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Science 18 August 2000:

Vol. 289. no. 5482, p. 1139

DOI: 10.1126/science.289.5482.1139b

LETTERS

Ancient DNA: Do It Right or Not at All

At the recent 5th International Ancient DNA Conference in Manchester, U.K., reportedby Erik Stokstad in his News Focus article "Divining diet and disease from DNA" (28Jul., p. 530), one presentation boldly opened with the claim that the field was nowmature and could move ahead with confidence. This optimism is unfounded, asdemonstrated by the notable absence of "criteria of authenticity" from manypresentations at the conference. Ancient DNA research presents extreme technicaldifficulties because of the minute amounts and degraded nature of surviving DNA andthe exceptional risk of contamination. The need to authenticate results becameobvious in the mid-1990s when a series of high-profile studies were shown to beunrepeatable (1). For example, DNA reputed to come from a dinosaur (2) was actually

contamination by a human mitochondrial gene insertion in the nucleus (numt) (3). Overthe ensuing years, criteria have been developed and put into practice by somepractitioners in the field. Regrettably, despite the recommendation that such criteria beroutinely applied (4-6), high-profile journals continue to publish studies that do not meetthe necessary controls (7), and many new researchers fail to utilize them. To publicizethese standards, we summarize the key criteria below.

Physically isolated work area. To avoid contamination, it is essential that, prior to theamplification stage, all ancient DNA research is carried out in a dedicated, isolatedenvironment. A building in which large amounts of the target DNA are routinelyamplified is obviously undesirable (8).

Control ampli fications. Multiple extraction and PCR controls must be performed todetect sporadic or low-copy number contamination, although carrier effects do limittheir efficacy (4, 9). All contaminated results should be reported, and positive controlsshould generally be avoided, as they provide a contamination risk.

Appropr iate mol ecul ar behavior. PCR amplification strength should be inverselyrelated to product size (large 500- to 1000-base pair products are unusual).Reproducible mitochondrial DNA (mtDNA) results should be obtainable if single-copynuclear or pathogen DNA is detected. Deviations from these expectations should beustified; e.g., with biochemical data. Sequences should make phylogenetic sense.

Reproducibility. Results should be repeatable from the same, and different, DNA

extracts of a specimen. Different, overlapping primer pairs should be used to increasethe chance of detecting numts (10) or contamination by a PCR product.

Cloning. Direct PCR sequences must be verified by cloning amplified products to

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determine the ratio of endogenous to exogenous sequences, damage-induced errors,and to detect the presence of numts. Overlapping fragments are desirable to confirmthat sequence variation is authentic and not the product of errors introduced when PCRamplification starts from a small number of damaged templates (11).

Independent replication. Intra-laboratory contamination can only be discounted whenseparate samples of a specimen are extracted and sequenced in independentlaboratories. This is particularly important with human remains or novel, unexpected

results.

Biochemical preservation . Indirect evidence for DNA survival in a specimen can beprovided by assessing the total amount, composition, and relative extent of diageneticchange in amino acids and other residues (12, 13).

Quantitation.* The copy number of the DNA target should be assessed usingcompetitive PCR (4, 11). When the number of starting templates is low (


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10. A. D. Greenwood, C. Capelli, G. Possnert, S. Pbo, Mol. Biol. Evol. 16, 1466(1999).

11. M. Krings et al., Cell90, 19 (1997).

12. H. N. Poinar, M. Hss, J. L. Bada, S. Pbo, Science 272, 864 (1996).

13. H. N. Poinar and B. A. Stankiewicz, Proc. Natl. Acad. Sci. U.S.A.96, 8426(1999).

The editors suggest the following Related Resources on Science sites:

In Science Magazine

NEWS FOCUS

5TH INTERNATIONAL ANCIENT DNA CONFERENCE:Divining Diet and Diseas e From DNA

Erik Stokstad (28 July 2000)Science289 (5479), 530. [DOI: 10.1126/science.289.5479.530] Summary Full Text

THIS ARTICLE HAS BEEN CITED BY OTHER ARTICLES:

Comm ent on "DNA from Pre-Clovis Human Coprolites in Oregon, North Americ a" .

H. Poinar, S. Fiedel, C. E. King, A. M. Devault, K. Bos, M. Kuch, and R. Debruyne (2009)Science 325, 148Abstrac t Full Text PDF

Prehistoric contacts over the Straits of Gibraltar indicated by genetic analysis of Iberian

Bronze Age cattle.

C. Anderung, A. Bouwman, P. Persson, J. M. Carretero, A. I. Ortega, R. Elburg, C. Smith, J. L.

Arsuaga, H. Ellegren, and A. Gotherstrom (2005)PNAS 102 , 8431-8435

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Evidence for a genetic discontinuity between Neandertals and 24,000-year-old

anatomically modern Europeans.

D. Caramelli, C. Lalueza-Fox, C. Vernes i, M. Lari, A. Casoli, F. Mallegni, B. Chiarel li, I.Dupanloup, J. Bertranpetit, G. Barbujani, et al. (2003)PNAS 100 , 6593-6597


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from the Alps.

F. Rollo, M. Ubaldi, L. Ermini, and I. Marota (2002)PNAS 99, 12594-12599


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