A Quantitative Approach to Detect and Overcome PCR Inhibition in Ancient DNA Extracts.
King, Christine E.,  Régis Debruyne, Melanie Kuch, Carsten Schwarz, & Hendrik N. Poinar.
Biotechniques 2009, 47(5):351-356.
doi 10.2144/000113244

Abstract

Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is undesirable. Two basic approaches to monitoring inhibition in such samples using real-time or quantitative PCR (qPCR) have been proposed. The first method analyzes the quantification cycle (Cq) deviation of a spiked internal positive control. The second method considers variations in reaction efficiency based on the slopes of individual amplification plots. In combining these methods, we observed increased Cq values together with reduced amplification efficiencies in some samples, as expected; however, deviations from this pattern in other samples support the use of both measurements. Repeat inhibition testing enables optimization of PCR facilitator combinations and sample dilution such that DNA yields and/or quantitative accuracy can be maximized in subsequent PCR runs. Although some trends were apparent within sample types, differences in inhibition levels, optimal reactions conditions, and expected recovery of DNA under these conditions suggest that all samples be routinely tested with this approach.

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New insights from old bones: DNA preservation and degradation in permafrost preserved mammoth remains.
Schwarz Carsten, Régis Debruyne, Melanie Kuch, Elizabth McNally, Henry Schwarcz, Andrew Aubrey, Jeffrey Bada, Hendrik N. Poinar.
Nucleic Acids Research 2009, Advance Online Access 1-15.

Abstract

Despite being plagued by heavily degraded DNA in palaeontological remains, most studies addressing the state of DNA degradation have been limited to types of damage which do not pose a hindrance to Taq polymerase during PCR. Application of serial qPCR to the two fractions obtained during extraction (demineralization and protein digest) from six permafrost mammoth bones and one partially degraded modern elephant bone has enabled further insight into the changes which endogenous DNA is subjected to during diagenesis. We show here that both fractions exhibit individual qualities in terms of the prevailing type of DNA (i.e. mitochondrial versus nuclear DNA) as well as the extent of damage, and in addition observed a highly variable ratio of mitochondrial to nuclear DNA among the six mammoth samples. While there is evidence suggesting that mitochondrial DNA is better preserved than nuclear DNA in ancient permafrost samples, we find the initial DNA concentration in the bone tissue to be as relevant for the total accessible mitochondrial DNA as the extent of DNA degradation post-mortem. We also evaluate the general applicability of indirect measures of preservation such as amino-acid racemization, bone crystallinity index and thermal age to these exceptionally well-preserved samples.

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Pour cet article de mon compère Carsten Schwarz, j’ai essentiellement réalisé l’analyse des données dans le cadre d’un modèle de dégradation stochastique de l’ADN (tel que proposé par Deagle et al. en 2007), ainsi que le traitement statistique des résultats. J’ai également participé à la rédaction du manuscrit et réalisé ses figures.

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Abstract

Gilbert et al. (Reports, 28 September 2007, p. 1927) reported that “hair shafts surpass comparably stored bone as an aDNA source [...] in regard to preservation and concentration of mtDNA.” When experimental parameters are carefully controlled for, including adequate sampling, quantitative polymerase chain reaction analysis, and modeling the decay of DNA, the general importance of this claim is not supported.

It is common to think of the land bridge that existed from time to time across what is now the Bering Strait as a one-way affair. After all, the route through the area known as Beringia is thought to be how many animals and humans made their way out of Asia and into North America.

But there were no “Eastbound Only” signs. Some animals — camel ancestors, for example — went the other way, from North America into Asia. And there is no reason that a species could not go both ways, if conditions were right. That appears to be the case with the woolly mammoth, according to a major phylogenetic analysis.

Hendrik Poinar of McMaster University in Hamilton, Ontario, and colleagues looked at mitochondrial DNA from 160 mammoth samples from across Eurasia and North America as a way of determining who was related to whom. They identified several groups, or clades — some endemic to Siberia and other parts of Asia and others to North America, having been separated after mammoths first migrated eastward more than 1.5 million years ago.

But as they report in Current Biology, the researchers found that at some point in the last 150,000 years, North American mammoths migrated back to Siberia. “When they’re coming back in, the endemic Siberian populations start to crash,” Dr. Poinar said, and by about 40,000 years ago, the North American mammoths had completely taken over.

Whether the Siberian mammoths died out on their own (because of what is called genetic drift) or were outcompeted and outfoxed by their North American relatives is not known, though Dr. Poinar suspects it is not coincidental that the Siberian animals began to die out when North American animals started arriving.

Either way, the mammoths that finally became extinct about 10,000 years ago were not of Siberian lineage. “I’m not sure the Russians would be happy that their iconic wooly mammoth has North American origins,” Dr. Poinar said.

Abstract

The Viverridae (Mammalia, Carnivora), one of the least studied groups of carnivorans, include two subfamilies of Asian palm civets: Hemigalinae and Paradoxurinae. The relationships between and within these two subfamilies have never been thoroughly tested using an extensive molecular sample set. In this study, we gathered sequences of four genes (two mitochondrial: Cytochrome b and ND2 and two nuclear: beta-fibrinogen intron 7 and IRBP exon 1) for eight of the eleven extant species representing these two subfamilies. The results showed that: (1) the Asian palm civets (Hemigalinae and Paradoxurinae) have a single origin and form the sister-group of the (Genettinae+Viverrinae) clade, (2) the Hemigalinae (including the otter civet Cynogale bennettii) are monophyletic, (3) the Paradoxurinae are monophyletic and (4) the small-toothed palm civet (Arctogalidia trivirgata) is an early offshoot within the Paradoxurinae. Using a relaxed molecular clock analysis, the differentiation of the (Hemigalinae+Paradoxurinae) was inferred to occur in the Late Oligocene/Early Miocene.

Abstract/Résumé

Nucleic Acids, which hold clues to the evolution of various animal and hominid taxa, are comparatively weak molecules from other cellular debris, and thus evolutionary biologists are in essence “time trapped”. Fortunately, DNA and protein fragments do exist in fossil remains beyond what theoretical experimentation do suggest. Sequestering of DNA molecules in humic or Maillard-like complexes likely represents a rich source of DNA molecules from from the past, that have yet to be tapped. These molecules were impossible to acquiredue to the selective nature of Polymerase Chain Reaction (PCR).
Recently, however, rapid parallel pyrosequencing techniques, such as those used in Metagenomics-based research, which allow for in theory, the identification of all short nucleotide sequences in a sample in a non-selective approach, has the potential to allow the identification of all nucleic acids in a sample and thus represents the way forward in ancient DNA. In theory this technology will allow the completion of genomes of extinct animals, plants and microbes. I will discuss the benefits and pitfalls of this metagenomic approach to ancient DNA, highlighting our recent efforts underway to sequence the woolly mammoth genome as well as other fossil remains.

Abstract

During a series of expeditions organized by CERPOLEX/Mammuthus to the Taimyr region in northern Siberia several mammoth (Mammuthus primigenius) carcasses were discovered and subsequently excavated and studied. The oldest specimen is the Arilakh Mammoth (ca. 55,800 BP). Much younger are the Jarkov Mammoth (ca. 20,380 BP) and the Fishhook Mammoth (ca. 20,620 BP), and still much younger are well-preserved Holocene mammoth remains (< 10,000 BP). Some paleobotanical data and descriptions of accompanying fauna provide insight into the ecology of the region during Late Pleistocene times. The faunal collapse at the end of the Pleistocene seems to be the result of increasing temperatures, greater humidity, increased snow cover, formation of insulating plant layers, later thawing of the soil, and a lower herbivore density amplifying the climatic effects.

Cet article est cité par:

1. van der Plicht J, Hogg A. 2006.  A note on reporting radiocarbon. Quaternary Geochronology 1(4): 237-240
2. van Geel B. et al. 2007.  Spores of coprophilous fungi from under the Dawson tephra (25,300 C-14 years BP), Yukon Territory, northwestern Canada
   Pal. Pal. Pal. 252(3-4):481-485
3. Ugan A. & Byers D. 2007. Geographic and temporal trends in proboscidean and human radiocarbon histories during the late Pleistocene. Quaternary Science Reviews 26(25-28): 3058-3080
4. Kienast F. et al. 2008. Continental climate in the East Siberian Arctic during the last interglacial: Implications from palaeobotanical records. Global and Planetary Change 60(3-4): 535-562
5. Kuzmin Y.V. 2008. Siberia at the Last Glacial Maximum: Environment and archaeology. J. Archael. Res. 16(2):163-221
6. van Geel B. et al. 2008. The ecological implications of a Yakutian mammoth’s last meal. Quaternary Research 69(3): 361-376
7. Ugan A. & Byers D. 2008. A global perspective on the spatiotemporal pattern of the Late Pleistocene human and woolly mammoth radiocarbon record. Quaternary International 191: 69-81

Abstract

Twenty years after the advent of ancient DNA studies, this discipline seems to have reached the maturity formerly lacking to the fulfilment of its objectives. In its early development paleogenetics, as it is now acknowledged, had to cope with very limited data due to the technical limitations of molecular biology. It led to phylogenetic assumptions often limited in their scope and sometimes non-focused or even spurious results that cast the reluctance of the scientific community. This time seems now over and huge amounts of sequences have become available which overcome the former limitations and bridge the gap between paleogenetics, genomics and population biology. The recent studies over the charismatic woolly mammoth (independent sequencing of the whole mitochondrial genome and of millions of base pairs of the nuclear genome) exemplify the growing accuracy of ancient DNA studies thanks to new molecular approaches. the number of mammoth nucleotides From the earliest publications up to now, was multiplied by 100,000. Likewise, populational approaches of ice-age taxa provide new historical scenarios about the diversification and extinction of the Pleistocene megafauna on the one hand, and about the processes of domestication of animal and vegetal species by Man on the other. They also shed light on the differential structure of molecular diversity between short-term populational research (below 2 My) and long-term (over 2 My) phylogenetic approaches. All those results confirm the growing importance of paleogenetics among the evolutionary biology disciplines.

Résumé

Après deux décennies de découvertes et de controverses, la paléogénétique semble, sinon avoir atteint l’âge de raison, du moins avoir délaissé les frasques de son impétueuse jeunesse. Si ses principes théoriques ont été à peine affinés en quinze ans, sa pratique opérationnelle a, elle, rapidement évolué, en bénéficiant de l’explosion méthodologique de la biologie moléculaire. C’est véritablement avec l’avènement de la méthode d’amplification de l’ADN par PCR que ce champ d’étude a pris son essor. Dès 1989, les travaux se multipliaient en s’intéressant à des groupes biologiques animaux et végétaux variés : espèces récemment éradiquées par l’homme (ratites), représentants disparus de la période glaciaire (mammouth laineux) ou, encore, espèces domestiques (cochon) définirent les entités qui restent aujourd’hui les cibles favorites de ces études. Les champs d’application se sont également multipliés, afin de mieux cerner l’évolution des espèces, des populations et des génomes : génétique des populations, phylogénie d’espèces, domestication, migration de populations, paléopathologie, paléogénomique et évolution moléculaire s’offrent désormais à une discipline décidément en plein essor.