2, Université Rennes 1, Rennes cedex, France
PCR analyses of ancient and degraded DNA suffer from their extreme sensitivity to contamination by modern DNA originating, in particular, from carryover contamination with previously amplified or cloned material. Any strategy for limiting carryover contamination would also have to be compatible with the particular requirements of ancient DNA analyses. These include the need (i) to amplify short PCR products due to template fragmentation; (ii) to clone PCR products in order to track possible base misincorporation resulting from damaged templates; and (iii) to avoid incomplete decontamination causing artifactual sequence transformation. Here we show that the enzymatic decontamination procedures based upon dUTP- and uracil-N-glycosylase (UNG) can be adapted to meet the specific requirements of ancient DNA research. Thus, efficiency can be improved to vastly reduce the amplification of fragments ≤100 bp. Secondly, the use of an Escherichia coli strain deficient in both UNG and dUTPase allows for the cloning of uracil-containing PCR products and offers protection from plasmid DNA contamination, and, lastly, PCR products amplified from UNG-degraded material are free of misleading sequence modifications.
Analyses of DNA from ancient and degraded samples and fossils are hindered by problems caused by the nature of the studied substrate; that is, the molecules are present at very low copy number, are fragmented, and contain modified bases. PCR amplification has made it possible to analyze genetic information from such material, but amplification of the degraded and modified DNA is not very efficient and sporadically contaminating intact modern DNA molecules can be preferentially amplified (1). Indeed, this contamination caused erroneous results (2,3). Furthermore, base modifications cause base substitution during PCR amplification, leading to erroneous sequence information (1)(4,5,6). These drawbacks led to the establishment of criteria to increase the likelihood of the authenticity of ancient DNA sequences (7). In particular, it is necessary to set up experimental conditions that prevent as nearly as possible any contamination by modern DNA and to clone PCR-amplified DNA to obtain sequence information from independent original molecules, to assess sequence heterogeneity, and to track possible base misincorporations (7,8).
There are four major sources of contamination by modern DNA: (i) the environment and the experimenters (archeologists, biologists, etc.); (ii) other ancient specimens; (iii) previous PCR products; and (iv) plasmids with the cloned target sequence. The latter two sources of contamination are usually controlled by physical containment. Nevertheless, they remain a major threat due to the high number of contaminating molecules. PCR itself produces in just one reaction up to 1011 copies of the ancient target sequence. These molecules are easily spread via aerosol transport. One aerosol droplet can contain many more DNA molecules than one gram of fossil material. Cloning is the second source of contamination because 1011 molecules containing the target sequence are produced in just one milliliter of bacterial culture. These contamination sources are common to all research areas that analyze degraded DNA in very small amounts [e.g., paleogenetics, forensic and conservation genetics, certain areas of medical research, and DNA traceability (analysis of DNA traces in food, soil etc.)]. The susceptibility to contamination increases with decreasing numbers of amplifiable target molecules.
Contamination with substantial amounts of DNA can be identified quite easily by running negative controls during the PCR, but low-level contamination leading to PCR amplification in only a few percent of the samples is more covert because it occurs with a frequency similar to bona fide amplification from ancient material and can be easily missed in conventional PCR assays when one is running only a few negative controls (8,9). Furthermore, contamination with previously amplified material can severely compromise the demonstrative value of data replication, even when performed in different laboratories. In many cases, these exchange material and may thus exchange contaminants as well. Rigorous practices in ancient or forensic DNA analyses should keep contamination to a low level that cannot seriously be guaranteed to be zero.
Quantitative real-time PCR (QPCR) is a useful quality control, which allows the quantification of both the target molecules in the extracts (10,11,12) and the inhibition by products contained in all fossil extracts (10). Procedures that lower the risk of contamination with amplified or cloned products, but are still compatible with both QPCR and specific requirements for ancient DNA studies, would further increase the reliability of the analyses.
Degradation of carryover contaminating molecules prior to amplification with various enzymes has been proposed. A current procedure makes use of dUTP incorporation instead of dTTP during amplification, which is preceded by treatment with a heat-labile uracil-N-glycosylase (UNG) to degrade uridine-containing carryover products from previous PCR amplifications (13). We refer to this approach as UPCR. Even though UNG treatment is sometimes used to remove deaminated cytosine residues from ancient DNA samples prior to amplification to minimize nucleotide misincorporation (4,14), UPCR is not in current practice in the ancient DNA field. Indeed, several issues limit its usefulness: (i) because ancient DNA is highly degraded, only small PCR fragments can be amplified, but it is unclear whether the current UPCR procedures are efficient enough to provide significant protection from carryover contamination in this type of setting (15); (ii) because “jumping PCR” between small fragments has been shown to cause nucleotide misincorporation and in vitro recombination, leading to sequence chimerism (1,6), it would be dangerous to favor jumping PCR events between subfragments of previously amplified PCR products that could be artifactually distinct from the known products and thus erroneously considered as authentic and novel; (iii) the desired cloning of the PCR products cannot be performed in current Escherichia coli strains used for cloning purposes because they degrade uridine-containing DNA.