How do we identify different species? One obvious way is morphology, the way an organism looks. This may include size, shape, and body color. Field guides or internet sites could be used to help identify a particular organism this way. However, what about organisms, like insects, that are often hard to identify strictly by morphology? That’s where a useful method called DNA barcoding comes into play.
We use the DNA barcoding technique in our lab to sample the biodiversity of insects, but we also use it to train undergraduate students in molecular biology. The purpose of this post is to describe the history as well as the science behind this technique. I will also describe how it works in our lab.
In 2003, Paul Hebert, a professor and current Director of the Biodiversity Institute of Ontario, came up with “DNA barcoding” as a way in which to identify species. DNA barcoding uses a short genetic sequence from an organism’s genome. For almost all animals, the gene region that is used is the mitochondrial cytochrome c oxidase 1 gene, or “CO1”. COI is a 648 base-pair region that codes for an enzyme that plays a major role in cellular respiration (how your body converts glucose to ATP), specifically the last step called oxidative phosphorylation. The COI gene is suitable for barcoding because its rate of mutation is fast enough to distinguish between animal species, even closely related species.
Here’s what our “pipeline” looks like:
Week 1: Students in our lab collect insects from sample plots. We have plots at a section of the city’s greenway trails as well as plots on campus. This way we can compare a disturbed urban area (campus) to a less disturbed area (greenway). After collecting in the field, the students attempt to identify the specimens using field guides and other internet resources. Here’s a great one. The insects are stored either in the freezer or in 70% ethanol.
Greenway field site
Week 2: The DNA is extracted from the insects. This process includes initially grinding up certain parts of the insects and then going through a series of steps to isolate the DNA. We use Carolina Biological’s protocol. The samples are then stored in the freezer until the next lab.
Week 3: The DNA is amplified, or copied. During this lab we use a procedure called Polymerase Chain Reaction, or PCR. The small amount of DNA that we have extracted from the insects is replicated. We use a COI primer, which are small pieces of DNA, to amplify the DNA of interest. The PCR machine can make about 68 billion copies! This is a step that also involves using extra deoxynucleotides, a cool enzyme called Taq polymerase, and a loading dye so that we can visualize the results during the next lab. Again, the sample are stored in the freezer until the next lab.
Week 4: The DNA samples are visualized on a gel using a procedure called electrophoresis. In our case, the DNA is separated according to molecular size with the aid of an electrical field. There are many interesting applications of using gel electrophoresis. We use it to determine whether or not we have our gene of interest.
Loading the gel
Week 5: In between week 4 and 5, our DNA samples are sequenced. During week 5, we use bioinformatics to determine our species based on our sequence with the help of a database. Students are able to compare the barcoding results with their original identification from the field guides during week 1. Students are also able to measure the biodiversity of each are (campus and greenway) using several biodiversity indices.
Two main objectives are met during this research. One, we are training undergraduate students to actually be scientists. Students need concrete examples of the scientific process from beginning to end. This research project uses a nice blend of field research and laboratory methods. The students are also given autonomy which increases learning. The second objective is to highlight the importance of biodiversity, even if it is local. Biodiversity is crucial for progress in ecological and evolutionary fields of science and also for conservation programs.
Stay tuned for our results.