. The rapid leles and loci during further cycles of Goudsmid and collaborators. (81 Retro- growth of this field was evident at a amplification. However, since these er- viruses that today are endogenous may recent meeting in Nottingham, Eng- rors happen more or less randomly, not have been so for very long. Indeed, land (July 8-10) organized by the Nat- they are averaged out in the direct se- one may speculate that the close co- ural History Museum and the Univer- quencing and are not detected. (s) For existence of species that resulted from sity of Nottingham--the first meeting practical reasons, ancient mtDNA se- the domestication of animals acceler- ever to be dedicated entirely to the quences are more easily accessible than ated the transfer of viruses between study of ancient DNA. Over 60 investi- are other old sequences; thus, it is a species. Thus, it may turn out to be gators convened in a friendly and fortunate coincidence for the field very interesting to study endogenous relaxed atmosphere for presentations that the mitochondrial genome is as well as exogenous retroviruses in dealing with subjects as diverse as ex- highly informative from a phylogenet- early domesticated species. tinct mammals and birds, the domesti- ic point of view. (6) mtDNA is likely to cation of plants, and endogenous remain the staple of conversation AUTHENTICITY retroviruses. among molecular archeologists. The power of the PCR to amplify a few However, because animal mtDNA is intact DNA molecules that can be ex- MITOCHONDRIAL AND NUCLEAR inherited maternally, it does not reflect tracted from ancient remains has SEQUENCES the male side of history nor processes opened up the field of molecular ar- Most work on ancient DNA to date has that occur in the nucleus. Therefore, it cheology. However, this power of the dealt with the amplification and se- is very stimulating that several groups PCR also causes severe problems in quencing of mitochondrial DNA now report the amplification of that the PCR will preferentially amplify (mtDNA), mainly because mtDNA ex- nuclear DNA sequences. For example, any modern undamaged DNA mole- ists in numerous copies per cell, which Lawlor et al. (7) have used conserved cules that contaminate an ancient are more likely than single-copy primers to amplify major histocompat- specimen or the PCR. Thus, the prob- nuclear sequences to survive the ibility class I heavy-chain genes as well lem of contamination has become a autolytic degradation of nucleic acids as the single-copy ~2-microglobulin spectre that haunts the entire field. that follows the death of an organism, from 7000-year-old human remains Undoubtedly, some of the data that as well as the effects of damage that ac- from a sinkhole site in Florida. In the were presented at the meeting, as well cumulate over centuries and millenia. case of the highly polymorphic heavy as some of the data that have been A further advantage of mtDNA is that chains, their primers amplified all of published, will turn out to be due to an individual usually contains only the 50 or so genes that exist for these contamination. It is important for this one type of molecule. Thus, when an proteins. The amplification product young field that investigators try to de- amplification product is obtained, it was then cloned and individual clones sign projects in which the authenticity can be sequenced directly rather than were sequenced. By comparing large of the sequences obtained can be eval-

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uated. One such area is the study of tions of their authenticity. Controls been claimed to be due to an accelera- animal remains. For example, when such as amplifications from extracts tion of the rate of DNA sequence Thomas et al. (gJ sequenced a short frag- devoid of tissue samples and amplifica- evolution. However, the 4500-year-old ment of the mitochondrial cytochrome tions without extract, as well as multi- maize alleles are as different from each b gene of the marsupial wolf and ple extracts from a specimen, can, and other as are modern alleles from each showed that this animal belongs in the should, always be performed to control other, and some ancient alleles are Australian radiation of marsupials, the for contamination. ~l l/ However, a very very closely related to modern alleles. sequences inspired confidence because small amount of contaminating DNA Therefore, the data clearly refute they clearly fell among marsupial may go undetected in such controls, claims of an acceleration of the evolu- animals but were not identical to any perhaps because the molecules become tion at the primary sequence level in extant marsupial studied. At the meet- bound to the walls of the reaction maize. The genetic basis for explosive ing, Carey Krajewski presented more tubes. What is disturbing in this regard morphological evolution of maize extensive sequences from a marsupial is our recent observation (A. Cooper, must therefore be sought elsewhere, for wolf. These new sequences partly in- M. HOss, S. Pafibo, unpubl.) that an- example, in the activities of trans- clude the previously determined se- cient extracts often contain molecules posable elements. quences of the cytochrome b gene but that will act as carriers, causing small The potential of the maize work for differ from these at two positions. amounts of contaminating DNA to be elucidating the history of humans is Thus, they provide a confirmation of amplified in the extract amplifications great. Once the different ancient maize an ancient sequence by an indepen- while no visible amplification products variants have been thoroughly charac- dent group. The data by Krajewski appear in the controls. Such a product terized by DNA sequence analysis, they futhermore throw new light on the may then be sequenced and considered can be used to study the spread of agri- natural history of Australia because to be ancient. Bryan Sykes pointed out culture and subsequent trade connec- they indicate that the marsupial wolf at the meeting that contaminants may, tions. This will most likely be success- together with the wombat were part of for example, also manifest themselves ful in the Americas where a dry climate an early radiation of marsupials, later as an animal sequence carried over to a in many regions facilitates corn cob to be eclipsed by a radiation including human bone extract from previous preservation. One hopes that attempts the Dasyuridae (e.g., the Tasmanian amplifications. to embark on the same route using Old devil and the tiger cat). World crops will also prove fruitful. DOMESTICATED SPECIES HUMAN REMAINS In view of these difficulties, it seems POPULATION STUDIES Results that may also lend themselves ironic that most work performed on One of the most stimulating develop- to controls against contamination are ancient DNA is devoted to ancient hu- ments in the field has been the the attempts to determine the sex of man remains. Our anthropocentricity demonstration by Hagelberg and human skeletal remains by the use of probably makes this unavoidable. We others 02-14) that bones in many in- Y-chromosome-specific sequences. This should not forget, though, that the stances may yield amplifiable DNA. topic is relevant to physical anthropol- history of humans can be followed, Thus, one can hope to retrieve old ogy because morphometrics provide not only by the study over time of our DNA sequences in regions of the world very limited help for sex determination own genes, but also of those of our where soft tissues are not preserved; for skeletons of nonadults as well as companion species, particularly do- also increased is the prospect of retriev- skeletal fragments. Hummel ¢1°~ showed mesticated animals and plants. The ing DNA sequences from many indi- that in some cases Y-chromosome- first results of this endeavor were pre- viduals. As a result, it may become pos- specific repeated sequences are amplifi- sented at the conference by Franco sible not only to study a few represen- able from archeological bones. This Rollo and Pierre Goloubinoff, who tatives of extinct species but to follow system offers the possibility of control- both study ancient maize. Goloubinoff populations over substantial time peri- ling for the frequency of false-positive has amplified and reconstructed alleles ods. To date, the most extensive such signals by the use of skeletons that are for the nuclear alcohol dehydrogense 2 study involves contemporary and mu- morphologically shown to be female. A (adh 2) gene from Peruvian and seum populations of kangaroo rats.OS) study of a large series of such bones, Chilean maize specimens that go back This work illustrates both the great performed in a blind manner, would 4500 years in time, i.e., more than potential and the problems of this ap- do much to increase the confidence in half-way toward the domestication of proach. Thomas et al. found that for work from ancient human remains in maize that took place in Middle Amer- populations where one mitochondrial general. ica around 7000 years ago. One of the lineage dominates--generally small With the exception of the Y-chro- enigmas of maize biology is the vast populations--it is possible to determine mosome sequences, the area in which morphological diversity that has been whether a certain population is the de- the problem of contamination is most generated over this very short history. scendant of another population. In vexing is the study of human remains. This seems to be paralleled by a large contrast, a larger rodent population ex- The reason for this is that the most amount of diversity at the DNA se- hibited such an amount of mitochon- likely source of contamination is hu- quence level, manifested, for example, drial diversity that samples on the or- man DNA and that the sequences among modern adh 2 alleles. Both of der of some dozens of individuals were themselves are unlikely to give indica- these phenomena sometimes have not able to confirm or reject the hypo-

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thesis that the populations living in lection on MHC alleles from European such diverse areas as behavior, inter- the area had changed over the last 60 infectious diseases could potentially be mediary metabolism, or cell motility years. followed. today find themselves studying the These studies point to a general molecular structure of genes. Now that problem of all studies that try to use FOSSIL DNA the PCR has made the retrieval of an- ancient DNA to address population ge- The result that has caused the most ex- cient DNA a feasible task if not an easy netic questions; that is, we will often citement in the field since the initial one, it is possible to envision that in be unable to obtain representative work showing that it was at all possible 10 years archeologists, anthro- samples of ancient populations. Even to go back in time is the report last pologists, and paleontologists will all when large numbers of individuals are year of amplification of a 790-bp DNA have come together at the same inevi- preserved, such as in the sinkhole sites fragment from a Magnolia leaf com- table meeting point--the double helix. in Florida, we do not know whether pression fossil that is 17-20 million the samples come from individuals years old. (t9) This finding is particular- that are related to one another, if they ly intriguing because the presence of REFERENCES are separated in time by several genera- water in the deposit would seem to in- 1. Higuchi, R., B. Bowman, M. Frei- tions, or if gene flow into the popula- dicate that spontaneous depurination berger, O.A. Ryder, and A.C. Wilson. tion has occurred. For humans, this should have degraded the DNA after 1984. DNA sequences from the problem is exacerbated by the demon- some tens of thousands of years. (2°) quagga, an extinct member of the stration that even small tribal groups Also, the high-molecular-weight DNA horse family. 312' 282-284. may contain large amounts of genetic that can be seen in many extracts from 2. P~i~ibo, S. 1985. Molecular cloning of diversity. (16) The only way out of this the Clarkia fossil bed specimens can be ancient Egyptian mummy DNA. dilemma may be to perform extensive shown to be of bacterial origin. (21~ Dr. Nature 314: 644-645. studies of present-day populations to Golenberg announced that other 3. P/~/ibo, S. 1989. Ancient DNA; Extrac- determine exhaustively their genetic groups have been able to determine se- tion, characterization, molecular variability. With such background in- quences from Clarkia leaf fossils, cloning and enzymatic amplification. formation, we may then be able to verifying that DNA can survive for mil- Proc. Natl. Acad. Sci. 86: 1939-1943. make use of even a fragmentary samp- lions of years. These results are eagerly 4. P/i~ibo, S., D.M. Irwin, and A.C. ling of an old population. awaited because they will determine if Wilson. 1990. Enzymatic amplifica- If this becomes possible, a number the field of ancient DNA will fulfill the tion from modified DNA templates. J. of questions concerning linguistic and recent promise of being able to go back Biol. Chem. 265: 4718-4721. c~dtural change in relation to popula- over geological time. 5. P~i/~bo, S. and A. Wilson. 1988. Poly- tion history may be addressed. In ar- merase chain reaction reveals cloning cheology, a very frequent question is ALLAN WILSON artefacts. Nature 344: 387-388. whether or not a change in material 6. Wilson, A.C., R.L. Cann, S.M. Carr, The only circumstance casting a som- culture detected by artefacts exhumed M. George, U.B. Gyllensten, K.M. bre atmosphere over this otherwise at excavations represents a replace- Helm-Bychowski, R.G. Higuchi, S.R. pleasant conference was the news of ment of one people by another or Palumbi, E.M. Prager, R.D. Sage, and Allan Wilson's disease. Since the meet- whether it simply reflects a change in M. Stoneking. 1985. Mitochondrial ing, we have been saddened by Allan economic structure or even fashion of, DNA and two perspectives on evolu- Wilson's death. No other individual for example, pottery making. The cur- tionary genetics. Biol. J. Linn. Soc. 26: scientist has done more to establish rent trend in archeology is to stress 375-400. the study of ancient DNA. The large continuity of populations, whereas ear- 7. Lawlor, D.A., C.D. Dickel, W.V. number of contributions by people lier, replacement was stressed (see, e.g., Hauswirth, and P. Parham. 1991. who presently or previously worked in ref. 17). Such issues may in some cases Ancient HLA genes from 7,500-year- his laboratory is a testimony to his be resolved by molecular biology (see, old archaeological remains. Nature seminal influence. In particular, Russell e.g., ref. 18). This will not only be of 349: 785-788. Higuchi's talk on his pioneering work great interest to historians but may 8. Goudsmit, J., J. Dekker, L. Smit, C. in Allan Wilson's laboratory with the also prove medically important. For ex- Kuiken, J. Geelen, and R. Petronius. quagga and his long-time involvement ample, the spread of disease alleles Analysis of retrovims sequences in circa with the mammoth provided a beauti- such as those for sickle cells must be 5300 years old Egyptian mummy DNA ful history of the field. Allan Wilson's understood as a result of selection as absence will be painfully felt for many obtained via PCR amplification and well as of population movements. molecular cloning (ed. W.V. Davies). years to come. However, without an understanding of British Museum Publications. (In the extent of past population move- press.) ments, it is impossible to distinguish CONCLUSION 9. Thomas, R.H., W. Schaffner, and A.C. these two factors from one another. In the few decades since biologists Wilson. 1989. DNA phylogeny of the Furthermore, if ancestor-descendant have learned to manipulate plasmids extinct marsupial wolf. Nature 340: relationships between ancient and and restriction enzymes, many areas of 465-467. modern populations can be demon- biology have converged at the molecu- 10. Hummel, S. and B. Herrmann. 1991. strated in the New World, effects of se- lar level. Thus, biologists interested in Y-chromosome-specific DNA ampli-

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Amplifying DNA from archeological remains: a meeting report.

S Pääbo

Genome Res. 1991 1: 107-110 Access the most recent version at doi:10.1101/gr.1.2.107

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