Week Three: New Optimized PCR Method Using Restriction Enzymes and Gel Electrophoresis

Hi everyone! This week was mostly prep for next week when I will be taking DNA samples and starting to sequence them. I wrote up protocols for both the DNA extraction as well as the PCR on Benchling. 

I will be taking DNA samples with a buccal (cheek) swab. The collection is pretty simple, participants just swab the inside of their mouth with a cotton swab and then submerge the cotton swab with their DNA into a tube with saline solution and swirl for about 30 seconds. Once this is done, I will transport the samples to the lab and store them on ice until I’m all prepped for sequencing. 

For the actual sequencing, I’ve decided to use a new optimized PCR method to detect the polymorphism within the COMT strand. This method was developed by scientists Bassam Lajin and Amal Alachkar, their study can be seen here. Simplified, this method uses a restriction enzyme to cut the DNA fragment at certain places. Based on the number of cuts of the DNA fragment and how long it is, we can conclude if there is a polymorphism or not. By using a method called gel electrophoresis, we can see the length of each DNA strand by using electrical currents.

Above is a picture of what my results could look like. Based on how many bands there are, the gel will then indicate which genotype and polymorphism the individual has. 

Understanding and breaking down this study was pretty difficult for me. I’ve had experience reading empirical studies before, but I’ve never tried to replicate one and use a method on my own. With help from my lab mentor, I’ve broken down the study and am thankful for this optimized method that uses gels rather than sending samples to a lab once I prepare them. This saves me money as well lets me gain more experience with lab work and gels! Next week is the collection and beginning sequencing. Thank you for reading!