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PubMed1994

Ancient DNA from Bronze Age bones of European rabbit (Oryctolagus cuniculus).

The European rabbit (Oryctolagus cuniculus) is now widely distributed throughout the world as a result of transportation by man. The original populations, however, were confined to southern France and Spain. In order to investigate the role of human intervention in determining the genetic diversity of rabbit populations, we are studying the origin of rabbits introduced onto a small Mediterranean island (Zembra) near Tunis over 1400 years ago, by examining ancient DNA extracted from rabbit bones found both on Zembra and on the European mainland. Ancient DNA was successfully extracted from rabbit bones found at two archaeological sites dated to at least the Early Bronze Age (more than 3500 years ago) in south-central France, and compared to that found in modern mainland and island populations using a small variable region of the cytochrome b gene. The results confirm that the Zembra Island population is descended from that present over 1400 years ago. The technical aspects of DNA extraction from bones and the implications of this type of research for determining the origin of introduced rabbit populations are discussed.

Animals↗

PubMed1995

Ancient DNA.

The investigation of molecular traits for evolutionary studies is a valuable alternative to the classic morphological approach. Since the mid-1980s molecular evolution has extended its field of investigation into the past. This has become possible with development of the polymerase chain reaction, which allows amplification of, and hence the study of, DNA from individuals long since dead. Many ancient, mostly extinct, animals and plants have been studied in order to determine their phylogenetic relationships with extant species. The biochemical properties of ancient DNA are also under investigation. The modifications that this molecule undergoes over long periods of time are of great interest as attempts are made to retrieve DNA from increasingly older remains.

Animals↗

PubMed1993

Going for the old: ancient DNA draws a crowd.

Approximately 170 researchers interested in reconstructing the past convened for the 2nd International Conference on Ancient DNA, held in Washington, D.C., from 7 to 9 October. While rejuvenated celluloid dinosaurs have grabbed headlines this year, these scientists were more concerned with topics such as tracing ancient human populations and understanding how DNA can survive the millennia.

Animals↗

PubMed2005

Ancient DNA from giant extinct lemurs confirms single origin of Malagasy primates.

The living Malagasy lemurs constitute a spectacular radiation of >50 species that are believed to have evolved from a common ancestor that colonized Madagascar in the early Tertiary period. Yet, at least 15 additional Malagasy primate species, some of which were relative giants, succumbed to extinction within the past 2,000 years. Their existence in Madagascar is recorded predominantly in its Holocene subfossil record. To rigorously test the hypothesis that all endemic Malagasy primates constitute a monophyletic group and to determine the evolutionary relationships among living and extinct taxa, we have conducted an ancient DNA analysis of subfossil species. A total of nine subfossil individuals from the extinct genera Palaeopropithecus and Megaladapis yielded amplifiable DNA. Phylogenetic analysis of cytochrome b sequences derived from these subfossils corroborates the monophyly of endemic Malagasy primates. Our results support the close relationship of sloth lemurs to living indriids, as has been hypothesized on morphological grounds. In contrast, Megaladapis does not show a sister-group relationship with the living genus Lepilemur. Thus, the classification of the latter in the family Megaladapidae is misleading. By correlating the geographic location of subfossil specimens with relative amplification success, we reconfirm the global trend of increased success rates of ancient DNA recovery from nontropical localities.

Animals↗

PubMed2005

How many clones need to be sequenced from a single forensic or ancient DNA sample in order to determine a reliable consensus sequence?

Forensic and ancient DNA (aDNA) extracts are mixtures of endogenous aDNA, existing in more or less damaged state, and contaminant DNA. To obtain the true aDNA sequence, it is not sufficient to generate a single direct sequence of the mixture, even where the authentic aDNA is the most abundant (e.g. 25% or more) in the component mixture. Only bacterial cloning can elucidate the components of this mixture. We calculate the number of clones that need to be sampled (for various mixture ratios) in order to be confident (at various levels of confidence) to have identified the major component. We demonstrate that to be >95% confident of identifying the most abundant sequence present at 70% in the ancient sample, 20 clones must be sampled. We make recommendations and offer a free-access web-based program, which constructs the most reliable consensus sequence from the user's input clone sequences and analyses the confidence limits for each nucleotide position and for the whole consensus sequence. Accepted authentication methods must be employed in order to assess the authenticity and endogeneity of the resulting consensus sequences (e.g. quantification and replication by another laboratory, blind testing, amelogenin sex versus morphological sex, the effective use of controls, etc.) and determine whether they are indeed aDNA.

Cloning, Molecular↗

PubMed2006

Recharacterization of ancient DNA miscoding lesions: insights in the era of sequencing-by-synthesis.

Although ancient DNA (aDNA) miscoding lesions have been studied since the earliest days of the field, their nature remains a source of debate. A variety of conflicting hypotheses exist about which miscoding lesions constitute true aDNA damage as opposed to PCR polymerase amplification error. Furthermore, considerable disagreement and speculation exists on which specific damage events underlie observed miscoding lesions. The root of the problem is that it has previously been difficult to assemble sufficient data to test the hypotheses, and near-impossible to accurately determine the specific strand of origin of observed damage events. With the advent of emulsion-based clonal amplification (emPCR) and the sequencing-by-synthesis technology this has changed. In this paper we demonstrate how data produced on the Roche GS20 genome sequencer can determine miscoding lesion strands of origin, and subsequently be interpreted to enable characterization of the aDNA damage behind the observed phenotypes. Through comparative analyses on 390,965 bp of modern chloroplast and 131,474 bp of ancient woolly mammoth GS20 sequence data we conclusively demonstrate that in this sample at least, a permafrost preserved specimen, Type 2 (cytosine-->thymine/guanine-->adenine) miscoding lesions represent the overwhelming majority of damage-derived miscoding lesions. Additionally, we show that an as yet unidentified guanine-->adenine analogue modification, not the conventionally argued cytosine-->uracil deamination, underpins a significant proportion of Type 2 damage. How widespread these implications are for aDNA will become apparent as future studies analyse data recovered from a wider range of substrates.

Animals↗

PubMed2003

Poly(A) tailing of ancient DNA: a method for reproducible microsatellite genotyping.

Microsatellites could be of great potential use in the analysis of ancient remains, but so far such analyses have failed to be reproducible mainly because of the high degree of ancient DNA (aDNA) degradation. During PCR, annealing of the primers to the complementary sequences of microsatellites occurs together with cross-annealing of partially degraded repeated sequences. This could create chimeric alleles that do not correspond to the authentic ones. Here we report a simple method for processing aDNA fragments prior to PCR that greatly reduces the production of chimeric alleles. This approach eliminates aDNA molecules broken within the repeats as targets for Taq polymerase by adding poly(A) tails at the 3(') ends of the DNA fragments, which disrupts the homology in the region and thus prevents annealing out of register. We have analyzed one dinucleotide- (D6S337) and two trinucleotide-containing loci (IT15 and SCA1) using poly(A)-tailed and the same untreated aDNA as template. aDNAs were isolated from 28 human remains, 600 and 7000 years of age. In repeated experiments with untreated aDNAs we obtained three to five times more alleles compared to poly(A)-tailed aDNAs. According to our results, modification of aDNA by poly(A) tailing is an efficient pretreatment for accurate genotyping.

Animals↗

PubMed2006

Comment on "Ancient DNA from the first European farmers in 7500-year-old Neolithic sites".

On the basis of analysis of ancient DNA from early European farmers, Haak et al. (Reports, 11 November 2005, p. 1016) argued for the Paleolithic ancestry of modern Europeans. We stress that the study is more limited in scope than the authors claim, in part because not all of the skeletal samples date to the time of the Neolithic transition in a given area of Europe.

Agriculture↗

PubMed2004

Genetic analyses from ancient DNA.

About 20 years ago, DNA sequences were separately described from the quagga (a type of zebra) and an ancient Egyptian individual. What made these DNA sequences exceptional was that they were derived from 140- and 2400-year-old specimens. However, ancient DNA research, defined broadly as the retrieval of DNA sequences from museum specimens, archaeological finds, fossil remains, and other unusual sources of DNA, only really became feasible with the advent of techniques for the enzymatic amplification of specific DNA sequences. Today, reports of analyses of specimens hundreds, thousands, and even millions of years old are almost commonplace. But can all these results be believed? In this paper, we critically assess the state of ancient DNA research. In particular, we discuss the precautions and criteria necessary to ascertain to the greatest extent possible that results represent authentic ancient DNA sequences. We also highlight some significant results and areas of promising future research.

Animals↗

PubMed2002

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

C/G-->T/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.

Base Sequence↗

PubMed2003

Enterobius vermicularis: ancient DNA from North and South American human coprolites.

A molecular paleoparasitological diagnostic approach was developed for Enterobius vermicularis. Ancient DNA was extracted from 27 coprolites from archaeological sites in Chile and USA. Enzymatic amplification of human mtDNA sequences confirmed the human origin. We designed primers specific to the E. vermicularis 5S ribosomal RNA spacer region and they allowed reproducible polymerase chain reaction identification of ancient material. We suggested that the paleoparasitological microscopic identification could accompany molecular diagnosis, which also opens the possibility of sequence analysis to understand parasite-host evolution.

Animals↗

PubMed2004

Authenticity of ancient-DNA results: a statistical approach.

Although there have been several papers recommending appropriate experimental designs for ancient-DNA studies, there have been few attempts at statistical analysis. We assume that we cannot decide whether a result is authentic simply by examining the sequence (e.g., when working with humans and domestic animals). We use a maximum-likelihood approach to estimate the probability that a positive result from a sample is (either partly or entirely) an amplification of DNA that was present in the sample before the experiment began. Our method is useful in two situations. First, we can decide in advance how many samples will be needed to achieve a given level of confidence. For example, to be almost certain (95% confidence interval 0.96-1.00, maximum-likelihood estimate 1.00) that a positive result comes, at least in part, from DNA present before the experiment began, we need to analyze at least five samples and controls, even if all samples and no negative controls yield positive results. Second, we can decide how much confidence to place in results that have been obtained already, whether or not there are positive results from some controls. For example, the risk that at least one negative control yields a positive result increases with the size of the experiment, but the effects of occasional contamination are less severe in large experiments.

Data Interpretation, Statistical↗

PubMed1999

Analysis of ancient DNA from a prehistoric Amerindian cemetery.

The Norris Farms No. 36 cemetery in central Illinois has been the subject of considerable archaeological and genetic research. Both mitochondrial DNA (mtDNA) and nuclear DNA have been examined in this 700-year-old population. DNA preservation at the site was good, with about 70% of the samples producing mtDNA results and approximately 15% yielding nuclear DNA data. All four of the major Amerindian mtDNA haplogroups were found, in addition to a fifth haplogroup. Sequences of the first hypervariable region of the mtDNA control region revealed a high level of diversity in the Norris Farms population and confirmed that the fifth haplogroup associates with Mongolian sequences and hence is probably authentic. Other than a possible reduction in the number of rare mtDNA lineages in many populations, it does not appear as if European contact significantly altered patterns of Amerindian mtDNA variation, despite the large decrease in population size that occurred. For nuclear DNA analysis, a novel method for DNA-based sex identification that uses nucleotide differences between the X and Y copies of the amelogenin gene was developed and applied successfully in approximately 20 individuals. Despite the well-known problems of poor DNA preservation and the ever-present possibility of contamination with modern DNA, genetic analysis of the Norris Farms No. 36 population demonstrates that ancient DNA can be a fruitful source of new insights into prehistoric populations.

Amelogenin↗

PubMed2003

"Ancient" protocols for the crime scene? Similarities and differences between forensic genetics and ancient DNA analysis.

We provide a short overview on some current issues in the fields of forensic genetics and ancient DNA (aDNA) analysis. We discuss about the existence of the possible points of contact between the two disciplines, in terms of open problems and the inherent approach to their solution. We mainly focus on the problem of results authentication, its theoretical and technical aspects.

DNA↗

PubMed2003

Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach.

The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR) inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.

Animals↗

PubMed2005

First systematic CGH-based analyses of ancient DNA samples of malformed fetuses preserved in the Meckel Anatomical Collection in Halle/Saale (Germany).

We present the first data on our comparative genomic hybridization (CGH)-based strategy for the analysis of ancient DNA (aDNA) samples extracted from fetuses preserved in the Meckel Anatomical Collection in Halle, Germany. The collection contains numerous differently fixed ancient samples of fetal malformations collected from the middle of the 18th to the early 19th century. The main objective of this study is to establish a "standard" aDNA extraction and amplification protocol as a prerequisite for successful CGH analyses to detect or exclude chromosomal imbalances possibly causative for the malformations described for the fetuses.

Congenital Abnormalities↗

PubMed2005

Ancient DNA analysis of human neolithic remains found in northeastern Siberia.

We successfully extracted DNA from a bone sample of a Neolithic skeleton (dated 3,600 +/- 60 years BP) excavated in northeastern Yakutia (east Siberia). Ancient DNA was analyzed by autosomal STRs (short tandem repeats) and by sequencing of the hypervariable region I (HV1) of the mitochondrial DNA (mtDNA) control region. The STR profile, the mitochondrial haplotype, and the haplogroup determined were compared with those of modern Eurasian and Native American populations. The results showed the affinity of this ancient skeleton with both east Siberian/Asian and Native American populations.

Base Sequence↗

PubMed2004

Prehistoric Sado Island populations of Sus scrofa distinguished from contemporary Japanese wild boar by ancient mitochondrial DNA.

Ancient mitochondrial DNA (mtDNA) mainly from Jomon Period Sus scrofa bone specimens (6,100-1,700 years old) was examined to clarify the genetic relationships between prehistoric and contemporary S. scrofa on Hokkaido, Honshu, Sado, and Izu islands of the Japanese Archipelago. Phylogenetic analysis of the mtDNA control region (574 bp) and analysis of pairwise nucleotide differences between prehistoric and contemporary S. scrofa sequences showed the following relationships between these groups: (1) a group genetically similar to contemporary Japanese wild boars was found mainly on Honshu Island, Hokkaido Island, and the Izu Islands, and (2) a monophyletic group distinct from contemporary Japanese wild boars was found on Sado Island. These results suggest that prehistoric people introduced S. scrofa from Honshu Island to Hokkaido Island and the Izu Islands. The estimated divergence times between the prehistoric Sado group and the other prehistoric S. scrofa is approximately congruent with the geological isolation of Sado Island from Honshu Island. Our results suggest that this extinct S. scrofa population was present on Sado Island as recently as around 2,000 years ago.

Animals↗