Whatever else he was, though, Mullis was a thinker and a problem-solver. The drive from Berkeley to Anderson Valley took two and a half hours, and it was a time for him to use that gift, to focus on difficult research problems, free from distractions. On this night, he was trying to come up with a quicker genetic test to tell if someone has a disease, such as sickle-cell, tied to a single base pair in their DNA. Somehow, he got to thinking about short sequences of bases—things that Cetus scientists had gotten very good at synthesizing in the lab—and how you could use them to latch onto a part of someone’s DNA that had the complementary string of bases, a GTTCCC in the synthetic sequence, for instance, matching up with a CAAGGG in the person’s genome. From the place of attachment, you could then get the DNA to start replicating itself, creating a new piece of DNA, in the same way that DNA replicates itself when a cell divides. These were known facts, things that could be done.
Like anyone who worked with DNA, Mullis knew that getting a large enough amount of any particular stretch of the genetic material to enable one to sequence it—to read the order of As, Gs, Cs, and Ts—wasn’t easy. The standard procedure was to insert the target DNA into bacteria and then get the bacteria to multiply on petri dishes, replicating the alien DNA along with their own. It was a messy process and far more time-consuming than the sequencing itself. In other words, generating a sufficient quantity of the targeted DNA was the rate-limiting step. Mullis knew that if you could solve this problem by coming up with a simple, fast way to create lots of DNA, it would be a huge methodological breakthrough. It could make DNA sequencing easy.
Suddenly, driving along Highway 128, with the long, pale, flowering heads of the buckeyes drooping down into the beams of his headlights, his mind full of synthetic bits of DNA, he realized how it could be done. It was a classic Eureka! moment, like Wallace, in a malarial fever, flashing on the mechanism of natural selection. Maybe it was Mullis’s tendency to think outside of the box at work. He has said that his use of LSD some years before probably helped, opening up new pathways in his mind. In any case, he could hardly believe his thoughts. He pulled off the highway, grabbed a pencil and an envelope out of the glove compartment, and scribbled down a few notes. A large buckeye loomed over the little Honda. His girlfriend stirred in her sleep. There was a small white highway marker where he had pulled out, mile marker 46.58.
What Mullis had realized was that, by using two different synthetic DNA sequences that would attach to areas that weren’t too far apart on a DNA strand, you could start in motion a process that would duplicate the target DNA—the stretch between the two attachment sites—over and over again. The key was that a new strand that had begun replicating from one of the bits of synthetic DNA would, in the next round of duplication, provide an attachment site for the other bit of synthetic DNA, which would then initiate replication in the other direction. Run the process long enough, round after round of duplication, and you’d end up with astronomical numbers of the target sequence. The first thing Mullis scribbled down was just the progression of increasing amounts of target DNA with each round of duplication. Starting from one molecule, you’d have two molecules after one round, four after another round, then eight, sixteen, and thirty two. Ten rounds would give you 1,024 molecules, and up and up exponentially. It would work, he thought. By the time he had driven another mile down the highway and pulled over again, he was already thinking about his Nobel Prize.
Mullis called his new method the polymerase chain reaction—PCR for short—after the enzyme, DNA polymerase, that catalyzes replication. When he described the new technique to his coworkers at Cetus, including his girlfriend, almost none of them saw that he was onto something groundbreaking. Maybe it was partly because he was known for being a bit flaky. (Did they know about his astral guardian angel?) Mullis himself has suggested that it was also the usual resistance to new ideas, even relatively straightforward ones, that kept them from recognizing the importance of his proposed method. In any case, it was some seven months later, and only after he had mucked around in the lab and generated some promising preliminary results, that many other scientists at Cetus got excited. With others on board, some of them much more careful experimental scientists than Mullis, it was soon apparent that PCR was a viable technique.
Mullis had another big breakthrough when he realized that the process could be streamlined by using a version of the DNA polymerase, the replication enzyme, from a bacterium called Thermus aquaticus that lives in the hot springs of Yellowstone National Park. In PCR, after a short period of DNA replication, the newly formed double strands have to be heated up to break them apart again to allow the next round of replication to take place; the polymerase from T. aquaticus (or Taq, for short), unlike the kind found in most organisms, stays intact when heated, and therefore does not have to be replenished in the test tube after each heating step. Using Taq and a heating block called a thermal cycler, programmed to heat up to break the DNA strands apart and then cool down to begin each period of replication, you could just toss in your ingredients and let the thing run, and after a few hours, a handful of copies of the targeted piece of DNA would turn into millions.
5.1 Kary Mullis invented the polymerase chain reaction (PCR), and thus changed the science of biogeography, among other things. Photo by Erik Charlton.
Mullis hadn’t been suffering from delusions of grandeur when he dreamed of a Nobel Prize that night under the flowering buckeyes. He shared the Nobel in Chemistry in 1993. While he was in Stockholm for the awards ceremony, the Swedish police came to his hotel room, responding to reports of a red laser beam, like those used on rifle sights, coming from his window. Sweden has a low crime rate, but someone had been murdered in Stockholm a year or so earlier by a sniper using one of those laser sights, so that red beam was making people nervous. When the police questioned him, Mullis had to confess: he had been playing with a new laser pointer, shining it near passersby on the street below to see how they’d react. It was just Mullis being Mullis.
The transformation that PCR brought about took place very quickly and happened to coincide with the few years when I was actually getting my hands dirty (and irradiated) in a molecular lab. In 1987, as part of my dissertation research, I wanted to construct a phylogenetic tree of garter snakes, so I started working in the lab of an evolutionary biologist named Rick Harrison to get the necessary genetic data. For my first year or so in Rick’s lab, I was screwing around with something called restriction fragment analysis, a standard method at the time. The technique required a long procedure for isolating the DNA from snake mitochondria—my main memory of this step is being afraid I would destroy an extremely fast and expensive ultracentrifuge by improperly balancing the samples in it—and then using enzymes that would cut the DNA where particular sequences of bases (say, GAATTC or AAGCTT) appeared. What you ended up with, after several more steps, wasn’t anything like the full DNA sequence, but instead just bands on a gel that very roughly showed the length, in base pairs, of the cut-up pieces of DNA (called restriction fragments). The process was time-consuming and complex, and, worst of all, it often wasn’t clear how to interpret the results. I guess it was fun in a way; there was some satisfaction in working through an elaborate procedure and seeing those bands of actual DNA appear. It was a little like following a difficult recipe to make a nice soufflé. Like a soufflé, though, it was mostly empty; the technique just wasn’t going to provide enough information about the differences between species to construct a reasonable garter-snake tree.
Luckily for me, in 1985 the description of PCR had been published. Once Mullis and other scientists at Cetus had worked out all the major kinks, it was obvious that the method would be enormously valuable. Among other things, it would be used to diagnose infections and genetic diseases and to analyze tiny DNA samples from crime scenes, from bits of frozen mammoth and Neanderthal tissues, and from feathers of extinct birds lying in museum drawers. Eventually, it would make possible the seq
uencing of the entire human genome. I don’t remember thinking about any of that at the time. What I knew was that a group of scientists—in the first PCR paper, Mullis’s name was buried in the middle of a list of seven authors—had invented a method that would let someone like me, who had no desire to become immersed in molecular biology, obtain actual DNA sequences.
And it did. A bright fellow grad student named Ben Normark was the first one in the lab to learn PCR, and he taught me all I needed to know over a few days. Even the first step showed the advantage of the technique. Since PCR would selectively find the target sequence in a mitochondrial gene and multiply it, there was no need to separate the mitochondrial from the nuclear DNA.25 No more balancing samples in that damned ultracentrifuge. Now the first step was just isolating all the DNA, the mitochondrial and nuclear components mixed together, from each garter-snake tissue sample, which was a fairly trivial task. After that I’d load the DNA samples into little test tubes along with the Taq polymerase, a bunch of free-floating bases, and some other ingredients, put the tubes into the Perkin-Elmer Cetus thermal cycler, and a few hours later I’d have, relatively speaking, a gargantuan amount of the target stretch of DNA, enough to get the sequences. Even though I understood what was happening in the test tubes, it felt like magic.
Unlike grad students today, who just send their PCR samples to an automated sequencing facility, I had to do the DNA sequencing myself, and that was a pain. I especially remember some unhappy moments with the thin sequencing gels, which tended to fold up on themselves when you were transferring them onto cardboard-like filters; I tossed at least one recalcitrant gel into the trash in disgust, several days’ work down the drain. However, at the end of this messy process, if the gel was cooperative, I had a picture with bands in four rows representing the four bases, from which I could read the actual sequences of As, Gs, Cs, and Ts. This result was far more useful to me than the old restriction fragments. The differences between garter-snake species were completely unambiguous—one species would have an A at a particular site while another species would have a T—and, compared to the restriction fragment data, there were lots of differences, which meant more information that I could use to sort out the evolutionary relationships.
With each new set of sequences, I would run a “tree-finding” program on the lab computer (I didn’t have my own computer—this was really the dark ages), and out would come a network of dots and lines, the program’s best guess for the garter-snake tree. For instance, the program told me that the two slender ribbon snake species (a subset of the garter snakes) were each other’s closest relatives (an expected result), and that the highly aquatic Mexican black-bellied garter snake was part of a group of terrestrial species (somewhat surprising). I have to admit that, examining that tree now, it doesn’t look too good. Compared to the tree from a far more extensive sequencing study that some colleagues and I did more recently, also using PCR, my initial tree was quite flawed: it was wrong in a few places, and just not all that informative overall. But it was a beginning.
I ran my first PCR in 1989, four years after the method was first described in print. In hundreds of labs, the same thing was happening right about then—that’s how fast the technique took off. Suddenly, everyone seemed to be using PCR to “amplify” tiny amounts of DNA into quantities that could be sequenced. In the Harrison lab alone, there were people sequencing crickets, swallowtail butterflies, bighorn sheep, crabs, beetles, and various fishes, all because of PCR. Elsewhere, the work extended to countless other animals along with plants, fungi, protists, and bacteria.
This wasn’t the first big pulse of DNA sequencing work: molecular biologists had started obtaining such sequences routinely in the 1970s, when new sequencing methods were invented. However, those studies usually focused on species that were medically important or were for some reason especially suitable for investigating basic issues in molecular biology, such as how genes work. The study species tended to be things like Drosophila melanogaster (the common laboratory fruit fly), yeast, Escherichia coli, the house mouse, and Homo sapiens. Mullis’s invention obviously helped such research. For our purposes, though, the key point is that PCR made it possible for evolutionary biologists—people who were interested in the diversity of living things, but, typically, had limited inclinations to learn difficult molecular techniques—to get DNA sequences from their favorite groups, whether they happened to be crickets, garter snakes, or maple trees. Thus, what PCR produced was an explosion of this most fundamental and unambiguous kind of genetic data, taken from many branches throughout the tree of life.
At the time I wrote my dissertation, including a chapter on the garter-snake tree, molecular dating analyses definitely were not in vogue. This was almost thirty years after Zuckerkandl and Pauling had first described the molecular clock, and by then it was clear that the pace of genetic change could be quite different in different lineages; the clock didn’t tick at a constant rate. For instance, mammals had a fast clock, sharks an exceptionally slow one. Within mammals, the rodent clock ran especially fast. Because of this inconstancy, the clock idea was considered something of a dinosaur, an idea that had to be jettisoned once the data were in. It was a nice thought, but it didn’t pan out. For my dissertation, I never even considered using the garter-snake DNA sequences to place ages on the branching points in the tree. This attitude was about to change, though. The clock was about to make a comeback.
A BIG PILE OF BOTTLES
In the Nevada ghost town of Rhyolite, not far from a cartoonish sculpture of a miner and his penguin and a more detailed one of the robes but not the bodies of Jesus and the apostles in their Last Supper poses, sits a house made almost entirely of old bottles embedded in mortar. The house is no artistic masterpiece, but it’s striking for the sheer number of bottles involved, reportedly about 30,000 of them. It turns out that in the early 1900s, when Rhyolite was in a gold-mining boom, lumber was scarce in the town, but the place had more than fifty saloons, and thus no shortage of beer and whiskey vessels. I picture some of the townsfolk sitting around at that point, saying, “We’ve got no wood, but look at all these damned bottles. We could make a house out of these things.” I envision these would-be architects a bit less than sober, drinking beers and (somewhat) carefully piling up the empties. In any case, however it came about, the idea of a bottle house seemed reasonable enough that the folk of Rhyolite ended up building three of them, although only the one survives.
I imagine a similar beginning to the explosion of molecular dating studies. Not boomtown rats in the desert this time, but a group of evolutionary biologists sitting around in a brewpub, pint glasses in hand, saying, “Hey, look at all these damned DNA sequences. They’ve gotta be good for something. Maybe we should use them to revive that old molecular clock idea.” This isn’t really how it happened. As far as I know, there was no particular get-together that rekindled interest in the molecular clock. However, it does seem that what led to the explosion was this overwhelming resource, all of these DNA sequences from crickets and crawdads, guinea pigs and alligators, mushrooms and magnolias. Typically, the point of this sequencing had been to figure out how groups are related to each other: Are animals more closely related to plants, or to fungi? (Fungi, unequivocally.) Where do whales fit into the tree of mammals? (Right next to hippos.) But now that these sequences were available, sitting in databases like GenBank that anyone could access with the click of a mouse, the old question about time started insinuating itself into people’s minds. It was a pile of bottles too big to ignore.
For someone interested in molecular dating, the pile of sequence data, in addition to being big, was enticing in ways that other kinds of molecular data were not. For one thing, as mentioned above, sequence differences between species are very clear and countable. You can know that the cytochrome b gene in a black-necked garter snake differs at exactly 92 nucleotide positions from the same gene in a checkered garter snake. In contrast, some other measures o
f genetic distances between species are far muddier, relying on indirect and sometimes very imprecise estimates of DNA differences. For instance, consider DNA-DNA hybridization, which I remember as a cutting-edge technique in the early 1980s. This method involved mixing the genetic material from two species, say a finch and a crow, so that double-stranded DNA formed in which one strand was from one species and the matching strand was from the other species. You then heated up this “hybrid” DNA and recorded the temperature at which, say, 50 percent of it had broken apart into the original single strands from finch and crow. That temperature was a measure of genetic similarity between the two species, because the closer the base-pair match between the strands of DNA, the stronger they stuck to each other and, therefore, the higher the temperature that was needed to jostle them apart. It was a measure, but it wasn’t a very precise measure. The breaking-apart temperature depended, for instance, on properties of the strands other than just the percentage of matching bases. Moreover, even with samples from the exact same specimens, the results could vary from lab to lab, or even from one trial to the next in the same lab, which was troubling. Some other measures of genetic distance have been similarly muddy. For evolutionary biologists thinking about how they might refine studies using the molecular clock, the perfect precision of DNA sequence differences was a refreshing change. It was a bit like going from a historical record based on hieroglyphics to one that uses a true written language with a large lexicon.
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