Ancient DNA

Ancient DNA /Wikipedia

Ancient DNA is DNA isolated from ancient specimens. It can be also loosely described as any DNA recovered from biological samples that have not been preserved specifically for later DNA analyses. Examples include the analysis of DNA recovered from archaeological and historical skeletal material, mummified tissues, archival collections of non-frozen medical specimens, preserved plant remains, ice and permafrost cores, Holocene plankton in marine and lake sediments, and so on. Unlike modern genetic analyses, ancient DNA studies are characterised by low quality DNA. This places limits on what analyses can achieve. Furthermore, due to degradation of the DNA molecules, a process which correlates loosely with factors such as time, temperature, and presence of free water, upper limits exist beyond which no DNA is deemed likely to survive. Allentoft et al (2012) tried to calculate this limit by studying the decay of mitochondrial and nuclear DNA in Moa bones. The DNA degrades in an exponential decay process. According to their model, mitochondrial DNA is degraded to 1 base pair after 6,830,000 years at −5 °C. Nuclear DNA degrades at least twice as fast as mtDNA. As such, early studies that reported recovery of much older DNA, for example from Cretaceous dinosaur remains, may have stemmed from contamination of the sample.

History of ancient DNA studies

The first study of what would come to be called aDNA came in 1984, when Russ Higuchi and colleagues at Berkeley reported that traces of DNA from a museum specimen of the Quagga not only remained in the specimen over 150 years after the death of the individual, but could be extracted and sequenced. Over the next two years, through investigations into natural and artificially mummified specimens, Svante Pääbo confirmed that this phenomenon was not limited to relatively recent museum specimens but could apparently be replicated in a range of mummified human samples that dated as far back as several thousand years (Pääbo1985a; Pääbo 1985b; Pääbo 1986). Nevertheless, the laborious processes that were required at that time to sequence such DNA (through bacterial cloning) were an effective brake on the development of the field of ancient DNA (aDNA). However, with the development of the Polymerase Chain Reaction (PCR) in the late 1980s the field was presented with the ability to rapidly progress.

Double primer PCR amplification of aDNA (jumping-PCR) can produce highly-skewed and non-authentic sequence artifacts. Multiple primer, nested PCR strategy was used to overcome those shortcomings.

Single primer extension (abr. SPEX) amplification was introduced in 2007 to address postmortem DNA modification damage.

Problems and errors

aDNA may contain a large number of postmortem mutations, increasing with time. Some regions of polynucleotide are more susceptible to this degradation so sequence data can bypass statistical filters used to check the validity of data. Due to sequencing errors, great caution should be applied to interpretation of population size. Substitutions resulting from deamination cytosine residues are vastly overrepresented in the ancient DNA sequences. Miscoding of C to T and G to A accounts for the majority of errors.

Antediluvian DNA studies

The post-PCR era heralded a wave of publications as numerous research groups tried their hands at aDNA. Soon a series of incredible findings had been published, claiming authentic DNA could be extracted from specimens that were millions of years old, into the realms of what Lindahl (1993b) has labelled Antediluvian DNA. The majority of such claims were based on the retrieval of DNA from organisms preserved in amber. Insects such as stingless bees (Cano et al. 1992a; Cano et al. 1992b), termites (De Salle et al. 1992; De Salle et al. 1993), and wood gnats (De Salle and Grimaldi 1994) as well as plant (Poinar et al. 1993) and bacterial (Canoet al. 1994) sequences were extracted from Dominican amber dating to the Oligocene epoch. Still older sources of Lebanese amber-encased weevils, dating to within the Cretaceous epoch, reportedly also yielded authentic DNA (Cano et al. 1993). DNA retrieval was not limited to amber. Several sediment-preserved plant remains dating to the Miocene were successfully investigated (Golenberg et al. 1990; Golenberg 1991). Then, in 1994 and to international acclaim, Woodward et al. reported the most exciting results to date — mitochondrial cytochrome b sequences that had apparently been extracted from dinosaur bones dating to over 80 million years ago. When in 1995 two further studies reported dinosaur DNA sequences extracted from a Cretaceous egg (An et al. 1995; Li et al. 1995), it seemed that the field would truly revolutionize knowledge of the Earth's evolutionary past. Even these extraordinary ages were topped by the claimed retrieval of 250 million-year-old halobacterial sequences from Halite. 

Unfortunately, the golden days of antediluvian DNA did not last. A critical review of ancient DNA literature through the development of the field highlights that few recent studies have succeeded in amplifying DNA from remains older than several hundred thousand years. A greater appreciation for the risks of environmental contamination and studies on the chemical stability of DNA have resulted in concerns being raised over previous reported results. The Dinosaur DNA was later revealed to be human Y-Chromosome, while the DNA reported from encapsulated halobacteria has been criticized based on its similarity to modern bacteria, which hints at contamination. Recent work also suggest that these bacterial DNA samples may not have survived from ancient times but may instead be the product of long-term, low-level metabolic activity.

Ancient DNA studies

Despite the problems associated with 'antediluvian' DNA, a wide and ever-increasing range of aDNA sequences have now been published from a range of animal and plant taxa. Tissues examined include artificially or naturally mummified animal remains, bone (c.f. Hagelberg et al. 1989; Cooper et al. 1992; Hagelberg et al. 1994), paleofaeces, alcohol preserved specimens (Junqueira et al. 2002), rodent middens, dried plant remains (Goloubinoff et al. 1993; Dumolin-Lapegue et al. 1999) and recently, extractions of animal and plant DNA directly from soil samples. In June 2013, a group of researchers announced that they had sequenced the DNA of a 560–780 thousand year old horse, using material extracted from a leg bone found buried in permafrost in Canada's Yukon territory. In 2013, a German team reconstructed the mitochondrial genome of an Ursus deningeri, proving that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost.

Ancient DNA studies on human remains

Due to the considerable anthropological, archaeological, and public interest directed toward human remains, it is only natural that they have received a similar amount of attention from the DNA community. Due to their obvious signs of morphological preservation, many studies utilised mummified tissue as a source of ancient human DNA. Examples include both naturally preserved specimens, for example, those preserved in ice, such as the Ötzi the Iceman (Handt et al. 1994), or through rapid desiccation, such as high-altitude mummies from Andes (c.f. Pääbo 1986; Montiel et al. 2001) as well as various sources of artificially preserved tissue (such as the chemically treated mummies of ancient Egypt). However, mummified remains are a limited resource, and the majority of human aDNA studies have focused on extracting DNA from two sources that are much more common in the archaeological record –bone and teeth. Recently, several other sources have also yielded DNA, including paleofaeces (Poinar et al. 2001) and hair (Baker et al. 2001, Gilbert et al. 2004). Contamination remains a major problem when working on ancient human material.

Pathogen and microorganism aDNA analyses using human remains

The use of degraded human samples in aDNA analyses has not been limited to the amplification of human DNA. It is reasonable to assume that for a period of time postmortem, DNA may survive from any microorganisms present in the specimen at death. These include not only pathogens present at the time of death (either the cause of death or long-term infections) but commensals and other associated microbes. Despite several studies that have reported limited preservation of such DNA, for example, the lack of preservation of Helicobacter pylori in ethanol-preserved specimens dating to the 18th century, over 45 published studies report the successful retrieval of ancient pathogen DNA from samples dating back to over 5,000 years old in humans and as long as 17,000 years ago in other species. As well as the usual sources of mummified tissue, bones and teeth, such studies have also examined a range of other tissue samples, including calcified pleura (Donoghue et al. 1998), tissue embedded in paraffin, and formalin-fixed tissue.

 (Wikipedia)