Welcome back to the Lab!
For the last several weeks, our Barcoding US Ants participants have been actively involved in the wet lab portion of our project. During the summer, each participant collected ant samples to bring in for DNA barcoding, and last weekend, we went through the necessary steps to prep our samples for DNA sequencing.
The wet lab portion is, in my opinion, the most fun portion of the entire Barcoding US Ants project. Participants must extract DNA from their specimen, amplify it using PCR, and analyze their results using gel electrophoresis before sending samples off to be sequenced.
Participants begin the process of DNA extraction by grinding tissue samples in Chelex resin. Chelex resin helps separate DNA molecules from other compounds in the tissue sample, including DNA-degrading enzymes that might otherwise destroy the sample. Once the tissue sample is finely ground in the vial of Chelex, it is placed in a hot water bath to help further break the cells open and expose the DNA. When the process is complete (after all of ten minutes), the specimen DNA will be left suspended in an aqueous solution while the Chelex beads and cellular debris sink to the bottom of the vial.
After successfully isolating DNA from their specimen, participants must amplify their DNA; DNA sequencing and other analysis requires millions to billions of copies of our target DNA strand, and in order to acquire that many copies, participants must perform DNA synthesis in an in vitro environment using a technique known as PCR.
PCR stands for Polymerase Chain Reaction. Polymerase is the enzyme that catalyzes the formation of new DNA strands, and a chain reaction describes a process by which the product of a reaction promotes further reactions to take place. In PCR, the product of the reaction is just more DNA.
The first step in PCR is denaturation: The DNA sample is heated to a point where the bonds between the nitrogenous bases in the DNA strand break apart, so you end up with two single strands of DNA (as opposed to one double-strand).
After the strands are broken apart, short, single-stranded DNA segments called primers will target the section of DNA that is to be amplified. This step is called annealing.
The last step in the process is extension. The polymerase will bind to the strands and begin adding nucleotides a single base pair at a time. The process is then repeated 20-40 times until you end up with billions of DNA copies.
Once all your ingredients are added – polymerase, primers, DNA and free nucleotides – the tubes can be added to your thermal cycler. The thermal cycler controls the temperature of the reaction. Denaturing occurs at ~95°C, annealing occurs at ~55°C, and extension occurs at 72°C. When this technology was developed, thermal cyclers were bulky, and PCR could not be performed outside of a laboratory setting. Now, I can hold a thermal cycler in the palm of my hand, and the entire process is automated. Just enter your temperature settings and the number of cycles you wish to perform and hit start. It’s truly that easy.
The final step to prepare our DNA for sequencing is to perform gel electrophoresis. Gel electrophoresis is a way to visualize the DNA strands and make sure PCR was successful. PCR isn’t foolproof, and sequencing can be a costly process; so there’s no point in sending in samples that potentially didn’t amplify.
Gel electrophoresis works like this: Amplified DNA fragments are stained using a special DNA-binding dye, and loaded into small “wells” or indentations at the top of the gel. An electric current is run through the gel, with the negative end of the current at the top, near the wells, and the positive end of the current at the bottom. The DNA molecules, which are negatively charged, will “run” towards the positively charged end of the gel.
It’s important to note that DNA molecules have the same charge per mass, meaning that larger DNA fragments will move through the gel at a slower rate than the smaller DNA fragments. This is how we are able to determine the size of the fragments; large fragments will show up as “bands” that are closer to the wells, while small fragments will show up as bands that have traveled further.
When the gel is finished running, the DNA fragments can be viewed with a UV light; they will show up as the neon green-yellow bands seen above. Using this method, we know which samples to send in for sequencing. The samples that successfully amplified are shown as distinct bands in the gel. Considering over half of the samples show banding (even faint banding in some cases), I would call our wet lab day a resounding success! Once our samples have returned DNA sequences, our participants will analyze the sequence data and identify their specimens. Onward, US Ants!
Until next time, thanks for visiting the lab!
Bug Wrangler Brenna
brenna@missoulabutterflyhouse.org
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