COLLECTING TICKS
The first phase of our project has involved a variety of collecting methods including tick drags and visual encounter surveys. For simple Borrelia prevalence data, our goals have been to collect a representative sample of ticks from each location as quickly as possible, and, because we are collecting adult Blacklegged ticks, this usually involves flagging understory vegetation within forests and along trail edges. Ticks are collected into small vials of alcohol for preservation and storage until we are ready to test them.
EXTRACTING DNA
In order to test ticks for pathogens, we need to extract DNA from within their bodies. The process involves 3 steps: 1) cutting the tick open with a razor blade, 2) freezing the tick in liquid nitrogen, and 3) chemical extraction. In the end, we are left with a solution that contains the DNA of the tick and the gazillions of bacteria, fungi, and other microbes on and inside of its body.
AMPLIFYING TARGET DNA
In order to see if a tick is carrying a pathogen like Borrelia, we have to have a way to see the pathogen's DNA within the mix of other DNAs. It would literally be like looking for a needle in a haystack!. To solve this problem, we use specific DNA sequences, called primers, that seek out the pathogen's DNA, and we use a specific process, called polymerase chain reaction (PCR), to make millions and millions of copies of that particular pathogen DNA. So, if Borrelia is present in a particular tick, the PCR process will make millions of copies of just that piece of Borrelia DNA. If Borrelia is not present, then no DNA will get copied.
SEEING DNA
DNA is invisible to us, and it's still all mixed together in little vials of fluid. In order to separate this mix of DNA, we use gel electrophoresis, which uses an electric current to drag DNAs through a gel. Larger molecules navigate the porous matrix of the gel more slowly than do smaller molecules, so this process separates DNA pieces by size. We add a chemical to the gel that causes the DNA to glow under ultraviolet light, and this allows us to see the DNA. This photo shows what the gel looks like under UV light. The wells into which the DNA samples were added are at the bottom of the photo. The sequence of bands at far left is a "ladder" that has DNA segments of known size. The 11 samples to the right of the ladder are ticks we tested, and the last 2 samples to the right of those are the Borrelia positive and negative controls. How many of the 11 ticks were positive for Borrelia? Did you say 9? Yes, that is scary!