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OF CHEMISTRY THE JOURNAL BIOLOGICAL Vol. 264, No. 17, Issue of June 15, p 9709-9712,1989 0 1989 by The American Society for Biochemistry and Moykular Biology, Inc.
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Immunological Distances-The first comparisons of the primary structure of a gene product of an extinct species to that of living species were achieved indirectly by using polyclonal antisera raised against a homogenate of mammoth muscle. Such an antiserum was tested for its ability to form precipiTHE EMERGINGFIELD OF MOLECULAR tates and complement-fixing lattices with a panel of native ARCHAEOLOGY* albumins from several living species (9, 10). The reactions were strong with Indian andAfrican elephant albumins, weak Svante Piiiibos, Russell G . Higuchii, and with sea cow albumin, and weaker still with other mammalian Allan C. Wilson albumins. Since the cross-reaction specificity of theantiFrom the Departmentof Biochemistry, University of mammoth serum matched exactly those of antisera LO the California, Berkeley, California94720 and the §Department pure albumins of elephants, the nativealbumins of these of H u m a n Genetics, Cetus Corporation, three species (mammoth, Indian elephant, and African eleEmeryville, California 94608 phant) are nearly identical in primary sequence (cf. Ref. 11). Radioimmunoassay, which does not require lattice formaMolecular evolution is a historic process through which tion and thus does not demand that a cross-reactive antigen genes accumulate changes due to stochastic events aswell as bear more than one antigenic site, confirmed the close relaselective processes. Students of molecular evolution suffer tionship of elephant and mammoth albumins (12); it also from the frustration of trying to reconstruct this historic placed the extinct Tasmanian wolf within the genealogical process from only a knowledge of the present-day structure tree for extant carnivorous marsupials (12) and Steller’s sea of genes. Until recently, there has been no hope of escaping cow within the tree for extant sea cows (13) on the basis of this “time trap.” However, advances in molecular biological tests with antisera to their albumins. However, this method, techniques have enabled us to retrieve and study ancient like other univalentmethods, can be less reliable as a predictor DNA molecules and thus to catch evolution red-handed. In of sequence divergence than is microcomplement fixation consequence, we can now study the genealogical relationships of extinct species and vanished populations. In addition, it (14). Immunological methods are especially likely to give misseems likely that we shall be able to monitor fast genetic leading results when employing antisera that are raised and processes such as recombinational events. Our review dis- later also tested against mixtures of poorly defined antigens. cusses older attempts to obtain molecular genetic data from For this reason, we consider most antigenic studies reported archaeological remains as well as recent achievements and on ancient materials to be of questionable genetic value. emerging vistas. Gene Frequencies-At the population level, anthropologists and paleozoologists are often interested in determining the Studies of Ancient Proteins frequencies of alleles at polymorphic loci in ancient populaThe first indications that molecular genetic information tions. Determinants of the AB0 system have been of most might persist in ancient materials were early demonstrations interest (15, 16) because they are present on nearly all cells that the peptide bond can last for up to lo8 years in fossil in mammalian tissues. However, blood group serology pershells and bones (1-3) and that subcellular detail implying formed on ancient tissues has many pitfalls because ‘Jf the the survival of ribosomes and chromatin is evident in insects possibility of differential degradation of polysaccharide antifrom 40 million-year-old amber (4). Indeed, these findings gens as well as contamination of the old tissues by plant and inspired the hope that genetic information should be retriev- microbial antigens which may cross-react with the antibody able from the amino acid sequences in ancient remains, and or bind to blood group determinants (cf.Ref. 17). In well substantial efforts over the past two decades went into such preserved human remains, serological typing of proteins enendeavors. coded in the major histocompatibility complex (HLA antiUnfortunately, the major proteins in bone (collagen) and gens) maybe more informative than ABO. HLA has the shell (conchiolin) are likely to be genetically uninformative additional advantage of fewer risks of anomalous cross-reacbecause collagen has a repetitious primary structure and is tions caused by proteins from other organisms (e.g. Ref. 18). encoded by multiple genes (5)’ whereas conchiolin is a com- Nevertheless, the inherent difficulty of interpreting tht, reacplex mixture of proteins whose genetic basis is unknown (6). tion of serological reagents with antigens that are modified Second, the proteins in ancient remains are structurally het- by unknown processes remains. erogeneous because of post-mortem modifications (7,8). Even in exceptionally well preserved remains, such as froDNA in Old Tissue Remains zenmuscle from an extinctSiberian mammoth, extensive Following the realization that DNA may survive in ancient modifications were evident from elemental analysis, electron microscopy, and amino acid analysis of the 40,000-year-old tissues (19-21), DNA has been extracted from a wide variety tissue (9, 10). In the case of albumin, one of the most stable of such remains (whether d r y , frozen, or preserved wet in globular proteins known in animal tissues (8), only about 2% peat), ranging in age up to 45,000 years (Table I). Although of the mammoth molecules could dissolve in water, and 80% DNA can be extracted from most soft tissue remains that are of the latter were modified in charge, size, or antigenicity (9). well preserved morphologically, post-mortem modifications made it hard to clone such DNA in bacteria. Higuchi et al. (22) succeeded in cloning mitochondrial DNA * Work done in the laboratory of A. C. W. received support from the National Science Foundation and the National Institutes of sequences from the extinct quagga, a member of the horse genus (Equus).This represented the first retrieval of phyloHealth. $ Supported by a long-term fellowship (ALTF 76-1986) from the genetically informative DNA sequences from a museum spec’ European Molecular Biology Organization. imen and allowed the quagga to be placed into aphylogeny of

Ancient DNA andthe Polymerase Chain Reaction

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9709

and the fourdeoxyribonucleotide triphosphates.Lqww S by A ATI CLC rn ~1A ATA nt ETA RQ m MT ATA m n c T n c ium bromidein electrophoreticgels and its activity as a template that C l a d q w u . some of the problems caused both thelow cloning efficienby R. andS. The specific hybridization test has not been directly sequencing the product obtainedwhen PCR was applied to a applied to the oldest plant remains or the insects in amber. oxidized pyrimidines. cient DNA. Bowman. Thomas. I / -/ Ancient DNA has usually been detected its staining with ethid. 29).. Right Natural animal mummies 10.. one is onlyminor lesions. unpublished obser.. part of a sequencing gel highlighting with an 5.. Kocher. Dots indicate identity with the amplified Dry remains quagga sequence.eachabout25bases long. which has no capacity for repair or misrepair... The amplification is done with two synthetic oligodeoxynucleotide L l . most damaged baseless sites. i.. Refs. P. 24. 26. Thomas... The arrow denotes a position at which a cloning 140 Refs. 22. such as deaminated bases... many of molecules will either not be replicated at all.. oftenby processes that are error-prone and thus brain Human 8.000 Refs... In additionto its abilityto detect extremely small The results argue for a close relationship between the quagga quantities of DNA... 43"*b Museum skins artifact (a T residue) occurs in the cloned quagga sequence.... or will be at a replicative dismodifications are so extensive that less than 1% of the DNA advantage because many lesions.. sequencingof two enzymatically ampli. related more closely to the domestic horse.. H. due to interwhich can be attributed to oxidative processes (27). T... during enzymatic amplification. DNA preparation from quagga skin.. " " can direct the incorporation of radioactive nucleotides into DNA in ~ l l ' nbn ..2.. .. Wilson.. ." 1I J primers. Mitochondrial DNA sequences obtained in two ways with DNA from a modern species is used to determine whether the DNA detected originates from the species under study or from a from skin of a 140-year-old museum specimen of the extinct quagga. Hybridization FIG. Since the 3' ends of the two base substitutions/hundred base pairs compared for any pair of the primers point toward each other. 27 Human mummies arrow the position in the amplified sequence at which a C (rather 45.down the DNA polymerase.. anexponential of whole mitochondrial genome(42). 45 Plants than a T) residue occurs in the quagga. fied segments (30). K. 21. unpublished data. The replacements observed in the cloned old DNA is heavily modified..000 Refs. Intact molecules will thus amplify be cal finds can expected to be undamaged.g... - The polymerase chain reaction (PCR)' can amplify preselected segmentsof DNA up to quantities which permit direct sequencing.. These positions in the quagga were later may persist for millenia. A.. while the second primer matches the the extinct quagga to mtDNAs from other members of the horse genus.000 Refs. the upper result comes from contaminating source.g.. R. inbacterialclones of quagga mitochondrial repetitive DNA sequences from an ancient Egyptian mummy DNA. This manifestsitself by a reduction sequences were thus due to cloning artifacts.. 30. 23)..9710 Minireview: Ancient DNA G A T C TABLE I c m OXIIYIT I 5 DNA from eight kinds of ancient remains . molecules.. unpublished data. bp.. the presence of DNA polymerase and random primers. since the majority the vectormolecules becomes ligated to base pair(s). Meyer. a thermostable DNA 8 7 6 5 4 3 2 1 0 Percent dwergence polymerase. and. of a' Ouagga Burchell Grew Mounfarn Wtld ASS Hall ASS Dornesftc Przewalskrr Zebras !l'he Polymerase Chain Reaction Molecular in Archaeology I . Insects in amber 26 million Ref.. Percent divergence refers to the estimated number of Crick strand at the other end.. resulting possibly in size down to an average of only a few hundred base pairs from misrepairof damaged molecules.. the zebras and asses (Fig. 46' damaged molecules whose modifications preclude replication Wet remains in bacteria. Higuchi and A.. such as baseless sites. Higuchi and B...some damaged molecules become repaired Pickled museum specimens 100 * in bacteria.32 likely to distort the information extracted from cloned sea R.. The middle result comes from Maximum sequencing a cloned fragment of unamplified quagga DNA (22). Some damaged molecules will of course have In attemptsto clone ancient DNA in living bacteria. FIG. and A... 25 and 26) and by an abundance lesions..T.. These or intramolecular cross-links. such as of In contrast. Recent advances stemming from Kary Mullis's invention of the polymerase chain reaction (28) have eliminated vations.. ancient ucts ((30) Fig. preferentially. Phylogenetic tree relatingmitochondrial DNA from The first primer matches part of the Watson strand at one end of the segment...1. 25.. 41.... PCR is an ideal tool to amplify a small number of intact ancient DNA molecules present in a vast excess of damaged the horse and its relatives.the two primers.. Second.. Modified from Ref. The Sample Study age lower result was obtained in the same way as the middle one except years that the DNA was cloned from highly purified mitochondrial DNA of a Burchell's zebra (23)..like when they were directly sequenced from amplification prodall macromolecules extracted from tissue remains..e. polymerase chain reaction.. PCR has the advantage of being a n in and Burchell's zebra and against the view that the quagga is vitro system. 1) (23). W. Left p a n e l (base sequences). shown not to differ from the general vertebrate sequence Little progress followed these initialsuccesses because.. Paabo.. andcross-links. slow molecules extracted from museum specimens or archaeologi. unpublished cies and cloning artifacts arising from modifications in andata... extremely low cloning efficiencies are obtained ' The abbreviationsuaed are: PCR... 2).which can confronted with this vast excess of damaged DNA in two ways. 44... in the quagga case... Wilson. (e.. see Ref. 21' Frozen remains Mammoth muscle 40.000 Refs.quences. The cloning of For example. e. repeated cycles of heating eight species.. Basasibwaki.... ... sequencing of two cloned segments synthesis of many copies of the specific segment bounded by (23). two replacement substitutions were found when the clonable quaggasequences were compared to othervertebrate se(24) showed that far older DNA may be preserved ain form and that nuclear DNA as well as mitochondrial DNA quences (22.. These estimates come from restriction mapping the and coolinglead to a chainreaction. First..000 p a n e l (photograph). starting from extremely small amounts of DNA Horses or evensinglemolecules (for a review...

36). shown to be absent in many Amerindians (34). part of the @-globin gene fromhuman mummies) have been unsuccessful. Although contaminating sequences of human origin are easily detected in ancient remains of nonhuman species. two primers ( A and B ) use undamaged parts of five damaged templates to amplify a mosaic product. Preserved brains exist in association with human skeletal remains at several sites in Florida (26) and owe their excellent state of preservation to anaerobic and neutral conditions in the waters of Florida peat bogs. Similarly. Another area where paleomolecular biology is producing information is zoology. provedto be closelyrelated to those of elephants? Since mtDNA is present in many copies/nucleated cell. contamination by nonhuman sequences may be harder to detect. Fortunately.”In this hypothetical example. It seems that the damage present in old DNA causes a strongly inverse correlation between amplification efficiency and size of the amplificationproduct. Therefore. An additional advantage of PCR is that its speed allows for easy reproduction of results. Furthermore.yellow). blue) to the other ( B . One is a direct repeat of 9 bp which occurs only one copy in many Asiansas well as some in Native Americans(32. Such errors would be encountered only if one were to clone individual molecules from the final population before carrying out the sequencing reactions (30. wherefragments of up to 1kilobase and longer amplify efficiently. I A *” A b Exponential phase: A 4 Amplification of the region boundedby A and B to make >lob copies ‘B FIG.3.33). the 40. The same procedures revealed DNA sequences from ancient humans that are preserved in the form of mummies. which is observed for ancient but not modern DNA.Minireview: Ancient DNA generate replication errors without retarding replication. Furthermore. the phylogenetic criterion of authenticity discussed above cannot be solely relied upon distinguish contaminatto ing DNA. archaeological remains generally do not yield any products above 150 bp in size. it can be assumed that this high copy number facilitates its survival and retrieval. However.g. 9711 Authenticity of Amplified Sequences Contamination by Modern DNA-The main concernpertinent to the amplification of ancient DNA sequences is contamination of the DNA extracts or reagents by contemporary DNA. and these studies can be expected to yield a fuller picture of the population structure and genealogical history of Amerindians. uiz. In addition to the quagga discussed above.It is not yet known if this was a common genotype in ancient Egypt and whether it is represented today in Egypt or elsewhere. Three informative mitochondrial polymorphisms were studied in the Florida brain. The strong inverse correlation between amplification efficiency and size of the amplification product (iii). the primers will be extended during first the PCR cycle up to points where lesions (green) or ends of fragments cause the polymerase to stop. manuscript in preparation. its contribution to the result. and the sequences obtained should be unambiguousand identical. . whereas better preserved specimens. After a sufficient number of cycles the two primers have grown so long that their 3‘ ends overlap and a full-length doublehs can serve as a strandedmolecule is formed. as expected. these exto tended primers can anneal other template molecules and be further extended. For example. Extracts from the archaic brain contained DNA that allowed amplification of 140-bp-long mtDNAfragments. T i molecule now template for a conventional chain reaction. the mammoth sequences wereseparable from the human sequences and.000-year-old Siberian mammoth (also referred to above) has yielded PCR products contaminated with human sequences. In an amplification reaction the where each template DNAis so damaged that no molecules allow DNA polymerase to proceed directly from one primer site (A. Lag phase: Ancient DNA Sequences Revealed via PCR Using PCR and direct sequencing.mtDNA sequences from the Siberian mammoth show the mammoth to be closely related to elephants but not identical to either elephant species. During subsequent cycles. presumablyoriginating from handling of the mammoth after its discovery. the authenticity of the DNA sequences obtained by bacterial cloning from the quaggawas demonstrated by the fact that they proved the quagga to be a close relative of extant zebras but not identical to any of those tested (22. In our experience. such contamination can often be detected by mere inspection of the sequences when a phylogenetic analysis fails to place the species under study close to its biological relatives. For example. attempts to amplify specific nuclear single-copy genes from ancient remains (e. to some extent can serve as a further criterion of authenticity. the fast evolution and maternal mode of inheritance of mtDNA makeit ideal for studying ancestordescendant relationships (35. R. the sequence determined on the whole descendant population of amplified molecules. Wehave found it imperative always to do control extracts (i) in parallel with the extracts of the old specimens in order to detect contamination in solutions and reagents (32. This is in sharp contrast to contemporary DNA. and a HaeIII site at position 8250 were amplified and sequenced. Higuchi and A. the following additional criteria must be set up and adhered to in order to detect any source of contaminating DNA. From the sequences obtained. Concept of “jumping PCR. which sets a limit to thelength of amplifications that can be performed.31). In fact.23). the archaic Floridian could be compared themtDNAs of more than 100 presentto day Native Americans. a Him11 site at position 13259. 37). Accordingly. presumably due to their lower abundance in DNA extracts. Furthermore. since each error is specific to one molecule in the starting population (and its descendants). Further work is in progress to characterize presentday Amerindian populations as well as further archaic Florida brains.islikely to benegligible. This ancient sequence does match not any of the three mitochondrial lineages found to date in America. it has been possible to obtain mitochondrial sequences from a 7000-year-old brain excavated in Florida (32). When extinct species are studied. several independent extracts (ii) from every individual should be prepared. Longer amplifications were unsuccessful. These errors as well as those introduced by the polymerase at undamaged sites will be present in the final population of molecules produced by PCR. a 4000-year-oldEgyptian priest was shownto carry an unusual D-loop sequence (27). Wilson.

J. I. If no template molecules spanning the entire segScience 209. and Kirk.. Immunol. Y.. M. Bremer. M. Leach. the ratio of silent to replacement changes in 3. 283-287 24. A. O. Rollo. Kocher. Amsterdam 37. P. A particularly important goal for the future is to to 500-bp pieces.G. c. Carr. B. E. J. Biochern. Sarich. 1 6 .9712 Minireview: Ancient DNA such as museum skins. (19%) NLlcletc A C Res. and Mikhelson. (1981) S h e w w u HUQH s w h S h e w wu L i Chin Chon 3 9 . (1975) Homo 2 6 . Manchester Unlversity Press. J. G. M. These 15. 379-382. E. Bristol. L. in press 38. Lowenstein. and Hauswirth.. Freiberger. J. Gillespie. J. T... (1986) cold Spring Harbor Symp. Physiol. (1981) Nature next cycle be further extended upon hybridization to other 291. R. Higuchl R. M. Jakobsson. eds) Academlc Press. K. G. A. (1983) Biochemistry 22.. Berg. A. V. (1989) Trends Genet. Mudryj. R... Hannum. Hou&:P. Flaherty. E. 2. Piiabo: s. (1984) Annu.774 39. George. O. C. E. G. the evolution of populations can be studied and will most often involve the comparison of ancestral and descendant populations. S... M. and Magor. J. Wrischnik.. Laipis. and Bendich. M. teeth. M. and sharing unpublished data with US. Scharf.20. D. A.M. A.. Sci. Other cases where rapid evolution may allow molecular processes to be observed directly can be provided by human parasites (especially viruses) and the evolution of domestic :. Schwartzfischer. G. Prager. Arnheim. W. D. C. A. R. W. Harrison.. G. A.. 1045-1051 22. M.. Wallace. 38) opens up the possibility of studying molecular evolution by actually going back in tirne and directly approaching DNA sequences that are ancestral to their present-day counterparts.104-105 42. 17. 487~491 32. (1b85) Biol. S. J. Sage R. i. and thepartially extended primers can in the 12. Phys. E.. A. Hansen. B k h m . H.. H. Wilson. E. Jr. c. Geor e M. Z.. Linh. S. S.. New York. Only when 13. in press 30. 81B. Natl. T. 6.. F.803-806 27.773-776 44. ed) pp.. and Wilson. George. Scharf.'Mullis'. 17. Lowenstein. and Wong. and Knowler. Palumbi S. S. T. C . Wrischnik. Quant. M. A. S. B..644-645 25.67-101 first extension. Berzofsky. T. Sninsky.545-568 erty Of PCR may affect the authenticity Of the amp1ified 9. and Arnheim. Wilson. Temrin. (1989) in The Hierarchy of Life (Fernholm. Vistas f o r Molecular Archaeology The recently achieved ability to study DNA from museum specimens and archaeological finds via PCR (27. (1988) Science 239. Stoffel. H.C. F... R.... W.. (1981) in Magadan Baby Mammoth (Vereschagin. and Wilson. R. D. 21. A... L. Wan% G.. Vogeli.. 178-190. Helm-Bychowskl.149-155 35. (1969) Nature 224.. extended primer products overlap with their 3' ends will a 14-18 conventional exponentialamplification reaction ensue. J. 0.. R. Weiner. H..and wilson.1350-1354 29. B. Arnbeim... (19%) Nature 3 3 4 . may allow for the amplification of up plants (41).1100-1102 Mosaic Sequences via Jumping PCR-An additional prop. Hoffmann.. Wilson. Gurd. G. Royle.. ~ 9775-9787 33.. E.8. R. A. K. K. N.. 2 6 . Prager. Erlich. J. Euol. Orrego. A. M. Higuchi. Manchester. (1983) Am. a ratio of 2 to 8 would have been M. DNA that are complementary to one or the other of the 11. 447which homoplasmy is the rule (35).. N. W. Erlich. Zimmer. News Letter 62. However. When the three above criteria (i. N. White. 61. M. A. T.287-289 C. E... G. Biol. B. Bowman. S. Oakes. C. Agee. 5. Piabo. H. andSarich. E. (Innis. Wilson..-A Moratory !anal.. amplification may in fact start from shorter fragments of the eds) pp. (1984) Nature 312. A.. M. promising avenues of research on molecular evolution may now be We refer to the study Of those molecular genetic processes that may occur rapidly enough to be studied On a time Of lo4 years' in contrast to the 'low accumulation of base substitutions. (1988) Maize Genetics COOP. G.2203-2214 41. A. Awedimento. Poinar. 7 3 . Doran. F. attributing changes in gene frequencies to factors such as variations in population size* and genetic drift. Rainey. J.. Sarich. 376-400 36. Acad. 3). E. E. A.. and Stoneking.. (1984) J. B. s. cules. Leningrad. (1970) Science 170. A. A... H. T. ~~~~~~& . ed) pp. Weiner. (1986) in Science in Egyptology (David. 40). Cann. J r and Ryder 0. and Jornvall. U.. 213-218 quences that result from recombination during amplification. S. Rogers.Mullis. 32.. C. Biol. Sensabaugh... 7 0 individuals.. S. (1976) h o c . and Hess. J. 15. (1986) Mol. N. (1982) Science 216. Amici.. (1989) Proc.. J.887-892 6. Knoxville. M.325326 initial steps may allow the amplification of regions that are 16. W. C. s.1241-1242 from the extant mountain zebra is 10 to 0... Garrison. Jr..Faloona. J. and'Braun.: 23. and Wilson. J. s. Salvi. H. K. Euol. P.p. Jeffreys. S O C . Piiiibo. and Garbuglia. in the case of 452 chromosomal genes from ancient remains of heterozygous 19.. Prager. Phys. C. V. v. M. In addition to Organisma' and population questions. B. Natl. In these cases. E. D.. Saiki. J.. H.. 4 3 . and Kocher. and iii) are fulfilled. G. V.. Taylor. 1557 .. and Richardson. distributed at random with respect to position within codons. J. Smlth-Gd1. H. and Giirtler. B.. (1988) Auk 105. K. T N DNA from the extinct quagga "'Ompared to modern DNA 4. D. E. the 10.. F. as well as E.C. (1984) D~ Altertum 3 0 . Miller. C. in particular C. K. H. (1984) Fed... Sercarz. F. Rosing. R. Paabo S. (1988) in Molecular Euolution and the ~ o ~ ~ i l(BroadRecord head. "jumping PCR' may generate erroneous se. M. Gyllensten.and Abdalla.V. even rare examples of single preserved specimens are of p e a t value. W. This allows us to address many questions involving the identity and relationship of extinct species to other extinct and extant species. eds) p 407 419. M. J. A. U. (1985) Science 230.. (1985) Plant Mol... If the diff~~ences 5. East.F. i n d Erlich... A. andLu.. Proc. M. R. L. U. A. 2541-2545 In fact. J. (1989) Nucleic Acids Res. (1987) J. ' ~ ~ . Gifford. D. M. p-bo. Second. Gelfand. D. Anthro 01 68.. T. 7. Benjamin. Stoneking. S.. and Whlte. V. and expected to predominate in an amplified population of mole. D. and Haigh..M. V. (1985) Nature 314. M. a n d v a n Regenmortel. G. Thomas. P. R. The substitutions would be nica..69-76 ( 1 9 a ) camp. Sequences longer than this have invariably refine the polymerase chain reaction so that it becomes POSproved to originate from contamination of the specimens by sible to study ancient nuclear single copy genes as well as modern DNA. ii. u..529-542 34. (1987) Nucleic Acids Res. Wyckoff. W.. S . specific errors stemming from post-mortem changes are not M. U. A. D. and Hood. S. AnthropoL 6 1 .586-588 enough extension steps have been performed so that the two 14. Horn. D. Higuchi. J. A. C. and Erlich. Ryder. A.. shells.. A.. Higuchi. M.409-411 fragments of the region that is to be amplified.. C. Helentjaris.H. J. and amber. B.. Curry. Ballinger. and Wilson... D. (1972) he Biochemktry o f ~ n i m l~ ~ ~ ~ i h Scientechbe affected in a predictable way. A. Mol.. had occurred at random. and de Crombrugghe. T. and Wischearth.. Beck. K. A. R. 148-153 longer than thelongest intact molecule in an extract. C.. Elsevier Science Publishers B. Gelfand D. G. 86. Higuchi. N. The building of a mosaic sequence via PCR poses no probR. H.. Tong. Nauka. T.Euol. H..E. Faster processes include recombination in minisatellite sequences which cause apparent mutation frequencies on the order of 10-2~locus~generation (39. Mol. X. Lowenstein. Ohkubo.. S. 3. N. (1986) Nature 323. Margoliash. (1980) Cell 22.. and Wilson. Pastan.. R..441-446 26. E.. J..3 and Wilson.. Connolly. R.. United Kingdom lem in the Of mtDNA sequences for 18.. R. a Acknowledgments-We thank numerous colleagues for discussion given sequence is considered likely to be of ancient origin. H. 43.. 2 1 .L. A. C.. Irani. T. c. the pattern Of f3ubstitution would 1. Acad. A.. Irwin.535-546 A..(1989) in PCR-Pmtocoka&Applicat. If they were to predominate and thus cause incorrect REFERENCES sequences to be determined. E. DNA sequences from bones. Dickel. (1971)Int. Sarich. Gyllensten.C. Prager. (1988) Nature 332.. K. Todd. H. United Kingdom 2. Pa&o. J..1939-1943 28. These fragments serve as templates in the Reichhn. Prager.282-284 1. 0. N. however. K. 278-281 40.4139-4145 expected. B. 2. Prager E M. G.. Villablanca. 20-33.Lowenstam.. (1988) Nature 336. (1980) sequences.. Saiki R. W... and Wilson. Biol. (1984) Naturwksemcha ten71.. Horn. B. L. Errors because of Post-mortem Chnges-As noted above. F. A.. primers (Fig. Yamada. s. and Wilson. which in nuclear and organelle genomes is generally less than 1% of sequence divergence/1O5years. M.. Higuchi. M. C. A. S. Reu. C. Wilson: A. J. ment defined by the primers exist in the old extract. F. R. (1985) Am. A. W. s. 30.. Jr.. Sei.. M. I. M. G. K. M. phabo. 387-388 31. Michael.. Myers for expert graphical work.. v.

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