8 thoughts on “C52 – Rick

    1. We chose our primers based on multiple diff criteria (which I explained in a different reply), and while we can’t know for sure, we must have simply overlooked something about our primer. For example, if the sequence of the primer allows it to bend and bind to itself, that is likely to happen rather than it binding to our gene. Glad that we had validated gene-specific primers to use!

  1. Why do you think there were no bands or intensity changes in the gene-specific genes? If there were bands or changes, what conclusions could you have reached?

    1. If you’re talking about during the gene expression analysis, the most likely reasoning that we saw no bands in our gel is because the primers did not anneal. This is strange though, because we used validated cDNA as well as validated gene-specific primers for the gene expression gel in order to ensure that we would see bands. It could have been simply human error when setting up the PCR tubes, or our cDNA may have degraded due to not being stored properly. We can’t really know for sure. If we had seen the bands that showed an increase in expression following DNA damage, we would have been able to assume that our gene is likely involved in the repair pathway without having to view the consensus gel of the Hyplrr gene. Good question, thanks!

    1. We used two programs: Blast and Primer3Plus. The Blast program allowed us to view the coding DNA sequence of our gene (Hyplrr), which we input into primer3plus. This program generates primers that are likely to anneal, but provides many quantitative values that we analyzed in order to chose the best one. We wanted a length of close to 20 nucleotides, and a melting temperature close to 55C. These are for optimal storage of the PCR tubes prior to running the gel (to ensure the primers won’t degrade). We wanted a low likelihood of forming homodimers and a low likelihood of the primers binding somewhere random in our sequence other than where we expected (so that we would know the exact length of the sequence made after they anneal). We also wanted a low chance of misprimering the ends of the primer, specifically the 3′ end, because this helps ensure that we know exactly where the primer should anneal in our genome. Thanks for the question!

  2. Amazing poster! I noticed that you mentioned to possibility to test if this mechanism is conserved in humans, how would you go about that?

    1. The same way that we can observe that double-peaked expression pattern of proteins involved in DNA repair of T. thermophila cells in order to look for genes that likely have the same functions, I think it would be plausible to do the same for the gene expression of genes involved in DNA repair of human cells. The major change in this course of action would be the model organism switching from T. thermophila cells to human cells, but T. thermophila was chosen for these experiments for a reason: their ability to quickly reproduce without a mate. It would take longer to do the testing on human cells, because mitosis would either have to be induced or the cells would need to be fertilized, but the same overall experimentation could be done based on the new expression pattern shown by human cells.

Leave a Reply