In your future directions you mention using different primers for better gene expression and data. Do you have any idea what those primers would be or how you would choose different ones?
Assuming that the results of your future experiments are consistent with your expectations (which supports that idea that Dicer-2 is involved in DNA damage repair,) what would be the next steps in confirming and identifying the repair mechanism? (This question may be beyond the scope of the course.) What were a few considerations you were aware of in the design and manufacturing of the Dicer-2 primers themselves, and how did you ensure that these primers wouldn’t attach to other sequences of DNA?
Thanks so much for the detailed questions Andy, it’s great to hear from you. Your first question is a really good one, and I’m not actually completely sure how I’d approach determining the specific repair mechanism experimentally. My first instinct is try to determine how crucial this repair actually is to cell survival by seeing whether the presence or absence of dicer-2 affects how well Tetrahymena cells are able to survive varying levels of exposure to DNA damage. Using that information I’d try to see if it was possible to group it with similarly important or unimportant siRNA’s in hopes that they might share a similar method of action.
For your second question, our most important consideration when designing our gene specific primers was that they would anneal to our gene and only our gene. That means we didn’t really want them to form secondary structures with themselves, or use a sequence that is found elsewhere in the Tetrahymena genome. The software we used was able to predict how prone they would be to forming structures with themselves and we used BLAST analysis to ensure that the primer sequences we chose were not found elsewhere in the genome.
The genes that are responsible for repairing this damage are the reason why it has little impact most of the time. When DNA damage repairing genes mess up or are defective, cell death or cancer is often the result.
It seems like the most likely source of error in running that PCR test was some kind of mistake made during loading. It’s possible that the Rad51 primers were nonfunctional or that either the forward or reverse primer was not added to the samples at all by mistake. Essentially I wouldn’t actually change anything other than making sure to use fresh primers and to be extra vigilant!
Your presentation really flowed great! I was wondering how confident you are in your conclusion that dicer-2 plays a role in the DNA damage repair without the DNA damage controls, how did you have to adjust your methods and conclusions to take the lack of controls into account?
Thank you Alyssa! Practically, I’m very confident that dicer-2 plays a role in DNA damage repair because other groups studied this gene as well and their slides supported the same conclusion that ours did (minus the error with control). Speaking only from the data our group collected themselves, it still seems by far the most likely conclusion. Our loading control makes it seem unlikely that the difference in brightness between the damaged and undamaged dicer-2 samples is due to anything other than a change in expression of that gene in response to that damage.
What other model organisms could be considered for continuation of this research and what results would be necessary to demonstrate Dicer-2’s role? You also mentioned that the exact mechanism could be investigated, how might you propose doing this?
curesymposium.org 
In your future directions you mention using different primers for better gene expression and data. Do you have any idea what those primers would be or how you would choose different ones?
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Assuming that the results of your future experiments are consistent with your expectations (which supports that idea that Dicer-2 is involved in DNA damage repair,) what would be the next steps in confirming and identifying the repair mechanism? (This question may be beyond the scope of the course.) What were a few considerations you were aware of in the design and manufacturing of the Dicer-2 primers themselves, and how did you ensure that these primers wouldn’t attach to other sequences of DNA?
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Thanks so much for the detailed questions Andy, it’s great to hear from you. Your first question is a really good one, and I’m not actually completely sure how I’d approach determining the specific repair mechanism experimentally. My first instinct is try to determine how crucial this repair actually is to cell survival by seeing whether the presence or absence of dicer-2 affects how well Tetrahymena cells are able to survive varying levels of exposure to DNA damage. Using that information I’d try to see if it was possible to group it with similarly important or unimportant siRNA’s in hopes that they might share a similar method of action.
For your second question, our most important consideration when designing our gene specific primers was that they would anneal to our gene and only our gene. That means we didn’t really want them to form secondary structures with themselves, or use a sequence that is found elsewhere in the Tetrahymena genome. The software we used was able to predict how prone they would be to forming structures with themselves and we used BLAST analysis to ensure that the primer sequences we chose were not found elsewhere in the genome.
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If DNA is always being damaged and repaired and has no impact, why is it relevant to understand how the genes work?
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The genes that are responsible for repairing this damage are the reason why it has little impact most of the time. When DNA damage repairing genes mess up or are defective, cell death or cancer is often the result.
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what would you do differently if you were to re-do the PCR test
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It seems like the most likely source of error in running that PCR test was some kind of mistake made during loading. It’s possible that the Rad51 primers were nonfunctional or that either the forward or reverse primer was not added to the samples at all by mistake. Essentially I wouldn’t actually change anything other than making sure to use fresh primers and to be extra vigilant!
LikeLike
Your presentation really flowed great! I was wondering how confident you are in your conclusion that dicer-2 plays a role in the DNA damage repair without the DNA damage controls, how did you have to adjust your methods and conclusions to take the lack of controls into account?
LikeLike
Thank you Alyssa! Practically, I’m very confident that dicer-2 plays a role in DNA damage repair because other groups studied this gene as well and their slides supported the same conclusion that ours did (minus the error with control). Speaking only from the data our group collected themselves, it still seems by far the most likely conclusion. Our loading control makes it seem unlikely that the difference in brightness between the damaged and undamaged dicer-2 samples is due to anything other than a change in expression of that gene in response to that damage.
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What other model organisms could be considered for continuation of this research and what results would be necessary to demonstrate Dicer-2’s role? You also mentioned that the exact mechanism could be investigated, how might you propose doing this?
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