PCR optimization for Kelp primers

Once we have obtained quality DNA from our kelp samples, it is time to look for viruses! But first, we need to optimize our PCR...

PCR stands for Polymerase Chain Reaction. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was not until 1985 that the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation (Lorenz, 2012). PCR was an uncommon word outside the scientific field until the current coronavirus pandemic arose. When I talk with family and friends about my work in the lab, now I can exactly name what I am doing: PCR. However, since most people only know this term because of SARS-Covid 2, they get confused and ask if I am analyzing coronavirus samples. No, I answer, PCR is a quite old technique used for many purposes, not just coronaviruses.

So, what PCR is?

PCR is an amplification technique that can generate many copies of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence) (Lorenz, 2012). PCR is a key technique in molecular biology, but also in medical fields, like within clinical diagnosis, since it is a fast and precise method to detect a broad range of microorganisms. Did you ever wonder how forensics can identify one person from millions just by grabbing a sample in a crime scene? Exactly, PCR. Tiny samples of DNA can be amplified by PCR in the lab and can be later compared with other sequences stored in DNA databases to determine if there is any match. The technical process is better explained in the following video by MiniPCR bio:

Therefore, PCR is key for this project as well. Remember that Phaeoviruses integrate their DNA into their host's DNA. Before screening for viruses, we need to make sure our algal DNA is good enough (we saw this step in the previous post), and it is worth to run a PCR test to detect specific algal genes that molecularly prove our samples are actually what we think they are: algae. In this case I used specific Phaeophyceae primers; meaning that if I have a sample with an algae belonging to that Algae Class, the result should be positive.

When we have good quality DNA extracted from our algal samples, we need to set the PCR reagents, and prepare again the "recipe". Once our "molecular cocktail" is ready, we will introduce it into the PCR machine (called thermal cycler), and we will set the thermal cycling conditions. After that, our PCR products need to be stained and loaded into an agarose gel, and after an electrophoresis procedure, we will visualize the final result under a UV lamp.

This is, again, a time-consuming procedure (not to say that, the more samples you have, the longer it will take to prepare), that can go wrong during any step of the whole process. It is, therefore, our responsibility to optimize our PCRs, in order to avoid problems. Even if we consider all the different parameters to avoid problems, there is always the possibility to forget something, to do something wrong, or just find a new problem that we didn't count with. It is all normal. It is frustrating sometimes as well (see comic below), but hei, we are here to investigate and to learn. So it is always possible to find out what went wrong, and to make further adjustments 🙂👍

If you are starting in the lab, or not, and you have problems with your PCRs, the work from Lorenz (2012) can be useful. My next step will be to screen for viruses with another PCR with specific primers for Phaeoviruses. I have already started and... guess what? It didn't work! So I am working in the optimization of my viral PCR. I will keep you posted!

Sources:

Lorenz, T.C. Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012).