Deciphering Molecular Mysteries

By Miyoko Chu

In Michael Crichton’s Jurassic Park, scientists extract dinosaur DNA from a fossilized mosquito that had fed on dinosaur blood. Fiction, yes. But if you walk into the Cornell Lab of Ornithology’s evolutionary biology laboratory, you won’t be disappointed by what biologists are really learning by consulting the DNA of animals.

On the surface, the setup looks fairly simple. Inside the laboratory are three long black tables called lab benches—the fireproof kind you’ll find in any college chemistry lab. Along the benches, undergraduates, graduate students, and Ph.D. biologists sit hunched over small trays filled with tiny plastic tubes. They’re in various stages of labeling them, or adding liquids using a micropipet—a slimmed-down, high-tech version of a turkey baster. The mysterious thing is that you can’t see any sign of the animals they’re studying—warblers, swallows, House Finches, todies from Hispaniola, Gray-headed Social-Weavers from East Africa, and even vampire bats from Latin America. The DNA from all of these animals is somewhere in the little tubes, as are the answers to a multitude of questions about social behavior, disease, conservation, and evolution.

Graduate student Rebecca Safran and undergraduate Colby Neuman sit at one of the lab benches with 48 small tubes in front of them. The contents of these tubes may answer an intriguing question about the lives of Barn Swallows: How do female Barn Swallows choose their mates? One of a Barn Swallow’s most conspicuous features is its long forked tail, and researchers in Europe have found that the males with the longest tails sire the most young. But Safran’s work shows that female Barn Swallows here in Ithaca, New York, seem to have an even greater preference for males who sport the most brightly colored ventral feathers. Using genetic analyses of blood samples to determine paternity in 70 Barn Swallow families, she and Neuman will soon find out whether males with darker feathers were cuckolded by their more brightly colored neighbors.

Photo credit: Jon Reis

Valentina Ferretti uses DNA to determine Tree Swallow paternity as Evolutionary Biology director Irby Lovette looks on.

In principle, the process of isolating DNA from blood is not much different from panning for gold. But instead of metal pans and sluice boxes, Safran and Neuman use 48 tiny columns, each with a piece of white filter paper in the bottom to catch the DNA. Using a micropipet, they add a miniscule amount of the blood sample to each filter—50 microliters to be exact (nearly 300 drops of this size would be required to fill a tablespoon). They add a series of solutions to trap the DNA on the filter while letting unwanted materials pass through. It takes a measure of faith, working with the invisible. Fortunately, their samples have so much DNA in them that, unlike panning for gold, virtually every sample is likely to yield what they’re looking for.

On the other side of the room, graduate student Valentina Ferretti carefully holds what looks like a flat square of transparent Jell-O on a paper towel. She’s already done her panning for Tree Swallow DNA and is about to see whether she actually got any. Half an hour earlier, she injected a bit of each sample into the gel and ran an electric current through it to pull any DNA down its length. A special stain will light up any DNA present, making it glow under ultraviolet light. Ferretti pops the gel into a box, a mini-darkroom hooked up to a digital camera and computer. Thin bands show up on the computer screen where the DNA is glowing in the gel. “Yes, there is enough,” Ferretti says with a relieved smile.

The next step is to use the DNA in a paternity analysis of Tree Swallows which, like Barn Swallows, are prone to mating with more than one partner. Though Tree Swallows are socially monogamous, researchers have found that as many as 80 percent of nests have at least one nestling sired by a male other than its social father, an extremely high incidence for any bird species. By analyzing DNA samples from Tree Swallows in the United States, Mangrove Swallows in Belize, and White-rumped Swallows in Argentina over the next few years, Ferretti will be sorting out why promiscuous behaviors vary, and the underlying ecological and social factors that contribute to this variability.

Meanwhile, undergraduate Megan Szymanski at the adjacent lab bench has already confirmed that her extractions of DNA from nine warbler species worked. Her next task is, in theory, no different from looking for dinosaur DNA in a mosquito. Though it’s the warblers she sampled, she’s really after the DNA of microscopic parasites that live in the warblers’ blood cells and cause avian malaria. In real life, extracting dinosaur DNA doesn’t work, because none is left after millions of years of degradation. But all Szymanski needs is a single fragment of parasite DNA in her sample, and she’ll be able to amass billions of copies in two hours, using a polymerase chain reaction (PCR) machine.

Photo credit: Irby Lovette

Cornell undergraduate Amy Dabrowski holds a Blue-winged Warbler she netted in Ithaca, New York. She will extract DNA from a sample of blood to study the genetic consequences of hybridization between Blue-winged and Golden-winged warblers.

The world’s most phenomenal copying machine is unremarkable in appearance—a box that superficially resembles a CD player with a capsule for inserting 96 tiny tubes instead of a disc. “In the little tubes we’ve put all the biological machinery necessary to make DNA,” says Irby Lovette, director of the Lab’s Evolutionary Biology program. “We put in the raw building blocks of DNA, the enzyme that links the building blocks into the DNA chain, and a couple of pieces that direct everything to happen on the segment of DNA we want.” The machine does nothing fancier than heat and cool this cocktail, a process that will create two copies, then four, then sixteen, and so on into the billions, until there is enough DNA for Szymanski to use.

This process allows Szymanski not only to detect whether the pathogens are present, but to provide enough DNA so that she can sequence their genes and identify different strains. In a pilot study last year, she found that 11 of 19 warblers were infected with three different strains of avian malaria. This year she hopes to gain a better understanding of how parasite strains have evolved in various warbler species over time.

Sequencing DNA is an impressive feat; it means being able to read an organism’s genetic code, one unit, or nucleotide base, at a time. If you could sequence the entire genome, as scientists have done for humans and other organisms, you’d have the genetic instructions needed to build an entire organism. But even a fragment of the genome, akin to a few sentences in a 1,000-page volume, contains enough information to sort out whether a particular male is the genetic father of the young it is raising, whether different populations of birds are genetically distinct, and how different species are related to one another.

Photo credit: Jon Reis

Researcher Helen Markland loads the DNA sequencing machine with samples from Vitelline Warblers in the Caribbean. She’ll compare DNA sequences to determine whether populations on different islands are unique.

Helen Markland stands in front of the machine that will read a 600-base-pair section of DNA from Vitelline Warblers, which are endemic to only a few remote islands in the West Indies. By studying their genetic variation, Markland hopes to find the answers to important conservation-related questions, including whether each island’s population is genetically unique and in need of protection. She’s already isolated the DNA, used the PCR machine to make several copies, and prepared her samples for the automated sequencing machine. At this point, the process is deceptively simple: she enters the sample numbers on a computer, puts her tubes onto a tray in the machine, and hits “Go.” The machine does the rest of the work, pulling her sample into tiny glass capillary tubes and running a laser beam over the sample to identify different base pairs, labeled with colored fluorescent dyes.

In a few hours, the machine is finished, and Markland transfers her data file to the laboratory’s computer room. “I still can’t believe that I put all those clear liquids into the machine and it came out with this,” Markland says. There on the computer screen is a beautiful sight: wavy lines representing nucleotide bases that were detected, and the genetic sequence for each Vitelline Warbler she sampled, with the letters A, C, G, and T representing each kind of base. When the computer lines up the sequences from the different birds, it shows that most are identical. But in a portion of the segment, one bird’s DNA has a G in place of an A. “This bird’s DNA sequence is different,” Markland says, pointing to the anomalous letter. And it’s from a different island than the others. That’s the kind of pattern I’m looking for.”

The DNA sequences that Markland and others find will be sent over the Internet to GenBank, an international DNA database maintained by the National Institutes of Health. They’ll be accessible to anyone who wants to use the sequences for other studies. Over the years, Lovette has contributed thousands of sequences to GenBank as part of his work in studying the evolutionary history of warblers and in sorting out how different species are related to one another. The new evolutionary biology lab has only been up and running for four months, but the young biologists in his crew are well on their way toward contributing to bird study and molecular biology in general.

Dozens of people use the Lab of Ornithology’s DNA laboratory. Some, like Markland, are exploring population genetics to inform conservation decisions about rapidly declining
Golden-winged Warblers and federally threatened Florida Scrub-Jays. Others, like Safran and Ferretti, are using DNA to understand the complex mating behaviors of birds. Researcher Sue McRae is studying the family lives of Gray-headed Social-Weavers, using DNA to sort out relationships among kin. Graduate student Dana Hawley is investigating whether the lack of genetic variation in House Finches in the East makes them particularly susceptible to mycoplasmal conjunctivitis, a deadly eye disease. Still others aren’t studying birds at all; undergraduate Gerald Carter is trying to isolate bird DNA from the stomachs of vampire bats to get a better understanding of what bats eat.

Remarkably, most of those who work here do not have any previous experience in molecular biology. “One of the beautiful things about our DNA analyses is that they all use the same tool box,” says Lovette. “You can train people in very basic skills that are universally applicable to all of molecular biology, whether they want to continue in ornithology or become cancer researchers.”

“My background is in field biology,” says Ferretti. “Before I came here, I never thought I could work with DNA. Now I’m learning the molecular techniques, and I’m enjoying it. It opens up so many possibilities.”