From DNA extraction to mitochondrial genes sequencing

My previous blog was focused on sampling Pacific Angel Sharks and my interaction with fishermen in the Gulf of California. Now, I will summarize the laboratory work and the protocol for knowing the genes of our species of interest from muscle tissue that we sampled previously.

Figure 1. Workflow in the laboratory, from DNA extraction to obtaining mitochondrial genes sequences. Figure © Massiel Alfonso

The first step is DNA extraction, which uses traditional methods such as phenol-chloroform or commercial kits developed for such purposes. The integrity of the DNA obtained is visualized in agarose gels (Figure 1), and its concentration and purity can be measured in spectrophotometers. Subsequently, we prepare the samples to amplify the genes of interest using the Polymerase Chain Reaction (PCR). This method uses primers that allow us to identify fragments and obtain multiple copies of them. The PCR product is visualized in agarose gels, detecting a band when the amplification is successful (Figure 1). The position of this band in the lane will depend on the size (in base pairs) of the amplified gene. In this project, we have been working with Cytochrome c oxidase, a gene involved in cellular respiration, and the Control Region of DNA, whose expected approximate sizes are 600 bp and 1000 bp, respectively. The PCR products were sent to a sequencing service to obtain the sequences using the Sanger method.

Finally, the files obtained were edited and aligned to search for homologies between the sequences. This alignment is our input for all diversity and genetic structure analyses. Interesting results will be shown in the next blog. Stay tuned!