 Winter 2005 |
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Putting a Grant into Practice: Ann Yezerski |
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In 2003, I wrote a proposal for the Laboratory Teaching Initiative Grant provided by ABLE. I had proposed the creation of a laboratory exercise for undergraduate genetics students to do a relatively high-throughput method of molecular genetic linkage mapping. It’s now 2005; has the exercise worked? It can be very difficult these days to design laboratory experiments that reflect what undergraduates might be presented with when they leave the very structured labs of their institution. This can be especially true when dealing with molecular biology. Molecular biology requires equipment and reagents that are very expensive (each micropipettor can be $250, and a tube of Taq polymerase can be $250 for 500 reactions). The field is also infamous for a constant influx of new techniques. Lately, one of the buzzwords of molecular genetics is “high-throughput.” Experiments in these fields often require huge datasets, which, of course, necessitate large numbers of replicate reactions. A genetic linkage map at the end of the century could be published with little over 100 markers. Now just half a decade later, closer to 1000 are regarded as minimal. How can a small undergraduate institution demonstrate such techniques with our financial, time, and space constraints? How can you do a thousand replicates without creating a room full of ennui? We have to face it: using fruit flies to demonstrate linkage is a nice little experiment, but you simply will not see a modern laboratory counting white-eyed, yellow-bodied flies -- or any phenotype for that matter -- to determine linkage. Scientists use genotype over phenotype whenever possible. Therefore, I decided to try to incorporate molecular genotyping in order to teach genetic linkage mapping like it may be done in a modern laboratory today. The exercise utilizes a lot of methods in order to best demonstrate modern methods. Instead of fruit flies, I use Tribolium beetles, because they are so great (actually it is because it was the organism I published a linkage map for and I think they are cute). However, these little guys did have a few advantages. The most important is that they do not fly. We all know the disadvantage to that in fruit flies (and we’re sick of taking the blame when our colleagues' offices get infested). Flour beetles are also cheap to maintain and freeze very well. I also happen to have several hundred recombinant inbred lines of them that I created several years ago. Therefore, the students do not have to be responsible for creating intercrosses and deal with the frequent failures and errors of these techniques. However, DNA extraction from these little guys requires a classic phenol-chloroform technique, which is longer and more dangerous than simple centrifuge methods now available. I do find that this method better illustrates how a DNA extraction works, as you must remove unwanted macromolecules one by one until only DNA is left. It is just a bit more informative than a simpler method. For the mapping itself, I use RAPD-PCR. There are several advantages to this. One is that you need only about 1/100th the DNA concentration of a typical PCR reaction. So, even a bad DNA extraction is usually plenty of DNA. If you choose not to use beetles, this method also has the advantage of not requiring any a priori information about your organism since the priming is random. The entire reaction itself is only 12 microliters, so large volumes of reagents are not necessary. The resulting banding patterns from these reactions are also relatively easy to interpret. It’s simply if a band is present on the gel or not (figure 1). One of the twists to this exercise is that we will be mapping using the data from eight different primers and 48 different beetles. That is a lot of work for undergraduates. This is where the high-throughput part comes in. Each group of students is only responsible for the extraction of six beetles, but then all of the groups share their DNA. This way the students start to get the feeling of sample sizes necessary without having the spend all of the hours necessary. Then each group uses the same 48 samples of beetle DNA with a different primer. This results in 384 pieces of data – enough to create a rudimentary map. One last issue with such an exercise is that, as most people know, molecular biology is a lot of “hurry up and wait.” How do you fill the time left empty while the centrifuge spins? This is the perfect spot to try some self-paced computer exercises in Bioinformatics, but we’ll leave that for the next grant proposal. So, that is the grand plan. Has it worked? Mostly. We always get some nice data, but sometimes entire groups end up with a blank gel after working so long. “Well,” we scientists say, “That’s real science!” But disappointment does not sit well with sophomores, so it is nice to have perfect pictures of the data on file for the unfortunate groups to analyze if things go terribly wrong. This way they’ve learned all of the techniques as well as the reality of research. Is this research? I’d say so. I use the data to add to my own genetic linkage map. So, I guess, I am really secretly using my students to collect data. Is that so horrible? No, it’s not really a secret, the students know they are adding to a real data set and sometimes it’s nice to feel that you are contributing to and practicing real science so that one day, you can do the same. Plus, it’s nice to see that this exercise has become the second favorite of the genetics students. The first? The DNA fingerprinting murder mystery lab. Thanks, “CSI.” Now, if we could just get a hit show about beetles…
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