wf_scat

1. Wolves deposit scat along hiking trails and at sites where they have killed moose. We look for these scats and collect samples of that which we find. We store the scat in tubes with alcohol until it can be analyzed in the lab. Each season we collect samples from several hundred different scats. By collecting so many scats, we just about guarantee collecting a scat sample from every wolf in the population.

From wolf scat to “DNA fingerprint”...

What is DNA?

2. In the lab, the scat samples are prepared by a series of chemical reactions that isolate the DNA from all the other material in the scat. This is called DNA extraction. Each scat’s DNA is held in a small vial. The actual amount of DNA is minute.

While moose scat tells us about moose diets, wolf scat tells us a completely different kind of story.

By combining the humble job of collecting wolf scat with the most sophisticated biochemical and statistical techniques, we use microsatellite DNA fingerprints to individually identify each wolf in the population, know when it is born, know when it dies, and know which pack territories it visits. We also use this genetic information to learn about the population’s genetic diversity.

3. DNA is a fragile molecule. These vials contain fragments of DNA from the wolf’s entire genome. For our purposes, we need to know about the DNA from certain loci, or locations within the genome.

The next step is to conduct a chemical reaction that produces many copies of only the DNA in which we have an interest. The chemical reactions are called polymerase chain reaction or PCR and this process is called DNA amplification.

With few exceptions, every cell in every organism contains DNA. DNA is a very long molecule that looks like a twisted ladder or, more precisely, double helix. The sides of the ladder are sugar phosphates. More importantly for our purposes, the steps of the ladder are pairs of nucleotides. The nucleotides come in one of four versions. We can call the four versions A, T, G, and C. The exact sequence of A’s, T’s, G’s and C’s is different for every individual - except clones and identical twins. The sequence of these nucleotides represents encoded information. Some of the information can be used to make proteins.

At a very fundamental level, all differences among all species and all differences among all individuals (of the same species) arise from differences in the kinds and relative amounts of proteins produced by an organism’s cells. These protein differences are just differences in DNA.

4. The next step is called DNA separation and visualization. Before getting to that, we need a little background...

The DNA that we amplify is called microsatellite-DNA. Each wolf has many microsatellite loci (places in the genome where one finds microsatellite-DNA). Microsatellite-DNA is a special kind of DNA that does not code for protein. Its purpose is not entirely understood, but its structure is simple. Microsatellite-DNA is typically just a pair of nucleotides repeated over and over again. For example, a common type of microsatellite-DNA is CACACACACA. This segment of DNA could be abbreviated CA-5, because the CA is repeated five times.

For any microsatellite-locus, a wolf inherits some of the microsatellite-DNA its mother had at that locus and some of microsatellite-DNA that its father had at that locus. So, at each microsatellite-locus each wolf has two copies of the microsatellite-DNA. These copies may be identical (e.g., both may be CA-5), or perhaps different (e.g., one copy could be CA-5 and the other copy could be CA-8).

Keeping this background information in mind, recall that we have each wolf’s DNA in separate tiny vials. The next step is to figure out which versions of the microsatellite-DNA each wolf has. To do this, we place a tiny bit of DNA into a machine that “pushes” the DNA through a gel that is contained in a tiny tube (the gel looks a bit like unflavored jello). The machine pushes the DNA with an electrical current. The electrical current pushes the different lengths of DNA different distances along the gel: smaller bits of DNA get pushed further and larger bits are pushed a lesser distance. That is, the DNA gets pushed along the tube according to the number of repeats it contained. The machine produces a graph (see below) showing how far along the DNA got pushed.

From DNA fingerprint to ecological knowledge...

You may find this next step difficult to understand. Give it a try; if you find it too difficult, skip to step 5.

These two graphs show the results for two different scat samples at the same locus. They represent different sets of alleles, the two scats must have come from different wolves.

For each wolf, we examine DNA at about 15 different m-loci. In this way, we are quite sure that two samples, should they have exactly the same genotype at all 15 loci are almost certainly the same wolf.

These results are also from different scat samples at the same locus. However, the samples have the same versions of the m-DNA. These scats may have been produced by the same wolf, or perhaps they are different wolves, and just by “coincidence” they happen to have the same versions of the m-DNA. To resolve this uncertainty, we could compare these two scat samples at a different locus (see below).

At this locus, the samples differ. Now we can be sure the samples were produced by different wolves.

5. Ultimately the result of conducting all these biochemical reactions on all these scat samples is a database listing the genotype of each scat sample at each of 15 loci. A sample of what that data base looks like is:

6. In the year 20XX, for example, we collected XXX scat samples. They represented XX different genotypes. That happened to be the same number of wolves that we counted from the airplane that year. X of those genotype were not detected in the previous year - they are presumed to be the new pups that had been born in the past year. Also, X of the genotypes from 200X were missing in 200X - these are presumed dead.

Some DNA does not encode for protein. And, the purpose of some DNA is not entirely clear, but is still useful to humans for telling individuals apart and for assessing certain aspects of genetic diversity. This is the kind of DNA that we use for studying wolves.

FROM HERE TO THE END OF THIS PAGE, THE MATERIAL IS STILL BEING DEVELOPED. IT SHOULD BE COMPLETED BY LATE SEPT 2007.

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