Multi-Step Ancient DNA Extraction Protocol For Bone And Teeth v1

protocols.io, 2019

Silica-based total DNA extraction protocol optimised for the recovery of ultra-short DNA molecules from skeletal material powder (e.g. bone, teeth), modified from Dabney et al. (2013) PNAS (doi: 10.1073/pnas.1314445110).

Medicine, Science and the Law, 2012

Bone is the most challenging tissue for DNA extraction and purification. Expensive commercial kits and specific equipments are often used in forensic and anthropology laboratories towards that goal. We present here an integrated procedure that gives satisfactory results for DNA preparation from fresh, ancient or weathered bones. Extraction is performed under simple but efficient vacuum-controlled conditions that greatly limit the risks of cross-contaminations. The whole process has been designed to minimize the need for expensive equipment and chemicals, and to be compatible with any molecular biology laboratory. In addition, no toxic reagents are necessary and the procedure is straightforward. Combined with quantitative polymerase chain reaction (qPCR), this method allows species identification and sex determination from subcellular amount of DNA (1-5 pg). In addition, enough DNA is generally obtained for human DNA profiling if necessary. The whole procedure from bone treatment to ...

The majority of ancient DNA studies on human specimens have utilised teeth and bone as a source of genetic material. In addition to the abundance of such samples, they are perceived to be a source of uncontaminated endogenous DNA. In this paper the levels of endogenous contamination * (i.e. present within the sample prior to sampling for the DNA analysis) are assessed within a range of European archaeological human bone and teeth samples. A series of experiments are also undertaken to artificially contaminate samples that have previously been assessed for biochemical and histological preservation. The findings demonstrate two important issues: a) although teeth are more resilient to contamination than bone, both are readily contaminated through handling or washing, and b) once contaminated in this way, both are difficult (if not impossible) to decontaminate. The specific biochemical characteristics of individual samples reveal that contamination is directly correlated to sample preservation, and in particular, levels of microbial attack and related increases in sample porosity. The implication is that the vast majority of the known archaeological record has been deeply contaminated. * The impetus behind this work was a series of incomprehensible aDNA results on human bone from Repton, Derbyshire, England. See Biddle and Kjolbye-Biddle, 2001, 45-96. 148

Philosophical Transactions of the Royal Society B: Biological Sciences, 1991

Journal of Archaeological Science, 2011

The recovery of genetic material from preserved hard skeletal remains is an essential part of ancient DNA, archaeological and forensic research. However, there is little understanding about the relative concentrations of DNA within different tissues, the impact of sampling methods on extracted DNA, or the role of environmentally-determined degradation rates on DNA survival in specimens. We examine these issues by characterizing the mitochondrial DNA (mtDNA) content of different hard and soft tissues in 42 ancient human and bovid specimens at a range of fragment lengths (77e235 bp) using real-time PCR. Remarkably, the standard drill speeds used to sample skeletal material (c. 1000 RPM) were found to decrease mtDNA yields up to 30 times (by 3.1 Â 10 5 mtDNA copies on average) compared to pulverization in a bone mill. This dramatic negative impact appears to relate to heat damage, and disappeared at very low drill speeds (e.g. 100 RPM). Consequently, many ancient DNA and forensic studies may have obtained false negative results, especially from important specimens which are commonly sampled with drills to minimize signs of damage. The mtDNA content of tooth cementum was found to be five times higher than the commonly used dentine (141 bp, p ¼ 0.01), making the cementum-rich root tip the best sample for ancient human material. Lastly, mtDNA was found to display a consistent pattern of exponential fragmentation across many depositional environments, with different rates for geographic areas and tissue types, improving the ability to predict and understand DNA survival in preserved specimens.

Genes

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly ...

Despite the widespread use of bones in ancient DNA (aDNA) studies, relatively little concrete information exists in regard to how the DNA in mineralised collagen degrades, or where it survives in the material's architecture. While, at the macrostructural level, physical exclusion of microbes and other external contaminants may be an important feature, and, at the ultrastructural level, the adsorption of DNA to hydroxyapatite and/or binding of DNA to Type I collagen may stabilise the DNA, the relative contribution of each, and what other factors may be relevant, are unclear. There is considerable variation in the quality of DNA retrieved from bones and teeth. This is in part due to various environmental factors such as temperature, proximity to free water or oxygen, pH, salt content, and exposure to radiation, all of which increase the rate of DNA decay. For example, bone specimens from sites at high latitudes usually yield better quality DNA than samples from temperate regions, which in turn yield better results than samples from tropical regions. However, this is not always the case, and rates of success of DNA recovery from apparently similar sites are often strikingly different. The question arises as to whether this may be due to post-collection preservation or just an artefact of the extraction methods used in these different studies? In an attempt to resolve these questions, we examine the efficacy of DNA extraction methods, and the quality and quantity of DNA recovered from both artificially degraded, and genuinely ancient, but well preserved, bones. In doing so we offer hypotheses relevant to the DNA degradation process itself, and to where and how the DNA is actually preserved in ancient bone.

2015

Poor DNA preservation is the most limiting factor in ancient genomic research. In the vast majority of ancient bones and teeth, endogenous DNA molecules only represent a minor fraction of the whole DNA extract, rendering traditional shot-gun sequencing approaches cost-ineffective for whole-genome characterization. Based on ancient human bone samples from temperate and tropical environments, we show that an initial EDTA-based enzymatic 'pre-digestion' of powdered bone increases the proportion of endogenous DNA several fold. By performing the pre-digestion step between 30 min and 6 hours on five bones, we identify the optimal pre-digestion time and document an average increase of 2.7 times in the endogenous DNA fraction after 1 hour of pre-digestion. With longer pre-digestion times, the increase is asymptotic while molecular complexity decreases. We repeated the experiment with n=21 and t=15-30', and document a significant increase in endogenous DNA content (one-sided pair...

A minimally destructive protocol for DNA extraction from ancient teeth

Ancient DNA sampling methods-although optimized for efficient DNA extraction-are destructive, relying on drilling or cutting and powdering (parts of) bones and teeth. As the field of ancient DNA has grown, so have concerns about the impact of destructive sampling of the skeletal remains from which ancient DNA is obtained. Due to a particularly high concentration of endogenous DNA, the cementum of tooth roots is often targeted for ancient DNA sampling, but destructive sampling methods of the cementum often result in the loss of at least one entire root. Here, we present a minimally destructive method for extracting ancient DNA from dental cementum present on the surface of tooth roots. This method does not require destructive drilling or grinding, and, following extraction, the tooth remains safe to handle and 28 These authors contributed equally to this work. Corresponding authors: [email protected],

ABSTRACTAncient DNA sampling methods—although optimized for efficient DNA extraction—are destructive, relying on drilling or cutting and powdering (parts of) bones and teeth. As the field of ancient DNA has grown, so have concerns about the impact of destructive sampling of the skeletal remains from which ancient DNA is obtained. Due to a particularly high concentration of endogenous DNA, the cementum of tooth roots is often targeted for ancient DNA sampling, but standard destructive sampling methods often result in the loss of at least one entire root. Here, we present a minimally destructive method for extracting ancient DNA from dental cementum present on the surface of tooth roots. This method does not require destructive drilling or grinding, and, following extraction, the tooth remains safe to handle and suitable for most morphological studies, as well as other biochemical studies, such as radiocarbon dating. We extracted and sequenced ancient DNA from 30 teeth (and 9 correspo...