In 1976, workers digging a tunnel for the Toronto subway discovered very old bones. Using radiocarbon dating, researchers determined that the partial skull and wood fragments were approximately 12,000 years old.
As anyone who has lived in Toronto can tell you, it’s not uncommon to wait more than a decade for a train. What was unusual, at least for today’s biologists, was that these fossils are the only known specimens of Torontoceros hypogaeus, a now extinct ungulate. Until recently, exactly how Torontoceros fit into evolutionary history remained a mystery. But in October, researchers discovered that the ungulate was a relative of the white-tailed deer, a discovery made possible by the wonders of DNA sequencing.
But how exactly do scientists extract DNA from bones that are thousands of years old? Well, it takes a sterile laboratory, some drilling and a little luck.
DNA is everywhere, and that can be a problem
We all live in a veritable soup of DNA. 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 all around us, all of which have their own DNA.
As Aaron Shafer, an associate professor at Trent University who led the Torontoceros sequencing research, explains, all that floating DNA requires setting up a lab that can be zapped with ultraviolet lights to kill any possible contaminants. Researchers wearing sterile “bunny suit” jumpsuits and N95 masks then blast the fossils again with UV, killing any viruses and bacteria clinging to the outer layer, before scraping that layer off as another measure of sterility. A drill is then used to penetrate inside the bone, generating a fine powder.
“We take the powder and you hope, fingers crossed, that there are cells in that powder that have fragments of DNA in them,” Shafer says.
Sometimes external DNA sources are useful
While outside sources of DNA may have been a problem for Shafer’s work on Torontoceros, for other researchers, these viruses and bacteria may be the focal point of their research. When Nicolas Rascovan, a biologist at the Pasteur Institute in Paris, examined the teeth in the mouths of soldiers in Napoleon’s army, he didn’t care much about the soldiers. Instead, he wanted to find out what killed them as they retreated from Russia in 1812.
In a recent article, he explained how DNA sequencing of the teeth revealed that the soldiers died of enteric and relapsing fever. In this case, they opened the teeth to access the dental pulp, the soft tissues supplied with blood. From this tissue, the team then extracted the DNA of the deadly bacteria carried by the unfortunate soldiers.
How to isolate DNA
Once researchers have collected their DNA dust, it’s time to isolate it. There are still a lot of things that aren’t DNA, like proteins, mixed in with the powder. For Rascovan’s research, he used chemical reagents to dissolve unwanted elements, while 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.
“A strand of DNA has multiple negative charges,” he explains. “That means if you have something that has a positive charge, it can work like a magnet.” This magnetism helps the DNA strands stick to the silicon so that the strands can then be read.
Scanning DNA with sophisticated machines
Then this physical DNA must be digitized so that it can be analyzed. Although there are several sequencers on the market, Rascovan says the most common is a machine from a company called Illumina.
These machines already have a library of artificially made DNA molecules, called adapters, that they can recognize. These adapters, so small that they are measured in angstroms, or a billionth of a meter, are then mixed with the original sample. The adapters act as tags to ensure that the Illumina machine can read the DNA strands that the adapters bind to.
DNA is made up of billions of pairs of building blocks called nucleotides. The sequencer acts as a kind of camera, taking photos of the samples and using the adapters to identify the base pairs and compiling 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 use
Although DNA sequencing now has a wide range of uses, from forensics to medical research, it can be particularly difficult to use for older samples, where the DNA strands may be damaged or incomplete. Fortunately, DNA has changed relatively little over millions of years. For Shafer’s research, this meant it was possible to use chemical reactions to fill in or repair missing or broken parts.
“​​If you look at the same region of DNA in a fresh sample and an old sample, you will find that some base pairs move apart in the old base,” he says. “This is a sign that something has happened to them that is not natural.”
There’s just one problem: The sequencer analyzes all the DNA in the sample, whether that’s what the researchers are looking for or not. For Rascovan, that means there could be DNA from the soldiers, and for Shafer, DNA from whatever microbes got into the Torontoceros fossil, as well as any other sources of DNA that might have gotten into the samples during their time in the earth.
Rascovan likened the procedure to taking a bunch of books, tearing out the pages, shuffling them all, and then trying to figure out the plot of one of them. Strand scanning means that each DNA fragment can be compared against an entire database, to determine not only which fragments are actually relevant, but also how they compare to known animals, bacteria and viruses. For Rascovan, that meant comparing the strands to the DNA of disease-causing bacteria like typhus to determine what caused the deaths of Napoleon’s soldiers more than 200 years ago. For Shafer, this gave him the ability to determine exactly where Torontoceros fit in the tree of life by comparing ancient DNA to contemporary animals like deer and caribou.
Rascovan and Shafer recognize that in addition to truly mind-blowing technology, there is another key ingredient to their success: luck. If too much time passes or samples are buried in conditions that are too hot or humid, the DNA being sought could degrade and become useless. However, the technology is continually improving and sequencing could be applied to more and more samples over time.
“The techniques have gotten a lot better,” Shafer says. “A study identified DNA from million-year-old fossils. If the DNA is there, if it’s well preserved, we can recover it now.”
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