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Molecular phylogenetics employing modern and ancient DNA

100%

Pusch C. M., Broghammer M., Blin N.

Journal of Applied Genetics

2003

vol. 44

issue 3

269-290

Comparative studies of DNA in recent populations and characterisation of ancient hereditary material have contributed very interesting facts to our understanding of evolution of modern mankind. Analysis of DNA homology in related species, assessment of mutations and polymorphisms in various populations and new DNA sequence data from prehistoric finds allowed ? via sophisticated DNA extraction techniques, PCR, sequencing and digitalised processing of genetic information ? insights into possible roots of Homo sapiens and related species, migration patterns and ancient cultural habits, thus enriching the palaeoanthropological discipline. However, a presentation of this development would not be complete without pointing towards the methodological limitations and manifold presentations burdened with artifacts, data misinterpretation and unjustified conclusions. Presently, this modern field of research is in its consolidation phase and new parameters for quality control and authentication are being implemented to avoid spectacular but unfounded reports. It is expected that most of the problems connected to old biomolecules may be closely related to fossilisation parameters. The future challenge will be the full understanding of the complex and multi-faceted processes underlying diagenesis, including the elucidation of nucleic acid ?postmortem damage?.

Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity

76%

Pusch C. M., Kayademir T., Prangenberg K., Conard N. J., Czarnetzki A., Blin N.

Journal of Applied Genetics

2002

vol. 43

issue 3

351-363

C/GT/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens.

Adaptor-mediated amplification of minute amounts of severely fragmented ancient nucleic acids

64%

Pusch C. M., Blin B. N., Blin N., Broghammer B. M., Broghammer M., Nicholson N. G. J., Nicholson G. J., Scholz S. M., Scholz M.

Journal of Applied Genetics

2000

vol. 41

issue 4

303-315

It has been repeatedly shown that high copy number mitochondrial DNA sequences can be recovered from ancient samples. A significant increase in the volume of information available to researchers will be observed when the amplification of nuclear DNA becomes commonplace and reproducible. To this end we established a modification of the Rapid Amplification of cDNA Ends (RACE) procedure normally used for the generation of cDNA ends from adaptor-ligated expressed sequence tag libraries. The modifications were designed to specifically address the problems associated with the highly damaged nucleic acids extracted from palaeontological specimens. For this study we used 6 human samples dating to 450 AD and ~6.500 BP that were refractory to reliable amplification of single copy loci by PCR. Racemate contents (ratio of D/L enantiomers) of aspartic acid, alanine, and leucine also indicated that no amplifiable DNA is present in 5 of the 6 samples. The proposed technique allowed us (i) to amplify four X-chromosomal loci from 5 human specimens, and (ii) to correct allelic drop-out phenomena at the amelogenin locus in one individual, thus showing that the threshold of 80 ? 10?3 for D/Lasp as a borderline for the presence/absence of amplifiable aDNA requires reassessment. Reliability of the proposed technique (i.e. amplification of DNA sequences endogenous to the find) was validated by the application of ?ancient RACE? (aRACE) to prehistoric animal samples.

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3 Journal of Applied Genetics

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3 Blin N.

3 Pusch C. M.

2 Broghammer M.

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1 Broghammer B. M.

1 Conard N. J.

1 Czarnetzki A.

1 Kayademir T.

1 Nicholson G. J.

1 Nicholson N. G. J.

1 Prangenberg K.

1 Scholz M.

1 Scholz S. M.

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Molecular phylogenetics employing modern and ancient DNA

Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity

Adaptor-mediated amplification of minute amounts of severely fragmented ancient nucleic acids