Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold... more Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to th...
Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a... more Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the Taq DNA polymerase during downstream applications. Here we present a simplified version of a protocol to extract DNA from paleofecal remains.
In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europ... more In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.
DNA that has been recovered from archaeological and palaeontological remains makes it possible to... more DNA that has been recovered from archaeological and palaeontological remains makes it possible to go back in time and study the genetic relationships of extinct organisms to their contemporary relatives. This provides a new perspective on the evolution of organisms and DNA sequences. However, the field is fraught with technical pitfalls and needs stringent criteria to ensure the reliability of results, particularly when human remains are studied.
Inhibition is problematic in many applications of PCR, particularly those involving degraded or l... more 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.
Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold... more Most genetic studies of Holocene fauna have been performed with ancient samples from dry and cold regions, in which preservation of fossils is facilitated and molecular damage is reduced. Ancient DNA work from tropical regions has been precluded owing to factors that limit DNA preservation (e.g. temperature, hydrolytic damage). We analysed ancient DNA from rodent jawbones identified as Ototylomys phyllotis, found in Holocene and Late Pleistocene stratigraphic layers from Loltún, a humid tropical cave located in the Yucatan peninsula. We extracted DNA and amplified six short overlapping fragments of the cytochrome b gene, totalling 666 bp, which represents an unprecedented success considering tropical ancient DNA samples. We performed genetic, phylogenetic and divergence time analyses, combining sequences from ancient and modern O. phyllotis, in order to assess the ancestry of the Loltún samples. Results show that all ancient samples fall into a unique clade that diverged prior to th...
Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a... more Paleofeces are the nonmineralized remains of dung from extant and extinct fauna. They represent a surprisingly large proportion of fossil remains recovered from cave sites across the world. Paleofeces contain the DNA of the defecator as well as the DNA of ingested plant and animal remains. To successfully extract DNA from paleofeces, a balance must be achieved between the minimization of DNA loss during extraction and the removal of coeluates that would otherwise inhibit the Taq DNA polymerase during downstream applications. Here we present a simplified version of a protocol to extract DNA from paleofecal remains.
In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europ... more In the 19th century, there were several major cholera pandemics in the Indian subcontinent, Europe, and North America. The causes of these outbreaks and the genomic strain identities remain a mystery. We used targeted high-throughput sequencing to reconstruct the Vibrio cholerae genome from the preserved intestine of a victim of the 1849 cholera outbreak in Philadelphia, part of the second cholera pandemic. This O1 biotype strain has 95 to 97% similarity with the classical O395 genome, differing by 203 single-nucleotide polymorphisms (SNPs), lacking three genomic islands, and probably having one or more tandem cholera toxin prophage (CTX) arrays, which potentially affected its virulence. This result highlights archived medical remains as a potential resource for investigations into the genomic origins of past pandemics.
DNA that has been recovered from archaeological and palaeontological remains makes it possible to... more DNA that has been recovered from archaeological and palaeontological remains makes it possible to go back in time and study the genetic relationships of extinct organisms to their contemporary relatives. This provides a new perspective on the evolution of organisms and DNA sequences. However, the field is fraught with technical pitfalls and needs stringent criteria to ensure the reliability of results, particularly when human remains are studied.
Inhibition is problematic in many applications of PCR, particularly those involving degraded or l... more 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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Papers by Melanie Kuch