In the early 1990s, the field of ancient DNA was just gaining traction as a serious scientific endeavor. DNA sequences had been recovered from a 170-year-old quagga (an extinct relative of the zebra), from human mummies that were thousands of years old, and from Neandertals and mammoths that were more than thirty thousand years old. Researchers were only beginning to appreciate what this ancient DNA could reveal.
The first applications of ancient DNA were taxonomic: to identify the living species that are the closest evolutionary relatives of extinct species. We now know, for example, that Asian elephants are more closely related to mammoths than are African elephants and that the closest living relative of the dodo is the ornate and beautiful Nicobar pigeon. Some of the taxonomic results from the analysis of ancient DNA have been surprising. In New Zealand, three different species of giant moa (Dinornis) had been described based on differences in the size of their bones. Ancient DNA isolated from these bones showed that, in fact, only one species of giant moa existed on each island. Size, in this case, had nothing to do with taxonomy; the biggest bones were all from female moa and the smaller bones were from males.
As techniques for isolating ancient DNA improved, the field progressed from asking taxonomic questions to asking more detailed questions about the evolutionary history of populations. DNA sequences could reveal cryptic patterns of local extinctions and long-distance dispersals that were invisible in the fossil record. For example, horses—the same species that humans would eventually domesticate—have been around as a distinct taxonomic lineage for at least a million years. Horses originated in North America and dispersed into Asia across the Bering land bridge, which connected the two continents intermittently during the Pleistocene ice ages. Throughout this period, horses dispersed between North America and Asia several times and in both directions, each time establishing new populations and/or hybridizing with populations that already existed. One might even consider the reestablishment of horses in North America by European colonists as the latest in a long history of local extinctions, dispersals, and recolonizations. Feral horses in North America represent, in essence, an unintentional experiment in rewilding that has been extremely successful.
Ancient DNA can identify genes for traits that don’t exist anymore, such as mammoth-specific hemoglobin, which makes red blood cells that excel at carrying oxygen around large bodies when it is very cold outside. Ancient DNA can also reveal precisely which genetic changes differentiate humans from Neandertals. To summarize, ancient DNA has turned out to be a very powerful technique for learning about the evolutionary processes that shaped existing biodiversity.
The research group that was leading discovery in ancient DNA during the late 1980s and early 1990s was Allan Wilson’s Extinct DNA Study Group at the University of California at Berkeley. This group of scientists was pioneering the development of protocols to recover fragments of DNA from the remains of dead organisms and, importantly, to distinguish authentic ancient DNA from contaminant DNA.
The science fiction potential for ancient DNA was very quick to catch on. In fact, Michael Crichton acknowledges the Extinct DNA Study Group as part of his inspiration for Jurassic Park. And, not long after the 1990 book, science fiction appeared to become scientific fact: several groups (but not the UC Berkeley group) reported sequencing DNA from stingless bees, honeybees, termites, and wood gnats that were tens of millions of years old and even a 120-million-year-old weevil. All of these sequences were generated by extracting ancient DNA from the bodies of insects preserved in amber.
It was too good to be true. In 2013, a team of scientists from the University of Manchester in England performed an experiment to see whether it is possible to extract DNA from bees preserved in copal. Copal, remember, is the precursor to amber and is not entirely fossilized. Copal is therefore much younger than amber. The Manchester team extracted DNA from two copal pieces that contained bees. One of the pieces was around 10,000 years old, and the other was less than sixty years old. They extracted DNA using the latest sample-preparation and DNA extraction techniques. In the end, however, they got nothing—just as we had gotten nothing from our 17-million-year-old piece of amber. They even got nothing from the copal specimen that was less than sixty years old.
This Manchester experiment was the second time that scientists tried to extract ancient DNA from copal-preserved bees. In 1997, a team of researchers from the Natural History Museum of London attempted to repeat—and therefore validate—the fantastical results of the early 1990s. These scientists gathered together a variety of amber and copal pieces from their museum’s collection and attempted to extract and sequence ancient insect DNA. They also failed to recover any authentic ancient insect DNA.
The absence of results is always challenging to interpret. It is possible that, if one were to generate more and more sequence data, a result might eventually manifest. However, the weight of evidence suggests that ancient DNA is not preserved in amber. Not much is known about what happens to insects once they become trapped in tree resin. Although they probably become quickly dehydrated, which is good for DNA preservation, other characteristics of amber make it an unlikely source of well-preserved DNA. Amber is permeable to gases and some liquids, for example, which means that the DNA may not be entirely isolated from the forces that destroy DNA over time. Also, fossilized amber might be subjected to very hot or high-pressure conditions over the course of its lifetime, both of which are terrible for DNA survival.
The failure to replicate these early experiments proves that DNA is not preserved in amber. What was it, then, that these researchers had been able to sequence in the early 1990s?
GETTING DNA FROM FOSSILS WHEN NO DNA IS PRESERVED
Insects are the most likely source of the insect DNA that was recovered from ancient amber in the early 1990s. Insects that are alive in the present day, that is.
Although I left this out above, the researchers at the Natural History Museum in London were sometimes able to isolate insect DNA from the amber from their collections. In fact, it is precisely this result that led them to conclude that amber was not a source of ancient DNA. In designing their experiment, they selected some pieces of amber that contained encapsulated insects and other pieces that did not. This provided a control: if the DNA was from the amber-preserved insects, then the amber with no insects should have no insect DNA. Their results did not support this hypothesis. They were equally likely to recover insect DNA from pieces of amber that contained insects as they were to recover insect DNA from pieces with nothing in them. The insect DNA must have been coming from some source other than the animals preserved within the amber.
This result points to a key challenge of working with ancient DNA. In order to recover DNA from specimens that have very little preserved DNA in them, one needs a very sensitive and powerful method for recovering DNA. But, the more sensitive and powerful the method is, the more likely it is to produce spurious results.
In these experiments, researchers were using a technique called PCR—the polymerase chain reaction—to amplify insect DNA (figure 6). PCR was developed in 1983 by Kary Mullis, who, at the time, was working as a biochemist for a company called Cetus Corporation. DNA-sequencing technologies were making it possible to learn the exact sequence of a fragment of DNA. However, to do so, these technologies required millions of clonal copies of the target fragment. Before PCR, this was achieved by enticing bacteria to capture random fragments of DNA within their genomes. These bacteria were then grown into colonies in which each bacterial cell contained an identical copy of the randomly incorporated DNA fragment: enough copies to sequence. PCR provided a much quicker way to copy DNA and, more importantly, a way to target specific parts of the genome to copy. PCR is now one of the most widely used and essential techniques in molecular biology.
Given its revolutionary implications, PCR is surprisingly simple. To walk through the process, imagine that we want to better understand the genetic differences between domestic and wild chickens. A gene th
yroid-stimulating hormone receptor, or TSHR, is thought to have played an important role in chicken domestication by making chickens reproduce more quickly. We propose to use PCR to amplify—make copies of—this gene from DNA extracts of both domestic chickens and from the preserved remains of ancient chickens that lived prior to the time that chickens were domesticated. We will then sequence the results of the PCRs to learn the sequence of this gene and determine whether domestic chickens have different versions than do their wild relatives and pre-domestic ancestors.
Figure 6. The polymerase chain reaction, or PCR. PCR is a common technique in molecular biology that is used to make billions of copies of a DNA sequence by repeatedly heating and cooling DNA in the presence of a DNA-copying enzyme, free nucleotides to build the copied DNA sequences, and DNA primers, which locate the part of the genome to be copied.
First, we need to have some way to target TSHR. We do this by designing two short DNA probes called primers, which will match the sequences of DNA that flank the ends of TSHR. We then make a mixture that contains these primers, the chicken DNA that we already extracted, free nucleotide bases, and a polymerase, which is an enzyme whose job it is to copy DNA. Then we can begin the copying process. We heat the mixture to break the hydrogen bonds that hold together the two strands of DNA. When everything is single-stranded, we cool it back down, which causes the strands to come back together. Because the primers are short and there are a lot of them in the mixture, the first thing that happens is that the two primers find precisely the places in the genome that they were designed to match—the regions flanking TSHR—and form double-stranded DNA with that part of the chicken genome. Finally, the polymerase fills in the missing sequence—the TSHR gene—between the primers, using the single-stranded DNA sequence as a template and the free nucleotides to fill in the missing sequence. When this is complete, the number of copies of TSHR has doubled. To make enough copies to sequence, we repeat the process thirty or forty times over the course of a few hours, eventually generating trillions of identical copies of TSHR.
PCR is extremely sensitive. Theoretically, PCR will work if only one copy of the target DNA sequence exists in the mixture of extracted DNA. On one hand, this is great news for ancient DNA, where very little DNA is expected to have survived. On the other hand, this is a recipe for potential disaster. If DNA can be PCR-amplified from only one fragment of DNA, then it takes only one fragment of contaminating DNA to ruin the experiment. Given this very high sensitivity to contamination, exceptional results, such as DNA from insects preserved in amber millions of years ago, require exceptional proof of authenticity. At the very least, the result should be able to be replicated. In the chicken experiment I described above, identical experiments were performed in ancient DNA laboratories at Durham University in the United Kingdom and at Uppsala University in Sweden. These identical experiments provided identical sequencing results from ancient chicken remains and, therefore, confirmed that the results were real and not due to contamination.
The main source of contamination in ancient DNA research is DNA from organisms that are alive in the present day. DNA is everywhere. It is on the glassware that is used in the lab. It is in the reagents and solutions that are used to extract DNA. It is on the laboratory benches and the floors and walls and ceilings. It is floating in the air in the labs and hallways. Even more problematically, this contaminating modern DNA is in great physical and chemical condition. Whereas pieces of ancient DNA tend to be broken into very small fragments, mostly of fewer than 100 base-pairs strung together (think “cat,” “ant,” “bug”), DNA from living organisms can be millions of base-pairs long (think “supercalifragilisticexpialidocious”). Ancient DNA is also broken. Ancient DNA fragments are often missing bases or have bases that are chemically damaged (“cyt,” “^nt,” “bg”). The polymerase enzymes used in PCR have trouble reading through these damaged sites and end up making mistakes when they copy the sequence (“cut,” “int,” “bog”). Further complicating things, ancient DNA fragments are frequently chemically linked to other pieces of DNA that are present in the DNA extract, forming knotted molecular structures that the polymerase doesn’t recognize as DNA. Because of these problems, the polymerase will preferentially find and make copies of clean, undamaged, freely floating, unbroken, contaminating DNA rather than broken, chemically linked, damaged, ancient DNA. In fact, a single intact fragment of DNA from a living organism can potentially outcompete many hundreds of damaged fragments of ancient DNA during PCR, leading to what looks like, but is not, a real sequence of ancient DNA isolated from a piece of amber. Or from a mammoth bone.
Contamination is not just an idle threat. Contamination comes in many forms, and has played an important role in shaping ancient DNA research. The first and only DNA sequences reported from dinosaurs were (no shock here) contaminants. In fact, many of them were human DNA sequences. Because no one believed dinosaurs were more closely related to mammals than they were to birds or reptiles, and almost nobody believed DNA could be preserved in these very old dinosaur fossils (which are, after all, rocks rather than bones), this result was easily recognized as contamination and rejected.
Contamination is often sneakier, however, and this is when it is most dangerous. DNA from modern pigeons (rock doves, the kind that eat leftover fast food and discarded cigarette butts in city centers around the world) somehow contaminated my very first ancient DNA research project, which was to sequence mitochondrial DNA—a type of DNA that is inherited only down the maternal line (figure 7)—from the dodo. The dodo is, as I mentioned, a pigeon, and I was lucky to have spotted the contamination before writing up my conclusions. In this case, spotting the contamination was pretty straightforward. Whereas most of my experiments failed to produce any DNA at all, one particular experiment produced a huge amount of very high quality DNA. This was a dead giveaway that it wasn’t real. I’m still not sure where the contamination came from, but, after that, I started leaving my shoes outside of the ancient DNA lab rather than just covering them up.
Figure 7. Two sources of DNA in our cells. In humans and other eukaryotic animals, each cell contains two types of genome. The nuclear genome, which includes both the autosomes and sex chromosomes, is found in the cell’s nucleus. The mitochondrial genome is found in the mitochondria, which are organelles in the cell cytoplasm. In most eukaryotic animals, mitochondria are inherited only along the maternal line.
In my experience and that of many of my friends and colleagues, there are particular contaminants that pop up from time to time no matter how clean the lab is. DNA sequences of domestic animals and house mice are pretty commonly observed contaminants. This is probably because most of our experiments are designed to amplify DNA from mammals, which, of course, these animals are. Contamination is something we have learned to live with, to expect, and to look for. Because of contamination, we as ancient DNA scientists have developed a healthy wariness of our own data and have set high standards of proof for the authenticity of our results.
This hopefully goes some way to explain the elaborate outfits we wear every time we enter the ancient DNA lab. We are not protecting ourselves from what genetic terrors might be preserved in fossils. Instead, we are protecting whatever DNA might actually be preserved in these fossils from ourselves.
Of course, no matter how careful we are not to contaminate our mammoth bones with our own or any other sources of high-quality DNA, we will likely never find a bone that contains only mammoth DNA. In fact, most of the DNA recovered from a randomly selected mammoth bone will be microbial. Which brings me to our next challenge.
THE SURPRISING DIVERSITY OF DNA IN FOSSILS
Let us assume we find a mammoth bone in Siberia and we want to extract DNA from that bone so that we can sequence the mammoth’s genome. First, we have to protect the bone from contamination. That means we don’t ever touch the bone with our bare hands, because DNA on our hands will get on the surface of the bone, and some of that will be absorbed into the surface layers. We a
lso don’t breathe on the bone, stick it in a bag that hasn’t been sterilized, or allow it to touch other bones. So, we wear gloves and facemasks and hairnets, and we store every sample separately. When we remove a chunk from the sample to take back to the lab (plate 5), we use sterile cutting instruments, work on sterile cutting surfaces, and clean everything with bleach between samples.
When we return to the lab from the field, we do not remove the sample from the sterile bag unless we are in the ancient DNA lab. There, while wearing our sterile and attractive ancient DNA outfits, we smash the bone into powder using sterile smashing equipment and perform DNA extraction using sterile solutions and sterile lab equipment. After we finish the DNA extraction, we have reduced a chunk of mammoth bone to the contents of a tiny, clear tube: an even tinier amount of liquid that looks indistinguishable from water. In that liquid, we should have mammoth DNA.
And bacterial DNA.
And fungal DNA.
And insect and plant and mouse and dog and human and other DNA.
These non-mammoth DNA sequences are, however, not contaminants. More accurately, they are not contaminants in the same sense that my DNA in that sample would be considered a contaminant. The non-mammoth DNA fragments in our DNA extract most likely got into the bone before it was excavated—sometime between when the mammoth died and when we dug up its bone. Bacteria living in the soil, fungi, insects, and plants are all organisms that colonize or grow around bone while it is decaying. Water percolating through the soil will also carry DNA, and this DNA will get into the bone. Even urine carries DNA. A few years ago we showed that sheep DNA can be recovered easily from the same layers of New Zealand soil in which moa DNA is abundant, even though sheep were not introduced into New Zealand until hundreds of years after moa went extinct. A lot of sheep live in New Zealand today. A lot of sheep produce a lot of sheep urine, which leeches through the soil to the deeper layers, commingling with the moa DNA.
How to Clone a Mammoth Page 7