Dna dating method
New technique provides accurate dating of ancient skeletons
Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material. This is achieved by releasing DNA from the bone matrix through incubation with either EDTA or phosphate buffer prior to complete demineralization and collagen extraction utilizing the acid-base-acid-gelatinization and ultrafiltration procedure established in most radiocarbon dating laboratories.
We also detect no skews in radiocarbon dates compared to untreated samples. Over the past 70 years, radiocarbon dating has become an important tool for archaeology due to its precision in dating organic material up to approx. More recently, advances in DNA sequencing technology have enabled the generation of genome-wide sequence data from hundreds of ancient remains, especially those of ancient humans 5 , 6 , 7 , 8 and their extinct archaic relatives 9 , 10 , 11 , providing insights into the history of human groups, their dispersals and interactions.
In contrast to AMS radiocarbon dating, genetic analysis of ancient bones and teeth is often feasible even from small amounts of sample material. This has been demonstrated, for example, in a series of genetic studies on fossil material from Denisova Cave, Russia. Both destructive methods, DNA analysis and radiocarbon dating, are invaluable tools for reconstructing past events and their timing, such as the colonization of Europe by anatomically modern humans AMH and Neanderthal extinction 7 , 16 , 17 , 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.
Since carbonates in the mineral fraction of hard tissues are exchanged with those present in the environment 19 , it is necessary to completely remove the inorganic component of bone or dentine during collagen preparation for AMS radiocarbon dating. This is typically achieved by acid-base-acid ABA treatment 20 , 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. 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 3 , Even though the exact mechanism of DNA preservation in ancient bones and teeth is not fully understood 24 , the binding of negatively charged phosphate groups in the DNA backbone to positively charged calcium ions on the surface of hydroxyapatite crystals is thought to play a major role 25 , 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.
Here we explored the feasibility of releasing DNA from ancient bones prior to collagen extraction using an ABA-gelatinization procedure followed by ultrafiltration. The second reagent is a neutral pH 7. Phosphate buffers are commonly used in liquid chromatography 29 and occasionally in ancient DNA research 30 to release DNA from hydroxyapatite. However, neutral phosphate buffers have been shown to preferentially release surface-bound microbial DNA rather than endogenous DNA from ancient bone For each of these reagents we evaluated the efficiency of DNA retrieval while monitoring possible losses of collagen and the accuracy of the resultant radiocarbon dates.
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. Schematic overview of the experiments performed in this study. DNA recovered from this buffer also showed a severe decrease in the relative abundance of endogenous vs. Comparing the suitability of EDTA, neutral and acidic phosphate treatments for DNA release prior to collagen extraction and radiocarbon dating.
N ratio horizontal line of the collagen preparation, and e uncalibrated AMS radiocarbon dates obtained from treated and untreated bone powder aliquots in years before present BP. Collagen yields from the powder aliquots used for DNA release are very similar to those that directly underwent collagen extraction Fig. Likewise, the percentage of carbon and the carbon nitrogen ratios C: N , which are routinely determined to assess the quality of collagen preparations 2 , 32 , were not substantially altered by DNA release Fig.
With the exception of one bone powder aliquot treated with neutral phosphate buffer, collagen extracted from sample B consistently yielded infinite dates. However, a phthalic acid C 8 H 6 O 4 blank that was measured on the same AMS magazine after a 14 C-rich sample yielded an age of 53, BP, providing no evidence for cross-contamination. We also observed no detectable radiocarbon carryover in the phthalic acid and bone background blanks on the AMS magazine containing the radiocarbon-rich sample A.
While it cannot be fully excluded that the observation in sample B was an isolated event of carbon carryover on the AMS, it is also possible that contamination with small amounts of modern carbon occurred during DNA release or collagen preparation. The experimental design was similar to the previous one, except that the DNA release was determined by comparing the number of DNA fragments released in the treatments to those released by full lysis of a separate powder aliquot Fig. Averaged across all samples, the ratio of endogenous to non-endogenous DNA obtained after each of the two treatments was similar to that obtained by full lysis of the untreated control sample powder.
However, if samples are considered individually, the percentage of endogenous DNA obtained by acidic phosphate treatment varies substantially when compared to the untreated control, ranging from an 8. This may be partially driven by differences in the size of DNA fragments recovered with acidic phosphate, which are shorter in most samples Supplementary Fig.
S4 , consistent with the results of the previous experiment. Loss of collagen is mostly driven by a single sample sample G , where insufficient yield after EDTA treatment compromised our ability to date the material. This result was reproduced when repeating EDTA treatment and collagen extraction for this sample. Acidic phosphate, on the other hand, did not reduce collagen yields.
N ratio of 3. Combining DNA and collagen extraction on 10 bones of various ages. N ratio horizontal line of the collagen preparation, and e the uncalibrated AMS radiocarbon dates obtained from treated and untreated bone powder in years before present BP. Technical replicates are shaded in grey. AMS radiocarbon dates of most treated aliquots were consistent with those obtained from the untreated controls Fig. We did, however, observe two outliers, both of which were shifted towards slightly older dates: Interestingly, the highest outlier was produced by bone powder treated with phosphate buffer, a reagent that may conceivably be contaminated by modern carbon from atmospheric carbon dioxide, but should be free of old carbon.
Furthermore, even if old carbon from an unknown source had been introduced during DNA release, dates from young samples would be expected to be particularly strongly affected by such contamination, a pattern not seen in our data. To evaluate whether outliers appeared stochastically in our data set, we repeated the DNA release and dating for samples F and L Fig.
This time, all dates were consistent with the untreated controls. Given that a total of 54 dates were generated in the course of our experiments, the occurrence of three outliers in samples B, F, and L is not unexpected, and indicates that DNA release, collagen preparation and graphitization together contributed only small and unsystematic error to the dating process. We thus conclude that DNA release using EDTA or acidic phosphate buffer has no detectable effect on the accuracy of radiocarbon dating.
By simultaneously recovering DNA sequences and radiocarbon dates from 12 ancient bones, we have successfully demonstrated that substantial amounts of DNA can be released from sample powder prior to radiocarbon dating without reducing the accuracy of dating. We identified two methods that are suitable for this purpose. These losses in collagen are small in most cases, but can occasionally reduce collagen yields below the minimum amount required for dating. While the reduced need for destructive sampling is the most obvious benefit of a combined collagen and DNA extraction workflow, it may also help to obtain samples of higher quality for DNA analysis.
The reconstruction of whole genome sequences from ancient bones and teeth is often hampered by contamination with microbial, human and other environmental DNA. This problem can in some instances be alleviated by enriching for parts of the genome by hybridization capture 36 or restricting analyses to DNA fragments that carry ancient DNA specific base damage 37 , These enrichment strategies can also be applied to DNA that was released from bone or dentine powder prior to collagen extraction.
However, it has also been shown that DNA preservation and contamination with exogenous DNA can vary greatly within one specimen, even in locations that are in close proximity. Extracting DNA from several sampling spots can thus help in obtaining DNA extracts that are richer in endogenous DNA and less contaminated, improving the scope of genetic analysis that can be performed on a given sample As material demands for radiocarbon dating are large, powder can be collected in multiple small sub-samples e.
DNA can then be released separately from these sub-samples before combining them prior to the ABA-gelatinization procedure for collagen extraction. We currently recommend that the DNA release step is performed in the radiocarbon dating laboratory not more than a week before entering the ABA procedure as further work is needed to determine the long-term stability of samples after DNA release. It is important to note that our work focused exclusively on methods that are compatible with collagen extraction using the well-established ABA-gelatinization procedure.
A less commonly used method for collagen preparation relies on decalcification of the bone matrix using EDTA instead of strong acids 28 , While it has been suggested that omitting the acid and base treatments prior to gelatinization in collagen preparation increases the yield of collagen 41 , 42 , disadvantages of this approach are the longer times required for complete decalcification and the possibility of skewing radiocarbon dates towards older ages However, it has been shown here and previously 28 that accurate dates can be obtained if EDTA is properly removed after decalcification.
In fact, our data suggests that the quality of isolated collagen may be higher with EDTA decalcification. Considering the substantially higher DNA yields obtained by EDTA treatment of bone powder compared to acidic phosphate, it seems that an EDTA-only collagen preparation might offer a more straight-forward and efficient approach for combining DNA analysis and radiocarbon dating. For the few radiocarbon dating laboratories that are already relying on EDTA decalcification for collagen preparation, this requires nothing else than storing the EDTA fraction for future DNA analysis.
For laboratories using the ABA-gelatinization procedure, pretreatment of bone powder with acidic phosphate provides a less efficient but safer method for releasing DNA from precious sample material prior to collagen preparation and radiocarbon dating. In summary, we have shown that two important ancient biomolecules, DNA and collagen, can be recovered from the same sample material. We hope that the work presented here will stimulate further research towards a deeper integration of the sample preparation workflows used for molecular analysis of ancient skeletal remains, leading to minimal destructive sampling of precious archaeological material.
Five samples were previously directly radiocarbon dated 21 , 43 , 44 , while the age of the other seven specimens was unknown or inferred from their chronological context 12 , 45 , Sampling was performed in a designated ancient DNA cleanroom. From each sample a large amount of bone powder 3. Leftover powder was stored at room temperature for further use if needed.
Following the experimental design summarized in Fig. Extracts from the remaining samples were converted into DNA libraries using a more recent implementation of single-stranded library preparation 49 automated on a Bravo NGS Workstation Agilent Technologies Negative controls buffers containing no sample powder or DNA extract were included during the initial bone powder treatment, DNA extraction and library preparation, and carried alongside the samples throughout all experiments.
The total number of molecules in each library was measured by digital PCR as described elsewhere Forward and reverse reads were overlap-merged to reconstruct full-length sequences 54 and assigned to the parent library based on perfect matches to expected index combinations. Where necessary, species identity of samples was assessed by analysing sequences mapping to mammalian mitochondrial genomes Libraries were then aligned to appropriate reference genomes cow, dog, dolphin, elephant, horse, human hg19 , polar bear and rhinoceros using BWA 56 with ancient DNA parameters 9.
Summary statistics were computed using custom Perl scripts Collagen was extracted for both radiocarbon and isotopic analyses following a previously established pretreatment protocol All samples were decalcified in 0. The acid treated portion was then rinsed with Milli-Q water and immersed in 0. The NaOH step was followed by a final 0.
Prior to use, the filters were cleaned to remove carbon-containing humectants 58 , To assess the preservation and amount of obtained collagen, C: N ratios and isotopic values were evaluated.
Current protocols for ancient DNA and radiocarbon analysis of ancient bones and teeth call for multiple destructive samplings of a given. A new way of dating skeletons by using mutations in DNA associated with The technique will enable a better understanding of historical.
Author contributions: We report a method for dating ancient human samples that uses the recombination clock. We show that this method provides age estimates that are highly correlated to radiocarbon dates, thus documenting the promise of this approach. By studying the linear relationship between the dates of Neanderthal admixture and the radiocarbon dates, we obtain, to our knowledge, the first direct estimate of the historical generation interval of 26—30 y. The study of human evolution has been revolutionized by inferences from ancient DNA analyses.
The molecular clock is figurative term for a technique that uses the mutation rate of biomolecules to deduce the time in prehistory when two or more life forms diverged. The biomolecular data used for such calculations are usually nucleotide sequences for DNA or amino acid sequences for proteins.
Interest in the origins of human populations and their migration routes has increased greatly in recent years. A critical aspect of tracing migration events is dating them. Inspired by the Geographic Population Structure model that can track mutations in DNA that are associated with geography, researchers have developed a new analytic method, the Time Population Structure TPS , that uses mutations to predict time in order to date the ancient DNA.
The first study of what would come to be called aDNA was conducted in , when Russ Higuchi and colleagues at the University of California, Berkeley reported that traces of DNA from a museum specimen of the Quagga not only remained in the specimen over years after the death of the individual, but could be extracted and sequenced. The laborious processes that were required at that time to sequence such DNA through bacterial cloning were an effective brake on the development of the field of ancient DNA aDNA. However, with the development of the Polymerase Chain Reaction PCR in the late s, the field began to progress rapidly. Multiple primer, nested PCR strategy was used to overcome those shortcomings. Soon a series of incredible findings had been published, claiming authentic DNA could be extracted from specimens that were millions of years old, into the realms of what Lindahl b has labelled Antediluvian DNA. Insects such as stingless bees,  termites,  and wood gnats,  as well as plant  and bacterial  sequences were extracted from Dominican amber dating to the Oligocene epoch.
A combined method for DNA analysis and radiocarbon dating from a single sample
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The use of DNA sequences to estimate the timing of evolutionary events is increasingly popular, although it is fraught with practical difficulties.
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Republish our articles for free, online or in print, under Creative Commons licence. And our DNA also holds clues about the timing of these key events in human evolution. When scientists say that modern humans emerged in Africa about , years ago and began their global spread about 60, years ago, how do they come up with those dates? Traditionally researchers built timelines of human prehistory based on fossils and artifacts, which can be directly dated with methods such as radiocarbon dating and Potassium-argon dating. However, these methods require ancient remains to have certain elements or preservation conditions, and that is not always the case. Moreover, relevant fossils or artifacts have not been discovered for all milestones in human evolution.
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