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1a for a schematic overview of the experimental design).Following DNA release, half of the aliquots were used for collagen extraction and dating, and half were incubated with an EDTA/proteinase K buffer commonly used in ancient DNA extraction to achieve full lysis of the bone powder and release any residual DNA.
(a) In an initial experiment, two bone samples were used to evaluate the suitability of EDTA as well as neutral and acidic phosphate buffers for releasing DNA prior to radiocarbon dating.
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.
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.
By comparing the number of endogenous DNA fragments recovered during initial DNA release to those obtained from subsequent full lysis of the same bone powder aliquots, we estimate that EDTA released 42% and 99% of the endogenous DNA from samples A and B, respectively, while acidic phosphate released 53% and 50% (Fig. In contrast, no more than 20% of the endogenous DNA was released by incubation in the neutral phosphate buffer from either sample. While the size distributions of DNA fragments retrieved from EDTA and neutral phosphate were similar, acidic phosphate showed an enrichment for short DNA molecules (Supplementary Fig. Prompted by these results we performed binding experiments of DNA to hydroxyapatite and bone powder, and found that when compared to long molecules, short molecules are both more efficiently released from hydroxyapatite by acidic phosphate and more efficiently retained from acidic buffers during subsequent silica-based DNA purification (Supplementary Figs S2 and S3).