However, the fossil record is often scarce and fragmentary, not only at Paleolithic sites, which limits the amount of material that can be sacrificed for molecular analyses.
More importantly, every effort possible should be taken to keep destructive sampling to a minimum in order to preserve the world’s archaeological heritage for future generations.
DNA was isolated from the EDTA, phosphate and lysis buffers by silica-based purification and converted into DNA libraries.
Yields of DNA library molecules were determined by digital PCR and the libraries characterized by high-throughput sequencing using Illumina’s Mi Seq platform (Fig. Horse and cave bear DNA fragments (endogenous DNA) were identified by mapping sequences with a length of at least 35 base pairs (bp) to a closely related reference genome.
Here we explored the feasibility of releasing DNA from ancient bones prior to collagen extraction using an ABA-gelatinization procedure followed by ultrafiltration.
Carbon 4 dating
More specifically, we tested three reagents that might enable the recovery of DNA without degrading the organic component of the bone/tooth matrix.Since carbonates in the mineral fraction of hard tissues are exchanged with those present in the environment, in which a first treatment with hydrochloric acid solubilizes carbonates and hydroxyapatite, the main inorganic component of bones and teeth, a second treatment with sodium hydroxide removes other organic molecules such as humic acids, and a third treatment with hydrochloric acid removes atmospheric carbon dioxide absorbed during the base treatment.The resulting collagen is then incubated in acid at high temperature to produce soluble gelatine.(b) In a second experiment, 10 additional bones were used to determine the efficiency of DNA release with EDTA and acidic phosphate buffer, and the impact of these treatments on collagen preparation and radiocarbon dating.(c) Overview of the sample preparation workflows used for radiocarbon dating and genetic analysis.Therefore, the biomolecules required for radiocarbon dating and ancient DNA analysis are presumably located in different fractions of the bone matrix, suggesting that it might be feasible to retrieve both from a single sample by targeting the inorganic and organic components of the bone/tooth matrix separately.Such a combined method for DNA and collagen extraction would not only reduce the number of samplings and thereby the amount of material required to perform both techniques, but also substantially increase the amount of material available for genetic analyses.Since carbon contamination may also arise from organic molecules that have entered the bone or tooth matrix through soil detritus, microbial invasion or post-excavation handling, ABA-gelatinization is often followed by ultrafiltration through membranes that separate high molecular weight collagen chains from shorter peptides, amino acids and other small molecules.DNA extraction, in contrast, is typically performed by lysis of the bone/tooth matrix using extraction buffers containing ethylenediaminetetraacetic acid (EDTA), a chelating agent that dissolves hydroxyapatite by means of sequestering calcium ions, and proteinase K, an enzyme that digests collagen and other proteins.To determine whether it is feasible in principle to extract DNA and collagen from the same sample material without affecting radiocarbon dates, we used a dentistry drill to remove 7 g of powder from a 300-year-old horse bone (sample A) close in age to the upper limit of radiocarbon dating, and 8 g of powder from a C isotopes (see Table 1 and Supplementary Table S1 for details on the samples used in this study).The powder from each sample was split into 500 mg aliquots, which were then either subjected directly to collagen extraction and dating, or incubated with EDTA, neutral, or acidic phosphate buffers to release DNA (see Fig.