In 1976, workers excavating a Toronto subway tunnel came across very old bones. Using radiocarbon dating, researchers determined that the partial skull and horn fragments were approximately 12,000 years old.
Anyone who has lived in Toronto can tell you that waiting over ten thousand years for a train is not unusual. What was unusual, at least to modern biologists, was that these fossils are the only known specimen from Torontoceros hypogeus, now extinct ungulate. Until recently, the way Torontoseros fit into evolutionary the story was a mystery. But in October, researchers discovered that the ungulate was a relative of the white-tailed deerThis discovery was made possible through the wonders of DNA sequencing.
But how exactly do scientists extract DNA from thousands of years old bones? Well, it takes a sterile lab, a little drilling, and a little luck.
DNA is everywhere and that could be a problem
We all live in a veritable DNA soup. Every sneeze and cough leaves pieces of ourselves floating in the air and settling on the ground, but there are also invisible bacteria and viruses around us, each with its own DNA.
As Aaron Schafer, an assistant professor at Trent University who led the Torontosequence study, explained, all that DNA floating around requires a laboratory that can be irradiated with ultraviolet light to destroy any possible contaminants. The researchers, wearing sterile “bunny suit” coveralls and N95 masks, then subject the fossils to another ultraviolet treatment, killing any viruses and bacteria stuck to the outer layer, and then scrape off that layer as another measure of sterility. The drill is then used to penetrate the bone, forming a fine powder.
“We take a powder, and you cross your fingers and hope that there are cells in that powder that contain DNA fragments,” Schafer says.
Sometimes external sources of DNA can be helpful.
While external sources of DNA may have been a challenge for Schafer's work on Torontoceros, for other researchers these viruses and bacteria may be the main target of their research. When Nicolas Rascovan, a biologist at the Pasteur Institute in Paris, examined the teeth of Napoleonic soldiers, he was not particularly concerned about the soldiers. Instead, he wanted to find out what killed them when they retreated from Russia in 1812.
In recent paperhe described how DNA sequencing of teeth showed that soldiers died of enteric and relapsing fever. In this case, they opened the teeth to gain access to the dental pulp, the soft tissue supplied by blood. From this tissue, the team then extracted the DNA of the deadly bacteria the hapless soldiers were carrying.
How to isolate DNA
Once the researchers have obtained the DNA dust, it's time to isolate it. There are still many non-DNA substances mixed into the powder, such as proteins. To study Raskovan, he used chemical reagents to dissolve the unwanted substances, leaving behind the DNA he was looking for. The solution was then mixed with silicon powder, which has a positive charge, and mixed with a centrifuge.
“DNA has a lot of negative charges,” he says. “What this means is that if you have something that has a positive charge, it can act like a magnet.” This magnetism helps the DNA strands stick to the silicon so they can be read.
Digitizing DNA using unusual machines
This physical DNA then needs to be digitized so it can be analyzed. While there are several sequencers on the market, Raskovan says the most common is Illumina's machine.
These machines already have a library of artificially created DNA molecules, called adapters, that they can recognize. These adapters, which are so small that they are measured in angstroms, or one billionth of a meter, are then mixed with the original sample. The adapters act as tags to ensure that the Illumina device can read the DNA strands to which the adapters are bound.
DNA is made up of billions of pairs of building blocks. called nucleotides. The sequencer acts as a kind of camera, photographing samples and using adapters to identify base pairs and compile them into a text file. The building blocks of life have now been converted into digital data that can be viewed, sorted, analyzed and compared.
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Ancient DNA can be difficult to work with
Although DNA sequencing currently has a wide range of applications, from criminal forensics For medical research, it can be particularly difficult to use on older samples where the DNA strands may be damaged or incomplete. Fortunately, DNA has changed relatively little over millions of years. For Schafer's research, this meant that chemical reactions could be used to fill in or repair any missing or broken parts.
“If you look at the same stretch of DNA in a fresh sample and an ancient sample, you will find certain base pairs that deviate from the ancient base,” he says. “That’s your clue that something unnatural happened to them.”
There's just one problem: the sequencer analyzes all the DNA in the sample, whether researchers are looking for it or not. For Raskovan, that means there could be DNA from the soldiers, and for Schafer, that means DNA from any microbes that ended up in the Torontoceros fossil, as well as any other sources of DNA that might have gotten into the samples while they were in the mud.
Raskovan compared the procedure to taking a bunch of books, tearing out the pages, mixing them up, and then trying to figure out the plot of one of them. Digitizing the strands means that each piece of DNA can be compared against the entire database to determine not only which pieces are actually relevant, but also how they compare to known animals, bacteria and viruses. For Raskovan, that meant comparing strands to the DNA of disease-causing bacteria such as typhoid to determine what exactly killed Napoleon's soldiers more than 200 years ago. For Schafer, this provided an opportunity to pinpoint where Torontoceros fits in the tree of life by comparing ancient DNA with that of modern animals such as deer and caribou.
Both Raskovan and Schafer admit that along with truly mind-boggling technology, there is another key ingredient to their success: dumb luck. If too much time passes or the samples are buried in conditions that are too warm or humid, the DNA they are looking for may become useless. However, the technology is constantly improving, and sequencing can be applied to more and more samples over time.
“The techniques have gotten a lot better,” Schafer says. “There's a study that has identified DNA from million-year-old fossils. If the DNA is there, if it's well preserved, we can get it now.”
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