Welcome Back to the Lab!

Onward, US Ants! Now that we’ve extracted and isolated DNA from our tissue samples, it’s time to amplify – or make copies of – our DNA. When performing analyses, you need many copies of the target DNA strands you wish to analyze; and isolating the same strand of DNA from millions of cell nuclei is an impossible task. That’s where PCR comes in.

This image shows the basic double-helix structure of DNA, including the nitrogenous bases. The bonds between the base pairs are broken during the first step of 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.

Image courtesy of Khan Academy

While the process may seem daunting at first, it is fairly simple when broken down into parts. Just like following a recipe!

The small white beads in the bottom of these tubes are dehydrated polymerase and free nucleotides. Remember, polymerase is the enzyme that builds the DNA strand, and the DNA strand can’t be built without nucleotides. Think of the polymerase as a zipper and nucleotides as zipper teeth. 

Only 2 μl (microliters) of DNA is needed for PCR.

Next we add our primers and our isolated DNA. The primers bind to the target region of the DNA strand; they essentially tell the polymerase where to start the extension process.

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.

Next week, we’ll cover how to determine the results of PCR, and take a look at the outcome of last summers collection efforts.

Until next time, thanks for visiting the lab!

Bug Wrangler Brenna

If DNA workshops sound like something you’d like to participate in, then stay tuned! After all, Barcoding US Ants is a community science project, and we want to involve you, the community. For more questions, email me at brenna@missoulabutterflyhouse.org and put “Barcoding US Ants” in the subject line.