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



eachabout25bases long....some damaged molecules become repaired Pickled museum specimens 100 * in bacteria. 23). and the fourdeoxyribonucleotide triphosphates.. the upper result comes from contaminating source. 27 Human mummies arrow the position in the amplified sequence at which a C (rather 45. Second. ancient ucts ((30) Fig. First.. of a' Ouagga Burchell Grew Mounfarn Wtld ASS Hall ASS Dornesftc Przewalskrr Zebras !l'he Polymerase Chain Reaction Molecular in Archaeology I . Since the 3' ends of the two base substitutions/hundred base pairs compared for any pair of the primers point toward each other. many of molecules will either not be replicated at all. Phylogenetic tree relatingmitochondrial DNA from The first primer matches part of the Watson strand at one end of the segment.. 43"*b Museum skins artifact (a T residue) occurs in the cloned quagga sequence...2. molecules. repeated cycles of heating eight species. preferentially.g. which has no capacity for repair or misrepair.. sequencingof two enzymatically ampli..... 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.. slow molecules extracted from museum specimens or archaeologi. such as of In contrast. 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). 29). Higuchi and B.g...quences.. extremely low cloning efficiencies are obtained ' The abbreviationsuaed are: PCR... unpublished obser.T.... andcross-links. anexponential of whole mitochondrial genome(42). and. such as baseless sites. The arrow denotes a position at which a cloning 140 Refs.which can confronted with this vast excess of damaged DNA in two ways.the two primers.. Percent divergence refers to the estimated number of Crick strand at the other end.. .. due to interwhich can be attributed to oxidative processes (27).. Some damaged molecules will of course have In attemptsto clone ancient DNA in living bacteria. 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. unpublished data. unpublished data. Recent advances stemming from Kary Mullis's invention of the polymerase chain reaction (28) have eliminated vations.. oftenby processes that are error-prone and thus brain Human 8. The middle result comes from Maximum sequencing a cloned fragment of unamplified quagga DNA (22).. some of the problems caused both thelow cloning efficienby R. . Thomas.... DNA preparation from quagga skin.. during enzymatic amplification.1. fied segments (30). 1) (23). starting from extremely small amounts of DNA Horses or evensinglemolecules (for a review. Right Natural animal mummies 10. 41. Bowman. 24. . The cloning of For example.. a thermostable DNA 8 7 6 5 4 3 2 1 0 Percent dwergence polymerase.. part of a sequencing gel highlighting with an 5.. Meyer.000 Refs... FIG. This manifestsitself by a reduction sequences were thus due to cloning artifacts. Insects in amber 26 million Ref. R.. Left p a n e l (base sequences).. such as deaminated bases.9710 Minireview: Ancient DNA G A T C TABLE I c m OXIIYIT I 5 DNA from eight kinds of ancient remains . W.. most damaged baseless sites.. or will be at a replicative dismodifications are so extensive that less than 1% of the DNA advantage because many lesions.. and A. (e. T. Higuchi and A. PCR has the advantage of being a n in and Burchell's zebra and against the view that the quagga is vitro system.. These estimates come from restriction mapping the and coolinglead to a chainreaction. Refs. the presence of DNA polymerase and random primers.. P.. bp. Intact molecules will thus amplify be cal finds can expected to be undamaged. andS... Hybridization FIG. when they were directly sequenced from amplification prodall macromolecules extracted from tissue remains. 30.. while the second primer matches the the extinct quagga to mtDNAs from other members of the horse genus. e. related more closely to the domestic horse.... in the quagga case. cient DNA....... one is onlyminor lesions. Basasibwaki.. 22. I / -/ Ancient DNA has usually been detected its staining with ethid.. These positions in the quagga were later may persist for millenia.. inbacterialclones of quagga mitochondrial repetitive DNA sequences from an ancient Egyptian mummy DNA.. Thomas.. K.down the DNA polymerase.. polymerase chain reaction... unpublished cies and cloning artifacts arising from modifications in andata. 45 Plants than a T) residue occurs in the quagga. Wilson.. Paabo. 21' Frozen remains Mammoth muscle 40.. 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. 44. These or intramolecular cross-links. H..... 2).. In additionto its abilityto detect extremely small The results argue for a close relationship between the quagga quantities of DNA. 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.. 25 and 26) and by an abundance lesions. the zebras and asses (Fig. 26. since the majority the vectormolecules becomes ligated to base pair(s). Wilson.000 Refs. " " can direct the incorporation of radioactive nucleotides into DNA in ~ l l ' nbn . shown not to differ from the general vertebrate sequence Little progress followed these initialsuccesses because....000 Refs.. The amplification is done with two synthetic oligodeoxynucleotide L l . - The polymerase chain reaction (PCR)' can amplify preselected segmentsof DNA up to quantities which permit direct sequencing. Kocher.... resulting possibly in size down to an average of only a few hundred base pairs from misrepairof damaged molecules. Modified from Ref. i.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 . 25.. 46' damaged molecules whose modifications preclude replication Wet remains in bacteria.32 likely to distort the information extracted from cloned sea R. 21.000 p a n e l (photograph).000 Refs. The replacements observed in the cloned old DNA is heavily modified. Dots indicate identity with the amplified Dry remains quagga sequence.." 1I J primers. oxidized pyrimidines. sequencing of two cloned segments synthesis of many copies of the specific segment bounded by (23). see Ref..

Therefore.Minireview: Ancient DNA generate replication errors without retarding replication. In fact. Wilson. presumably due to their lower abundance in DNA extracts. the following additional criteria must be set up and adhered to in order to detect any source of contaminating DNA. Longer amplifications were unsuccessful. and these studies can be expected to yield a fuller picture of the population structure and genealogical history of Amerindians. two primers ( A and B ) use undamaged parts of five damaged templates to amplify a mosaic product. The same procedures revealed DNA sequences from ancient humans that are preserved in the form of mummies. archaeological remains generally do not yield any products above 150 bp in size. shown to be absent in many Amerindians (34). since each error is specific to one molecule in the starting population (and its descendants). a 4000-year-oldEgyptian priest was shownto carry an unusual D-loop sequence (27). However. part of the @-globin gene fromhuman mummies) have been unsuccessful. In our experience. blue) to the other ( B . Although contaminating sequences of human origin are easily detected in ancient remains of nonhuman species. One is a direct repeat of 9 bp which occurs only one copy in many Asiansas well as some in Native Americans(32. Furthermore. these exto tended primers can anneal other template molecules and be further extended. as expected. provedto be closelyrelated to those of elephants? Since mtDNA is present in many copies/nucleated cell. For example. the phylogenetic criterion of authenticity discussed above cannot be solely relied upon distinguish contaminatto ing DNA. 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. the 40. 37). Fortunately. wherefragments of up to 1kilobase and longer amplify efficiently.yellow). it can be assumed that this high copy number facilitates its survival and retrieval. Higuchi and A. Lag phase: Ancient DNA Sequences Revealed via PCR Using PCR and direct sequencing.g. This ancient sequence does match not any of the three mitochondrial lineages found to date in America. Three informative mitochondrial polymorphisms were studied in the Florida brain. several independent extracts (ii) from every individual should be prepared. Similarly. The strong inverse correlation between amplification efficiency and size of the amplification product (iii). 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. 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. During subsequent cycles. uiz. From the sequences obtained. the sequence determined on the whole descendant population of amplified molecules. This is in sharp contrast to contemporary DNA. 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. whereas better preserved specimens.23). 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. a Him11 site at position 13259. its contribution to the result. 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. An additional advantage of PCR is that its speed allows for easy reproduction of results. R. attempts to amplify specific nuclear single-copy genes from ancient remains (e. Further work is in progress to characterize presentday Amerindian populations as well as further archaic Florida brains. manuscript in preparation. which sets a limit to thelength of amplifications that can be performed. Concept of “jumping PCR. 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.”In this hypothetical example. Furthermore. In addition to the quagga discussed above. 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.36). Another area where paleomolecular biology is producing information is zoology. the archaic Floridian could be compared themtDNAs of more than 100 presentto day Native Americans. It seems that the damage present in old DNA causes a strongly inverse correlation between amplification efficiency and size of the amplificationproduct. and a HaeIII site at position 8250 were amplified and sequenced.33). Furthermore. I A *” A b Exponential phase: A 4 Amplification of the region boundedby A and B to make >lob copies ‘B FIG.3. 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. When extinct species are studied. it has been possible to obtain mitochondrial sequences from a 7000-year-old brain excavated in Florida (32). to some extent can serve as a further criterion of authenticity. and the sequences obtained should be unambiguousand identical.31).000-year-old Siberian mammoth (also referred to above) has yielded PCR products contaminated with human sequences.It is not yet known if this was a common genotype in ancient Egypt and whether it is represented today in Egypt or elsewhere. presumablyoriginating from handling of the mammoth after its discovery. contamination by nonhuman sequences may be harder to detect. the fast evolution and maternal mode of inheritance of mtDNA makeit ideal for studying ancestordescendant relationships (35. T i molecule now template for a conventional chain reaction. Accordingly. Extracts from the archaic brain contained DNA that allowed amplification of 140-bp-long mtDNAfragments.mtDNA sequences from the Siberian mammoth show the mammoth to be closely related to elephants but not identical to either elephant species. which is observed for ancient but not modern DNA. Such errors would be encountered only if one were to clone individual molecules from the final population before carrying out the sequencing reactions (30. the mammoth sequences wereseparable from the human sequences and.islikely to benegligible. . For example.

V. O. Zimmer. Saiki R. E. K... J. and'Braun. H. Hoffmann. i n d Erlich. M. in press 30. R. B.. and Wilson. S O C . 4 3 .. Garrison.. W. 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. Lowenstein. Rainey.. J. C. s. F. the 10.. R. (1989) Proc.149-155 35. R. andSarich. D. 2. Benjamin. L. shells. Stoneking. U.. (1983) Biochemistry 22. Royle. When the three above criteria (i. Wrischnik. ment defined by the primers exist in the old extract. A. K.. 278-281 40. S . J. These 15.409-411 fragments of the region that is to be amplified. Ohkubo. V. S. (1987) Nucleic Acids Res. J. S. R. Michael. J. in particular C. W.. If no template molecules spanning the entire segScience 209. 148-153 longer than thelongest intact molecule in an extract. A. Piiiibo. M. Amici. D. a ratio of 2 to 8 would have been M. c.. J. Faster processes include recombination in minisatellite sequences which cause apparent mutation frequencies on the order of 10-2~locus~generation (39.. 7.4139-4145 expected. amplification may in fact start from shorter fragments of the eds) pp. (1986) cold Spring Harbor Symp. (1985) Plant Mol. (1984) J. Doran.282-284 1. Knoxville. 86. Carr. M. and Kocher. Erlich. (1b85) Biol.69-76 ( 1 9 a ) camp. B. and Knowler. F. attributing changes in gene frequencies to factors such as variations in population size* and genetic drift. 487~491 32. may allow for the amplification of up plants (41). and Wong. B.. Weiner. Phys. E. S. Higuchl R. G. W... B. Second. T. (1984) Naturwksemcha ten71. K.803-806 27.. Piiabo: s. S. E. 387-388 31.. S. A. T.67-101 first extension. ii.. J.. and Wilson. S. C. A. Sage R. s.. Horn. R.. Piabo. Jakobsson. Helentjaris. A. Scharf. Weiner. A. J. L. C . and Haigh. ed) pp.L. Wallace. Jeffreys. Thomas. A. S. and Hauswirth.. Kocher. Gifford. and Magor. Margoliash.9712 Minireview: Ancient DNA such as museum skins. (1983) Am. Jr. Euol. M. W. (1985) Nature 314. (1988) Nature 336. Physiol. J. Freiberger.1241-1242 from the extant mountain zebra is 10 to 0... had occurred at random. J. C.. (1988) Nature 332.773-776 44. W.. and Kirk. G.. M. Taylor. A. Todd. Irani. Wilson: A... J r and Ryder 0.20. R. Wilson. 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 :. and Wischearth. Poinar. H.. J. 283-287 24. A. H. and Stoneking. Stoffel. R. G. Awedimento.. A... F. C. and Bendich. (1984) Fed. Lowenstein. a Acknowledgments-We thank numerous colleagues for discussion given sequence is considered likely to be of ancient origin. Ryder. (1972) he Biochemktry o f ~ n i m l~ ~ ~ ~ i h Scientechbe affected in a predictable way. c. M. Gyllensten. Wilson.. R.'Mullis'. R.. H.. C. Scharf.. F.C. A. I. A. ' ~ ~ . H. X. Sarich. Only when 13. In addition to Organisma' and population questions. (19%) NLlcletc A C Res. specific errors stemming from post-mortem changes are not M. In these cases.. and de Crombrugghe. Natl. K. E. J. A. E. and expected to predominate in an amplified population of mole. Higuchi. E. Wilson... Wan% G. T... C. O. Leach. R. A. E. This allows us to address many questions involving the identity and relationship of extinct species to other extinct and extant species. D. D. (1986) Nature 323. Dickel.. Myers for expert graphical work. R. Biol... A. and Arnheim.. A.. (1988) Science 239.Lowenstam. (1969) Nature 224. Wrischnik.. T. V. H. 178-190. Jr. Vistas f o r Molecular Archaeology The recently achieved ability to study DNA from museum specimens and archaeological finds via PCR (27... (1982) Science 216. J. N. M. Mol. A. 30.and wilson. Elsevier Science Publishers B. N. C.. (1987) J.. Quant. v. B.644-645 25. Arnheim. and Erlich. Helm-Bychowskl. and Wilson.. Arnbeim.. (1984) Annu. F. Paabo S. H. Sarich.. Higuchi. K.. A.. E. Linh.. J. Mudryj. K. 3). M. S. C. 3. phabo. Beck. Higuchi. R. J. and Whlte. and Wilson. United Kingdom 2. the ratio of silent to replacement changes in 3. Sarich. eds) p 407 419. Gurd. Lowenstein. M.C. which in nuclear and organelle genomes is generally less than 1% of sequence divergence/1O5years. C. L. N.8. (1971)Int. T.586-588 enough extension steps have been performed so that the two 14.. News Letter 62. G. D.G. Biol. D. (1986) Mol.3 and Wilson. Prager. G. even rare examples of single preserved specimens are of p e a t value... W. A. c.. and Giirtler. V. M.. Smlth-Gd1. 7 0 individuals. A. Curry.774 39. S. A. F. 0. Biol. 7 3 . George. Euol. Bowman. H. L. 20-33. 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..529-542 34. Wyckoff..M. Villablanca. A.. P. Higuchi. G. Mol. N. (1985) Am. Immunol. andLu. 2 6 . J. T. "jumping PCR' may generate erroneous se.887-892 6.. N. E. however. Pastan. and Wilson. 40). Phys. 43.. Sei. C. i. A. (1980) Cell 22. M. V.441-446 26. Natl.. A. A.-A Moratory !anal. The substitutions would be nica. (1989) Trends Genet. Cann.. Prager. extended primer products overlap with their 3' ends will a 14-18 conventional exponentialamplification reaction ensue. (1970) Science 170. M. M... Salvi. Rogers.. J. H. teeth. M. Hou&:P.. A. Prager. Biochern.. W. p-bo. Pa&o. T. F. M.. 17. Errors because of Post-mortem Chnges-As noted above. H. in the case of 452 chromosomal genes from ancient remains of heterozygous 19. (1984) Nature 312. Anthro 01 68. 376-400 36. T. and Jornvall. Acad. as well as E. N. B. 1 6 .p.. J. Higuchi. If the diff~~ences 5. M. Manchester Unlversity Press...C. and Wilson.. R. Horn. Prager. and Garbuglia. Prager E M. u. D. 21.. A. eds) Academlc Press.. I.. White. Rosing. Orrego. s. J. Leningrad. Berzofsky. Vogeli.. H.535-546 A. and Richardson. G. G. 15. (1981) Nature next cycle be further extended upon hybridization to other 291. Agee.1100-1102 Mosaic Sequences via Jumping PCR-An additional prop. However.1350-1354 29. (1981) in Magadan Baby Mammoth (Vereschagin. U.2203-2214 41.... Gelfand D.. ed) pp.. and Hood.. H. If they were to predominate and thus cause incorrect REFERENCES sequences to be determined. 2.F.: 23. W. (19%) Nature 3 3 4 . (1980) sequences. D. H... G. s.. New York. Erlich. T N DNA from the extinct quagga "'Ompared to modern DNA 4. B k h m . AnthropoL 6 1 ..M. and sharing unpublished data with US. Proc. 213-218 quences that result from recombination during amplification. V.. G. 61. the pattern Of f3ubstitution would 1. A particularly important goal for the future is to to 500-bp pieces. B. ~~~~~~& . s.. Berg.Faloona. Bristol. C. and iii) are fulfilled. and thepartially extended primers can in the 12. Amsterdam 37. A. M. (1984) D~ Altertum 3 0 . (1989) Nucleic Acids Res. U.. Sercarz... Sci. Oakes. (1976) h o c . E. Bremer. Gelfand.Mullis. 447which homoplasmy is the rule (35). Reu.. Prager. Sninsky. J. distributed at random with respect to position within codons.. J.287-289 C. S.. 81B. cules. M.1939-1943 28. S. Acad. Flaherty.. Ballinger. M. D. and Hess. A. (1988) in Molecular Euolution and the ~ o ~ ~ i l(BroadRecord head. Rollo.. M. Palumbi S. B. Temrin.. Nauka. primers (Fig. 5. M. and amber. Geor e M.. East. R. (1988) Auk 105. P. 0.. B. 32. A. Hansen. C. A.Euol. M. DNA that are complementary to one or the other of the 11. United Kingdom lem in the Of mtDNA sequences for 18. G. K. The building of a mosaic sequence via PCR poses no probR. (1989) in The Hierarchy of Life (Fernholm. T. D. Sensabaugh. Irwin.. v. George.H. E. 17. Y.. ~ 9775-9787 33. K. (1975) Homo 2 6 . 2 1 . C. D. 1045-1051 22. E. 379-382.. K. (1986) in Science in Egyptology (David. These fragments serve as templates in the Reichhn. S. 6. 1557 . B.. Tong. T... P. Hannum. Yamada. R. E. Laipis. Harrison. Saiki. 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.104-105 42.. DNA sequences from bones. S.and Abdalla.545-568 erty Of PCR may affect the authenticity Of the amp1ified 9. W. the evolution of populations can be studied and will most often involve the comparison of ancestral and descendant populations.. N.. Gyllensten. Miller. U.. M. G. (1988) Maize Genetics COOP. G. a n d v a n Regenmortel. M.325326 initial steps may allow the amplification of regions that are 16. Manchester.. E. Z. Jr. E. (1985) Science 230. J. and Mikhelson. in press 38. (1981) S h e w w u HUQH s w h S h e w wu L i Chin Chon 3 9 . Connolly.E.. Wilson. 2541-2545 In fact. Gillespie. K. M.. and Wilson.. J.(1989) in PCR-Pmtocoka&Applicat. H... (Innis. Schwartzfischer.. A.

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