Late Pleistocene North America hosted at least two divergent and ecologically distinct species of mammoth: the periglacial woolly mammoth (Mammuthus primigenius) and the subglacial Columbian mammoth (Mammuthus columbi). To date, mammoth genetic research has been entirely restricted to woolly mammoths, rendering their genetic evolution difficult to contextualize within broader Pleistocene paleoecology and biogeography. Here, we take an interspecific approach to clarifying mammoth phylogeny by targeting Columbian mammoth remains for mitogenomic sequencing.
The discovery of antibiotics more than 70 years ago initiated a period of drug innovation and implementation in human and animal health and agriculture. These discoveries were tempered in all cases by the emergence of resistant microbes. This history has been interpreted to mean that antibiotic resistance in pathogenic bacteria is a modern phenomenon; this view is reinforced by the fact that collections of microbes that predate the antibiotic era are highly susceptible to antibiotics. Here we report targeted metagenomic analyses of rigorously authenticated ancient DNA from 30,000-year-old Beringian permafrost sediments and the identification of a highly diverse collection of genes encoding resistance to ?-lactam, tetracycline and glycopeptide antibiotics. Structure and function studies on the complete vancomycin resistance element VanA confirmed its similarity to modern variants. These results show conclusively that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use.
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.
Gilbert et al. (Reports, 9 May 2008, p. 786) analyzed DNA from radiocarbon-dated paleofecal remains from Paisley Cave, Oregon, which ostensibly demonstrate a human presence in North America predating the well-established Clovis complex. We question the authenticity of their DNA results and argue that in the absence of intact stratigraphy and diagnostic artifacts, and in view of carbon isotope anomalies, the radiocarbon dates of the oldest specimens are unreliable.
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.
Researchers sorely need markers and approaches for biodiversity exploration (both specimen linked and metagenomics) using the full potential of next generation sequencing technologies (NGST). Currently, most studies rely on expensive multiple tagging, PCR primer universality and/or the use of few markers, sometimes with insufficient variability.
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