The use of real-time PCR in aDNA studies
Focusing on the population level requires careful evaluation of the molecular techniques used to answer each specific question. Real-time pcr might become a standard in the study of aDNA (Pruvost & Geigl, 2004), since it combines several advantages as outlined below.
Real-time PCR provides a reliable method to quantify the DNA concentration originally present in an extract of a target organism (Poinar et al., 2003). By using highly specific primers and an oligonucleotide probe complementary to an internal segment of the target sequence, the amplification of any nontarget – ancient or recent – DNA present in the extract (e.g. resulting from microorganismal contamination) is excluded a priori. Like the primers, the probe anneals to the template sequence, but is successively cleaved by the polymerase during the elongation of the newly synthesized DNA strand. Probe cleavage emits a fluorescent signal that is recorded during each amplification cycle. The original template DNA quantity added to the reaction mix can then be calculated. At the same time, it is possible to identify a single-nucleotide polymorphism (SNP) or a short insertion/deletion (indel) by adding two probes, labeled with different fluorescent dyes, which are complementary to either of the two sequence variants (allelic discrimination). Depending on which probe hybridizes to the target sequence, the respective dye, or both in case of heterozygosity when analysing a nuclear marker, emits a fluorescent signal. Thus, both organelle or nuclear markers can be screened (Alonso et al., 2003). The SNPs or presence/absence of short indels can be identified, targeting short DNA fragments of about 60–300 nucleotides, a size compatible with degraded DNA.
However, such an allelic discrimination approach has some limitations when working with aDNA. Base modifications, which may by detected by a thorough, but laborious, PCR amplification and cloning/sequencing approach, could lead to wrong allele assignment. Using uracil N-glycosylase (UNG; Longo et al., 1990) could serve as a loophole (Hofreiter et al., 2001a). This enzyme removes uracil from DNA molecules, which produces an abasic site and thus leads to a break in the DNA strand. It may be routinely applied in real-time PCR, where thymine (dTTP) is replaced with uracil (dUTP) in the PCR mix for incorporation in the elongating DNA strand. At the start of the PCR cycling, UNG activity destroys any formerly amplified DNA molecules before being heat-inactivated – a positive side-effect in aDNA work because it helps to prevent cross-contamination with PCR products from earlier amplifications. However, UNG equally affects aDNA templates that contain uracil instead of cytosine because of deamination (Lindahl, 1993). While UNG treatment might thus prevent the potential genotyping of a wrong probe-compatible sequence, it may also destroy too many of the few authentic DNA molecules left in the extract of a fossil sample. As a consequence, preliminary real-time PCR without UNG could be run in order to evaluate the likelihood of finding sufficient templates to risk partial decomposition.
This technique not only corroborates credibility of the results obtained, but also allows for high-throughput genotyping since no post-PCR treatment, such as gel electrophoresis, is necessary. The PCR tubes remain closed throughout the analysis, thereby minimizing the risk of contamination predominantly by PCR products. Because PCRs beginning with < 1000 template molecules are likely to provide inaccurate results (Cooper & Poinar, 2000; Hofreiter et al., 2001b), the quantification of the initial amount of DNA in the sample could help evaluate the authenticity of the sequences obtained. Ultimately, however, additional evidence is necessary for distinguishing aDNA from recent, contaminating DNA, and at least part of the samples have to be cloned and sequenced (Cooper & Poinar, 2000).
Potential applications of real-time PCR analyses in aDNA studies are abundant. In general, the combination of universal primers (e.g. rbcL, trnL-F) and species-specific probes represents a promising strategy. For example, postglacial migration of plant species, or even colonization routes of particular genotypes or lineages, could be integrated over space and time, particularly where present-day patterns of genetic variation based on easily accessible polymorphisms are clear-cut. Suitable model systems are manifold. White oaks in Europe exhibit ancient divides between several maternal lineages that survived in different glacial refugia, characterized by SNPs or small indels in cpDNA (Ferris et al., 1993; Dumolin-Lapègue et al., 1999; Deguilloux et al., 2002; Petit et al., 2002). These cpDNA variants could be traced across chronosequences (i.e. the changes over time at a given location). Similarly, Norway spruce (Picea abies) shows a clear differentiation between two mitochondrial lineages, distinguished by several SNPs (Sperisen et al., 2001). These lineages point to separate glacial histories. There is further evidence for two SNP-based mitotypes occurring in populations of the Carpathian mountains (F. Gugerli et al. unpublished), a region considered to be a glacial refugium for P. abies. A third example is provided by a SNP found in a cpDNA gene of extant silver fir (Abies alba) that shows a cline across central Europe (Liepelt et al., 2002).
The choice among genomes and genes
As in any population genetic survey, selecting the appropriate molecular markers to address a particular question is essential. The choice depends on the type of question as well as the spatial and temporal resolution envisaged. While no general recommendation can be given, cpDNA and mtDNA represent promising targets because they are present in multiple copies within each plant cell. Consequently, they are most likely to be retrieved from fossil tissues. In the case of aDNA, the choice of markers is limited by constraints inherent in the use of highly degraded DNA. It has been demonstrated that hydrolytic and oxidative damage will degrade aDNA to short fragments no longer than 200 nucleotides (Yang, 1997; Pääbo et al., 2004). Targeting such short fragments will decrease the information content of each analysis, compared with those typically employed with contemporary material. The inverse relationship between amplification success rate and the size of the amplicon has been confirmed with DNA isolated from dry wood, with effects still detectable for fragments shorter than 100 nucleotides (Deguilloux et al., 2002). The same study confirmed that sequences present in multiple copies per cell (i.e. cpDNA, mtDNA and ribosomal DNA sequences) yield more reliable amplification compared with single-copy nuclear sequences. This is relevant since uniparentally inherited markers, such as cpDNA or mtDNA, are generally more strongly structured in space than are nuclear markers and have consequently been extensively used in phylogeographic studies (Petit et al., 2003, 2005). Instead, microsatellite markers are prone to slippage during amplification, a problem that could be exacerbated when working with highly degraded templates, making the cloning of each genotype a crucial and necessary step to support allelic data. Surprisingly, anonymous markers such as random amplified polymorphic DNA (RAPDs) have also been used in a few instances, although these techniques are known to be prone to inconsistencies, even when working with DNA isolated from fresh tissues. Advantages and limitations of particular marker types should be considered when planning palaeogenetic studies.