A Complete mtDNA Genome of an Early Modern Human from Kostenki, Russia (original) (raw)

Ancient DNA studies: new perspectives on old samples

2012

In spite of past controversies, the field of ancient DNA is now a reliable research area due to recent methodological improvements. A series of recent large-scale studies have revealed the true potential of ancient DNA samples to study the processes of evolution and to test models and assumptions commonly used to reconstruct patterns of evolution and to analyze population genetics and palaeoecological changes. Recent advances in DNA technologies, such as next-generation sequencing make it possible to recover DNA information from archaeological and paleontological remains allowing us to go back in time and study the genetic relationships between extinct organisms and their contemporary relatives. With the next-generation sequencing methodologies, DNA sequences can be retrieved even from samples (for example human remains) for which the technical pitfalls of classical methodologies required stringent criteria to guaranty the reliability of the results. In this paper, we review the methodologies applied to ancient DNA analysis and the perspectives that next-generation sequencing applications provide in this field.

Ancient DNA – Pitfalls and Prospects

Indian Journal of Science and Technology, 2015

Ancient DNA (a-DNA) studies in past three decades has been uplifted from short stretch archaeological mtDNA retrieval to reconstruction of complete mitochondrial genome sequence using next generation sequencing. Study of a-DNA acts as validating tool to test the assumed hypothesis used in origin, evolution, diversification, distribution, domestication and extinction of species; although, scientific progression in the field is frequently hindered by erroneous reports due to contamination and other technical bottlenecks. With the advent of appropriate methodologies such as 3'-C3 spacer tagged oligonucleotide blocking, shotgun sequencing, amplicon sequencing Polymerase Chain Reaction, single primer extension methodology, development of broad spectrum ancient sequence primer, slippage proof amplification methods, use of multiplexing strategy in PCR (Polymerase Chain Reaction) amplification, etc. in a-DNA research try to minimize the difficulties associated with it. Present review highlighted the pitfalls as well as prospects of a-DNA study and tries to assess the significance and acceptability of a-DNA research for its future application in understanding of evolutionary biology.

Advances in ancient DNA studies

2006

The first aDNA studies used bacterial cloning to amplify small sequences retrieved from skins of animal and human mummies, and revealed the inefficient reaction kinetics of this technique (Higuchi et al., 1984; Paabo 1985, 1989). These studies demonstrated that the genetic material surviving in ancient specimens was often principally microbial or fungal in origin. Endogenous DNA was generally limited to very low concentrations of short, damaged fragments of multi-copy loci such as mitochondrial DNA (mtDNA). The invention of the polymerase chain reaction (PCR) made it possible to routinely amplify and study even single surviving molecules, allowing the number and range of aDNA studies to diversify rapidly (Paabo 1989; Paabo & Wilson 1988; Paabo et al., 1989; Thomas, 1989). However, the enormous amplifying power of PCR also means that contamination from modern DNA becomes a major problem. Contamination is especially likely when previously amplified PCR products are present. False posi...

The future of ancient DNA: Technical advances and conceptual shifts

BioEssays, 2014

Technological innovations such as next generation sequencing and DNA hybridisation enrichment have resulted in multi-fold increases in both the quantity of ancient DNA sequence data and the time depth for DNA retrieval. To date, over 30 ancient genomes have been sequenced, moving from 0.7Â coverage (mammoth) in 2008 to more than 50Â coverage (Neanderthal) in 2014. Studies of rapid evolutionary changes, such as the evolution and spread of pathogens and the genetic responses of hosts, or the genetics of domestication and climatic adaptation, are developing swiftly and the importance of palaeogenomics for investigating evolutionary processes during the last million years is likely to increase considerably. However, these new datasets require new methods of data processing and analysis, as well as conceptual changes in interpreting the results. In this review we highlight important areas of future technical and conceptual progress and discuss research topics in the rapidly growing field of palaeogenomics.

A highly divergent mtDNA sequence in a Neandertal individual from Italy

Current Biology, 2006

Experimental procedures The site The site of Riparo Mezzena, a rockshelter in Monti Lessini near Verona (Italy), is situated at 250 m above sea level, at the base of the Middle Eocene rock formation Nummulites complanata. Investigations near Mezzena brought to light a stratigraphical sequence approximately 1.5-1.7 m deep. Archaeological deposits produced lithic, faunal and human remains. Taphonomic and archaeozoologic analyses are currently in progress. The approximately 5400 artefacts unearthed at Riparo Mezzena place the site within the Charentienne, Eastern Ferrassie, cultural complex. Analysis of the microfauna shows an environment characterised by continental steppe (represented by species/remains of Microtus and Pitymys), broken up by broad-leaved forests (confirmed by the presence of Neomys, Sorex and Arvicola) within a non-microthermic and moderately humid climate Preliminary tests on DNA preservation The stereoisomeric D/L ratio observed for the three diagnostic amino acids are D/L Asp = 0.074, lower than the proposed limit of 0.10 under which there are good probabilities to retrieve intact DNA [S1], D/L Glu = 0.028 and D/L Ala = 0.015. The amino acid content was 37,188 parts per million. It has been possible to amplify endogenous DNA from Pleistocene remains when this value was higher than 30,000 parts per million [S2]. Therefore, overall, these results do not suggest that the endogenous DNA be highly degraded. An indirect indication of the state of preservation of the sequence is the rate of the substitutions occasionally observed in single clones, which reflect nucleotide misincorporation during the action of Taq polymerase. In previous studies, the misincorproration rate was 0.27% on average for 27 2,500-years-old Etruscans [S3] and 0.39% for the 4,500-year-old "iceman" of the Alps [S4]. For the much older MLS Neandertal sample, we had 0.32%. Real-time PCR amplification was performed using Brilliant® SYBR® Green QPCR Master Mix (Stratagene, La Jolla, CA-USA) in MX3000P 2 (Stratagene, La Jolla, CA-USA), using 0.5µM of appropriate primers (forward primer located at H 16107 and reverse primer located at L 16261). Thermal cycling conditions were 95°C for 10 min, 40 cycles at 95°C for 30 s, 53°C for 1 min and 72°C for 30 s, followed by SYBR® Green dissociation curve step. Two tenfold serial dilutions of the purified and quantified standard (the region between H16107 and L 16261 HVRI of an ancient DNA sample [S3]) were included in the experiment to create the standard curve in order to know the number of initial DNA molecules in the samples. At least two no-template controls were included for each experiment. Quantitative PCR analysis showed a conspicuous copy number of mtDNA molecules (approximately 1700), higher than the minimal required to avoid irreproducible results. Genetic analysis About 2 mg of bone powder were removed by drilling the skull fragments of MLS; the stereoisomers of aspartic, glutamic, and alanine amino acids were determined by high performance liquid chromatography [S1]. DNA was extracted in two laboratories, both exclusively dedicated to ancient DNA work (Florence and Barcelona). All most stringent protocols and procedures for the analysis of ancient DNA have been followed [S5-S7]. Briefly, 1.5 g of bone powder was processed in Florence and a sample of 0.5 g of bone was sent to Barcelona for independent replication. Extraction was performed in both laboratories as described in [S 8], with UNG treatment [S9] in order to minimize post mortem damage. In both laboratories, mtDNA sequences were generated by using 60 cycles of PCR and 5 µl of extract. The strong inhibitory effect of the extract required a final 1 to 100 dilution to obtain positive amplifications. Different primer pairs were used, some of them designed to match Neandertal-specific substitutions at the 3' end (Supplemental Figure S1). In Florence PCR products were cloned using the TOPO TA Cloning kit (Invitrogen), according to the manufacturer's instructions. Screening of white recombinant colonies was accomplished by PCR. The colonies were transferred into a 30 µl reaction mix (67 mM Tris HCl [pH 8.8], 2 mM MgCl2, 1 µM of each primer, 0.125 µM of each dNTP, and 0.75 U of Taq polymerase) containing M13 forward and reverse universal primers. After 5 min at 92°C, 30 cycles of PCR (30 s at 90°C, 1 min at 50°C, 1 min at 72°C) were performed and clones with an insert of the expected size were identified by agarose-gel electrophoresis. After purification of these PCR products with Microcon PCR devices (Amicon), a volume of 1.5 µl was cycle-sequenced, according to the BigDye Terminator kit (Applied Biosystems) supplier's instructions. The sequence was determined using an Applied BioSystems 3100 DNA sequencer. In Barcelona the PCR products were cloned with the pMOS Blue cloning kit (Ammersham) and multiple clones for each fragment were subsequently sequenced in an ABI 3700 DNA sequencer