Releasing secrets bound to ancient remains with modern DNA extraction techniques: an interview with Elena Essel

You recently published an article on ancient human DNA recovered from a Paleolithic pendant [1]. Could you provide a brief overview of this work?

One of the big questions in many archeological sites is, who made a tool or artifact? This is a question that many of our collaborators from the archeological field ask us. When people think about ancient DNA, they often assume skeletal remains, but at many sites there are no skeletal remains of ancient humans, so no clues about who made or used these artifacts or tools.

We took a big step forward with sediment work, we can now go to a site and analyze the sediment for DNA, even if there are no human remains [2–4]. Even in the absence of skeletal remains, at least in some sites, we can detect human DNA in the sediments and can give more insights into who made the artifacts. However, it is indirect proof, so we were really excited about the challenge of directly associating an artifact with the DNA of the person who handled, made or used an artifact. Such questions were driven by our collaborators in the field of archeology.

We started our sediment work by focusing on stone tools because stone is an easy substrate to deal with, being more dense, more solid, more robust, and less likely to break during the extraction process compared to artifacts made of osseous material. The results were disappointing; it was much more challenging than we expected. We realized that we had to switch focus, so we went back to the material that we know best, skeletal remains. In the field of ancient DNA, we have been working with skeletal remains for many years. We know a lot about how DNA is preserved in bone and the chemical interactions between bones or teeth and DNA.

The challenge with bones and teeth is that they are very fragile, and much more porous and brittle than a stone tool. It is crucial to ensure that the integrity of the objects is maintained during the extraction of any DNA- this was where I joined this project. We had to think completely differently because the standard procedure in the ancient DNA workflow involves drilling a little hole into the bone. This was not an option for the bone and tooth artifacts, because you would lose crucial information that is stored on the surface. The first challenge was to develop a protocol that preserved the integrity of the artifacts without altering the surface.

We first used material that was similar to the artifacts, or material that could have been the raw material from those artifacts, to develop the protocol. We applied different DNA extraction techniques and analyzed the surface before and after the extraction to evaluate their effects. We found that one of the methods could extract DNA from objects made from bones and teeth, and also did not alter the surface, so we started to delve into this method.

The method was reported in a previous publication in BioTechniques as a pretreatment for decontaminating bone powder [5]. In this method, we drilled a small hole before collecting powder, so we had to tweak the method a bit for these objects, which can range from just a few centimeters up to 10 or 20 cm. After optimizing the method, we arrived at a protocol that allowed us to extract DNA from the objects while preserving their structure and integrity.

How do the extraction techniques differ for different specimens, for example bone vs. artifact?

For the bone material that we often work with, skeletal remains, we are interested in the DNA of the animal or the human being that the remains came from. We usually take a dentist's drill, and we collect a bit of bone powder while drilling, then we dissolve the powder in ethylenediaminetetraacetic acid (EDTA), which is a strong decalcifying agent. EDTA is used in laundry or dishwasher detergents to decalcify the water and protect your washing machine; it actually dissolves calcium.

This is great for bone powder because we want to dissolve the bone matrix to release the DNA into solution, but it is a nightmare for the artifacts because if we use a decalcifying agent, we actually dissolve the informative surface of the bone or tooth artifact, so we had to use a different approach.

From previous experiments and publications, we know that one can use a trick to release the DNA from the bone matrix without actually dissolving the bone matrix [5,8]. The bone matrix consists of about 70% of a mineral called hydroxyapatite, which is mostly calcium, while the DNA backbone contains phosphate. The calcium is positively charged, and the phosphate is negatively charged so DNA binds very tightly to the bone matrix via electrostatic interactions. We can add an excess of free phosphate ions using a phosphate buffer, then these free phosphate ions start to compete for the calcium-binding sites in the hydroxyapatite, pushing out the DNA and replacing the DNA that was formerly bound to the bone matrix. The DNA is then released into solution, so it is accessible for us to purify it, enrich it and work with it. Thus, we can get the DNA out without dissolving the bone matrix, keeping the bone intact.

How important was temperature-controlled extraction of DNA from ancient bones to your research?

Carrying out the phosphate extraction at room temperature did not yield as much DNA as with the EDTA-based approaches, where we dissolved the entire bone matrix. The temperature-controlled DNA extraction that we published in BioTechniques was a game changer for this project [2]. We are making use of a very simple physical principle: applying heat increases the movement of the molecules; more heat means more movement, and with more movement, we can push more DNA molecules from the bone matrix into solution. This also allows us to release DNA that is bound tightly or deeply in the bone matrix into solution without dissolving the bone.

Once you have extracted the DNA, how do you analyze it?

We use two different approaches. In the first approach, we sequence everything. For the pendant and similar artifacts, we expect to find at least two sources of DNA: the DNA of the animal that the pendant or the artifact was made from – in this case a cervid (deer) – and contaminant DNA from all the microbes that inhabited the bone after it was deposited. If we are lucky, we also find a third component, the ancient human DNA of the person who handled the object.

In the second approach, we take this DNA ‘soup’, which contains the cervid DNA, the human DNA, and the microbe DNA, and we specifically enrich the DNA of interest. If we are looking for ancient human DNA, we use so-called probes, which are artificial DNA fragments bound to magnetic beads. Since DNA is complementary, with its double-stranded structure, these single-stranded probes attract the complementary strand in our DNA. As the probes are bound to magnetic beads, we can use a magnet to collect them and wash off all the DNA that we are not interested in. In the case of the pendant, we obviously wanted to look for ancient human DNA, so we used probes containing artificial pieces of human DNA to enrich for the human fraction. This allows us to sequence the human DNA fraction in greater depth compared to what we could do with shotgun sequencing, enabling us to look at population genetic aspects or determine which biological sex the DNA comes from. It would be too costly to sequence the sequences of interest when sequencing all the DNA because the human fraction would be less than a percent.

What temperatures are animal, human, & microbe DNA extracted at?

Microbial DNA is predominantly released in the low-temperature fractions. We think this is because the microbe DNA is mostly surface bound, and it hasn't penetrated too deeply into the bone matrix. We find the animal's DNA in all the temperature fractions, which makes sense because during the life of the organism, there was DNA surrounding the bone in bodily fluids.

Interestingly, human DNA was only extracted in the very high-temperature fractions, above 90°C. As an artifact is handled, skin cells get left behind. For example, sometimes I might chew on my pen, so saliva can be deposited, or you sweat or cut yourself, leaving blood. The bone matrix is porous, it acts like a sponge, it absorbs liquids and carries the DNA with the bodily fluids into the inner parts of the bone matrix of the artifacts. When an object is handled, used a lot or worn in close contact with skin and sweat and saliva, the DNA of that person can penetrate deeply into the artifact. The DNA is then bound, sitting in the matrix, so we need high temperatures to be able to extract the DNA.

That's incredible & it's remarkable how this DNA is still there, even though it's 20,000 years old

It really is incredible! As a methods person I was very excited about the method, but I think the most exciting finding of the paper is that DNA of the person handling an osseous object can preserve over such great time periods. When we started this whole project, we knew that it could be a helpful method for non-destructively extracting DNA from objects, but the chances of finding ancient human DNA were very slim. We thought that it might not work out and we kept our expectations low to avoid disappointment, so it was really mind-blowing for us to find ancient human DNA.

Forensics people might find it less surprising as they have common techniques for extracting contact DNA. For example, they can actually extract DNA from a fingerprint. For me, it was amazing that this is still possible after 20,000 years. The DNA is still there and there's still enough for in-depth population and genetic analysis.

How long would the DNA take to break down? Could it be there for hundreds or even thousands more years & is there any way of predicting that?

This is a tricky question. I think the preservation mechanism of ancient DNA is likely to be similar for all bone objects. Independent of being an artifact of an unmodified bone or tooth DNA is bound to the bone matrix. Since the DNA preservation mechanism is the same, I think the rules are the same. Whether the DNA comes from the animal itself or from the maker or user, the preservation conditions are important. The oldest DNA that we know from bones was recovered from a 1,000,000-year-old mammoth coming from permafrost. If we found an object that was a million years old under permafrost, I would love to try our DNA extraction method on it.

You mentioned that you work with a lot of archeologists. Did they uncover any other objects that they were interested in you sampling?

We are working closely with our collaborators excavating archeological sites and we do have some samples that we are currently working on. The objects are still super rare, so if you are on a dig for, let's say four weeks, finding five or six objects would be a huge success. And finding the one that was handled intensely enough and preserved the DNA of the person handling feels like winning in the lottery.

You've already mentioned quite a few challenges when it comes to extracting DNA. Were there any other challenges that you faced?

The biggest challenge that we faced was contamination with modern human DNA. We started this project with artifacts from collections that were excavated decades ago, in the 1970s, 80s, and 90s. Back then nobody knew about ancient DNA yet and the problem of leaving modern human DNA on them. So, the objects were excavated without gloves, without any precautions to limit the introduction of modern DNA. They have been heavily studied, so people were handling them intensely.

Even worse, it used to be common to lick a sample to test whether it was bone or stone. If it's stone, the liquid saliva will stay on the surface, but if it's bone, it will penetrate. I was a bit shocked when I first heard this since this is exactly how we think the ancient DNA of the person handing this thing got into the sample, so then the modern human DNA is competing with the DNA that was absorbed 20,000 years ago. The modern DNA is perfectly preserved and there are millions of copies of it: we were just drowning in modern human contamination, so even if there was ancient human DNA, we would have a very hard time detecting it.

How did you separate the ancient human DNA from the modern human DNA?

We can use some characteristics that are typical for ancient DNA. Over time, DNA degrades and, especially on the ends, we have an accumulation of what we call C to T substitutions, whereby cytosine is converted to uracil over time, so that when we sequence the DNA, cytosine shows as a thymine.

We look at the C to T substitution patterns, we actually call it a smiley plot because if you look at your DNA fragment in the interior part, you see the normal frequency of C to T substitutions, while at the outer ends of the plot, you see these elevations, so it really looks like a smiling face. Fragments that carry these C to T substitutions at the end can be considered authentic ancient molecules. DNA fragments without these signals at the ends can be put into the modern category and disregarded.

How long does it take for that degradation to happen?

This is not easy to answer: It really depends on the preservation conditions. You can already see these patterns quite strongly in forensic samples that are only 50 years old, but then also you have samples that are several thousand years old, but come from favorable preservation conditions, such as permafrost, so the degradation rate is a lot slower. You can't put a timestamp on it, but if you see the C to T substitutions then you know you have something that underwent degradation – whether that be 50 or 20,000 years – then you have to take a careful look.

How did you confirm the DNA you found was in fact ancient?

We conducted several tests to determine the age of the DNA. First, we looked for C to T substitutions, which are a common marker of ancient DNA. We found these substitutions, which gave us confidence that we were indeed looking at ancient DNA. We then used a genetic dating approach to estimate the age of the DNA. This approach involves counting the number of mutations in the mitochondrial DNA and comparing it to the number of mutations in known DNA samples. We found that the DNA we were studying was approximately 20,000 years old. Finally, we compared the DNA to known ancient populations. We found that it closely matched two samples from the same time period and geographical area. These samples were from a region that is slightly further east of Denisova Cave, but they are still within the same geographical area.

Can you tell me about how your research interlinks with the archeologists. How are the discoveries you made with your team important in understanding the past & how ancient humans lived at that time?

As I mentioned earlier this is the first time that we have been able to directly link a historical object from Paleolithic times to a genetic profile directly. When you think about medieval times, you have a burial and you have grave goods, and you can directly associate these grave goods with the person who was buried in this grave.

It is more difficult when studying Paleolithic times. I'm not aware of a case where we have this direct burial context in Paleolithic times, so it is really tricky to make any assumptions about connections between artifacts and humans at a site. This is really the first time that we have been able to start looking into those connections.

In the case of the Denisova pendant, we were able to identify that the DNA that was left on this pendant came from a woman. Now we cannot say much from that one sample about society structure or division of labor. We would need many more samples to start seeing patterns, but maybe we could start looking into such aspects of behaviors and sociality. We might see that one type of tool contains predominantly male DNA while predominantly female DNA is on another tool.

We might be able to see how people were traveling, or how ideas were traveling. If we find a specific type of tool at one site, and then we see it 5000 years later at a different site, then we can start linking them genetically and see how these objects or ideas moved geographically and timewise.

By extracting DNA from more samples, we hope to find more ancient human DNA that allows us to open the window to the past and get a better idea about these ancient societies. It would be awesome to help shed light on aspects like the social structures. Until now, this has been tricky, if not impossible, for the Paleolithic period.

What other aspects of your research are you excited by & what are your hopes for the future of your research?

The first goal is to reproduce the method on other artifacts. We want to confirm that the method doesn't just work for one pendant from the Denisova cave but that it works systematically for samples that have been freshly excavated with precautions to limit modern human contamination.

Once we have confirmed that the Denisova cave case was not a single lucky shot, then, as a methods person, I'm really interested in digging into the technical aspects. Is DNA preserved better in one type of artifact than another? We have the DNA from a pendant, which is a personal object. Are personal objects more promising sources of ancient DNA than tools because they were worn in close body contact over many years?

Something that I would really like to look into more closely is, can we overcome the problem of modern DNA contamination? There are so many exciting artifacts sitting in collections that have been contaminated with modern DNA but I'm sure they have exciting stories to tell. I would love to improve the methods so that we can also use contaminated samples for this type of study. I'm looking forward to getting back into the lab to find some parameters that can help to overcome the problem of modern human contamination.

If you could sample any artifact in the world to extract DNA, what would you chose?

Rather than a specific object, I am more interested in objects from a particular time frame: the transitional phase where we know that Neanderthals and modern humans coexisted. There are some technological complexes where it is unclear if they were made by modern humans or Neanderthals, or maybe they lived there together. If we could extract the DNA of the people handling, making and using such objects, we can maybe contribute to answering the question of who made them.

I would love to read about that if you managed to find out more! I can see why you get so excited about all of the work that you do

I'm a methods person, so when I started working here, I wasn't terribly interested in Neanderthals or early modern humans. When I came here, it was the technical challenges that excited me and the possibilities to recover DNA that is 400,000 years old [6,7]. I find it incredible to see that with these rather technical, very chemical methods, we can then tell something about humankind. I think it's a unique place here in Leipzig, where we can really combine these two things: science and history. And as a bonus, celebrate the methods and the biochemistry behind them.

It is interesting how this sort of research has been used to overcome those challenges. Without the techniques that you spend hours developing in the lab, we might never be able to understand the history of ancient humans

I never thought that I would be excited about Neanderthals, but the team here in Leipzig changed my mind! Sometimes, we're interested in one sample so we specifically develop a method for this one sample, but then it also works on other samples, making the techniques more sensitive, helping to squeeze out the teeny tiny bits of DNA that are there.

A new method might be developed to answer one specific question for one or a handful of samples, but then it can be applied to a broader set of samples, helping us gain so much more insight into the history of humans. There is a lot of crossover between the forensics field and the ancient DNA field, so some of these methods for studying the past might help to do something good today as well.