THAT TIME FORGOT
Researchers use genetics to detect what's in water
By HOLLY FOX
It is possible to detect mutations, diagnose diseases and infections, determine the sex of an unborn child or get a genetic fingerprint of a crime suspect from evidence collected at the scene — all from a single cell. The technology that makes this possible — the polymerase chain reaction — is being used today by UM researchers in their conservation efforts.
In the laboratories of UM's Division of Biological Sciences, and in streams in western Montana and Alaska, UM's researchers are applying the principles of genetics to the problems of conservation. Most of their research deals with salmon and trout, although they also work with grizzly bears, bighorn sheep and plants.
Sidebar: Of fish & human fertility
Research Specialist Kathy Knudsen has been studying fish for more than 20 years. Her latest project, funded by a grant from the National Science Foundation, has been to determine whether techniques used in human forensics and conservation genetics can be used to determine what species of fish are present in a stream from a small sample of stream water.
"People are familiar with the polymerase chain reaction (PCR) technique used in human forensics because it is the technique that allows researchers to determine who a small sample of blood or semen came from in crime investigations," Knudsen says. "Our goal was to see if we could use that technique on a sample of stream water to determine which fish species are present."
PCR was invented in 1988 by Kary Mullis, who used a DNA polymerase enzyme from the heat-stable organism Thermus aquaticus or "Taq," which he obtained from the hot springs of Yellow-stone National Park in Montana and Wyoming. Starting with small quantities of DNA — as small as one target molecule — PCR can generate millions of copies of a specific target DNA sequence in about three hours.
First, the double-stranded DNA sample is denatured, made into a single strand, by heating it. The sample is cooled to allow two primers to connect to the regions on either side of the target sequence. The primers flag the beginning and end of the DNA sequence to be copied. The sample is then reheated slightly to allow the enzyme "Taq" polymerase to read the code of the desired sequence and build a copy, creating two double-stranded copies of the original DNA sample. The process is repeated 30 to 40 times until the millions of copies necessary for analysis of the DNA sequence variation have been created.
The technique itself is nothing new, Knudsen says, the research is just starting from a different point.
"We've been doing this technique with fish for several years," she says. "But instead of starting with a small piece of fin or a piece of liver, we're starting with some water that we hope has fish cells in it."
To get the cells from the water samples, Knudsen designed a simple sampling apparatus consisting of a vacuum flask with a filter holder above it and a small hand pump. The entire contraption is the size of a small cooler, and thus, easily transported. All a researcher needs to do is scoop up some water, which gets sucked through the filter with the pump. The water is discarded and the filter, which contains the cells, is stored in a vial filled with alcohol until it reaches the lab.
In the lab the DNA is extracted from the cells in the filter. PCR is used to make enough copies of the DNA so that researchers can look for certain species-specific fragments. If a bull trout-specific band shows up, for example, researchers know that bull trout live in the stream where they took the sample.
Knudsen says this new method of inventorying streams is a big improvement over the traditional method, electrofishing.
"In electrofishing researchers use electrodes to stun the fish so that they can scoop them up and identify them," she says. "That process is still useful and effective, but it's very labor-intensive and a lot of streams are completely inaccessible, especially with all the equipment that is needed, in addition to the crew of people necessary. Also, in electrofishing, the researchers need to be very close to the fish — the fish have to swim between the electrodes. With our new technique, you just have to get a sample of the water downstream from the fish."
The research on the new inventorying technique began in Division of Biological Sciences' fish tanks. "We started there because we knew, with the density of the fish in the tanks, there would be a lot of cells in the water," Knudsen says. "It worked there, so we tested it in Rattlesnake Creek, where we knew there was a large population of fish, and it worked there, too."
The research continues in streams all over Montana, as well as in Alaska, including many waterways that would be difficult to access for researchers who want to electrofish.
There are still some questions left to answer, Knudsen says. "We need to do a lot more research to determine how many fish need to be in the stream before this technique will work, or how close we need to be to the fish to pick up their cells. We haven't determined the sensitivity of this method yet."
Regardless, research thus far has yielded some interesting results that point to the importance of this technique.
"It's really clear that where fish populations are doing well are places where there are no roads," says UM Professor Fred Allendorf, who works with Knudsen on this project. "It's obvious from our research that roads have a major harmful effect on fish populations, so it is really important to have a way of accessing and inventorying roadless areas."
The technique is also hugely important in conservation efforts, Knudsen says. "Two of the main questions you have to answer in conservation are what species do we have and where are they living? The great thing about this technique is that it can provide that information with much less effort and field equipment than electrofishing, which means you can sample streams that would otherwise be inaccessible."