Hi Paige! We first designed our primers using online programs Primer3Plus and BLAST which were then created and sent to us. We validated the designed primers by PCR and was visualized using gel electrophoresis. The purpose of our research was to determine whether or not the Rsp1 gene is involved in DNA damage repair of Tetrahymena thermophila. We next induced DNA damage by adding hydroxyurea (DNA damaging reagent) to cells . Followed by RNA extraction to then isolate it by cDNA synthesis. Which was a reverse transcriptase mixed with the extracted RNA. Resulting in separating the RNA which led to creating another PCR and visualizing the results by gel electrophoresis. This allowed us to see that DNA damage was induced and conclude that Rsp1 is involved in DNA damage repair.
Hi Roxie, further experiments using CRISPR cas9 can be used to edit a cells DNA. It would precisely cut DNA and let natural DNA repair to take over. Since we concluded that Rsp1 is involved in DNA damage repair pathway. Rsp1 with CRISPR cas9 editing could be used in different organisms to correct DNA sequences that originally would be unable to correctly repair DNA damage that would’ve led to mutations. Further experimentation will lead to repairing DNA damage and developing future therapeutics.
Hi Lauren,
I’m curious about the process of designing the primers and your experience using Primer3Plus and NCBI blast, would you mind giving your input on these tools?
Hi Elle, so Primer3Plus and BLAST were used to design primers that could be used to amplify our gene Rsp1 with PCR. There was a lot of information to decipher but that led us to be confident in choosing our primers to use.The primers with Primer3Plus were compared to a set of parameters we were given to find the best primers. Primer3Plus gave us info on where the primers binds in the template DNA, the length, and much more info. We determined which formed the least stable structures. We then used BLAST to ensure our designed primers were complimentary to our gene Rsp1. BLAST gave us info to ensure primers only annealed to our gene and is ultimately unique.
Thank you Connor!! The new and rapidly developing technology of CRISPR-Cas9 is very exciting to see how far and life-altering it has come. Now having a better understanding of how crucial DNA repair is from our research. It’s exciting to see what new developments will arise in helping so many affected by diseases and ailments.
curesymposium.org 
Could you please explain your methods a little bit more clearly in how you did your experiment?
LikeLike
Hi Paige! We first designed our primers using online programs Primer3Plus and BLAST which were then created and sent to us. We validated the designed primers by PCR and was visualized using gel electrophoresis. The purpose of our research was to determine whether or not the Rsp1 gene is involved in DNA damage repair of Tetrahymena thermophila. We next induced DNA damage by adding hydroxyurea (DNA damaging reagent) to cells . Followed by RNA extraction to then isolate it by cDNA synthesis. Which was a reverse transcriptase mixed with the extracted RNA. Resulting in separating the RNA which led to creating another PCR and visualizing the results by gel electrophoresis. This allowed us to see that DNA damage was induced and conclude that Rsp1 is involved in DNA damage repair.
LikeLike
Could you elaborate on your rationale for continuing to CRISPR?
LikeLike
Hi Roxie, further experiments using CRISPR cas9 can be used to edit a cells DNA. It would precisely cut DNA and let natural DNA repair to take over. Since we concluded that Rsp1 is involved in DNA damage repair pathway. Rsp1 with CRISPR cas9 editing could be used in different organisms to correct DNA sequences that originally would be unable to correctly repair DNA damage that would’ve led to mutations. Further experimentation will lead to repairing DNA damage and developing future therapeutics.
LikeLike
If time and resources were not a problem, what is one additional experiment you would like to do?
LikeLike
Hi Lauren,
I’m curious about the process of designing the primers and your experience using Primer3Plus and NCBI blast, would you mind giving your input on these tools?
LikeLike
Hi Elle, so Primer3Plus and BLAST were used to design primers that could be used to amplify our gene Rsp1 with PCR. There was a lot of information to decipher but that led us to be confident in choosing our primers to use.The primers with Primer3Plus were compared to a set of parameters we were given to find the best primers. Primer3Plus gave us info on where the primers binds in the template DNA, the length, and much more info. We determined which formed the least stable structures. We then used BLAST to ensure our designed primers were complimentary to our gene Rsp1. BLAST gave us info to ensure primers only annealed to our gene and is ultimately unique.
LikeLike
do you think CRISPR would have made this experiment significantly easier?
LikeLike
Great presentation! What are applications of DNA repair are you most excited about?
LikeLike
Thank you Connor!! The new and rapidly developing technology of CRISPR-Cas9 is very exciting to see how far and life-altering it has come. Now having a better understanding of how crucial DNA repair is from our research. It’s exciting to see what new developments will arise in helping so many affected by diseases and ailments.
LikeLike