Box 1 Authenticity of ancient DNA sequences
Ancient DNA – unlocking plants’ fossil secrets
Version of Record online: 23 DEC 2003
Volume 161, Issue 2, pages 335–339, February 2004
How to Cite
Parducci, L. and Petit, R. J. (2004), Ancient DNA – unlocking plants’ fossil secrets. New Phytologist, 161: 335–339. doi: 10.1111/j.1469-8137.2004.00987.x
The extraction and preparation of the PCR required for amplification of ancient DNA (aDNA) must be done in a laboratory that is rigorously separated from work involving modern DNA, equipment should be regularly treated with bleach and UV irradiation, and protective clothing should always be worn (Handt et al., 1994; Höss et al., 1994). In addition to these basic lab procedures, a number of criteria have been developed to determine the ancient origin of DNA sequences (Cooper & Poinar, 2000):
Physically isolated work area To avoid contamination, it is essential that, before the amplification stage, all aDNA research is carried out in a dedicated, isolated environment. A building in which large amounts of the target DNA are routinely amplified should be banned.
• Control amplifications Multiple extraction and PCR controls must be performed to detect sporadic or low-copy number contamination, although carrier effects do limit their efficacy. All contaminated results should be reported, and positive controls should generally be avoided, as they increase contamination risk.
• Appropriate molecular behaviour PCR amplification strength should be inversely related to product size (large 500- to 1000-bp products are unusual) and sequences should make phylogenetic sense.
• Reproducibility Results should be repeatable from the same, and different, DNA extracts of a given specimen.
• Cloning Direct PCR sequences must be verified by cloning amplified products to determine the ratio of endogenous to exogenous sequences, or damage-induced errors. Overlapping fragments are desirable to confirm that sequence variation is authentic and not the product of errors introduced when PCR amplification starts from a small number of damaged templates.
• Independent replication Intra-laboratory contamination can only be discounted when separate samples of a specimen are extracted and sequenced in independent labs.
• Biochemical preservation Indirect evidence for DNA survival in a specimen can be provided by assessing the total amount, composition, and relative extent of diagenetic change in amino acids and other residues.
• Quantitation The copy number of the target DNA should be assessed using quantitative RT-PCR. When the number of starting templates is low (< 1000), it may be impossible to exclude the possibility of sporadic contamination, especially for human DNA studies.
• Associated remains Evidence that DNA has survived in associated material from other species is critical supporting evidence. Such remains could also make good negative controls for PCR amplifications of the target species.
- Issue online: 23 DEC 2003
- Version of Record online: 23 DEC 2003
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