Hello! I know that you mentioned in your future directions that you would like to see how Twi-7 is expressed in other types of breaks – based on your research this semester, would you expect Twi-7 to have low expression in single stranded DNA breaks as well or has there been research that Twi-7 acts differently with different breaks? Thank you!
Great question! There is not much literature currently present on this specific gene which is why we included it in our research. It is difficult to say whether or not we would get more expression in the case of a single-strand break, but it is definitely possible!
That’s a good question. Gel electrophoresis is known as a qualitative method, meaning that it provides information about the presence or absence of DNA fragments, but it does not provide quantitative information about the amount of DNA present. Gel electrophoresis also has a certain threshold for this qualitative information, so when a low amount of DNA is present, it may not be visible on the gel, or it may appear as a faint band that is difficult to distinguish from background noise. This can be particularly problematic for genes that are not highly expressed, as the amount of DNA they produce may be very small.
I mentioned qPCR as a potential alternative experiment because qPCR is a quantitative experiment that can be much more sensitive to relative levels of DNA expression compared to gel electrophoresis.
Great question! The procedure we conducted to find double strand breaks used Hydroxyurea as our DNA damaging reagent. For a single strand break, we would need to use a different reagent that would only damage DNA on one strand. Hope that helps!
Great question! We used the same primers for both the gDNA and cDNA. We had 2 reasons to test on both of these strands. First, testing the primer on gDNA helps to confirm its specificity to the target gene and ensures that it does not amplify any other regions of the genome. Secondly, testing the primer on cDNA helps to ensure that it can amplify the target gene from the transcribed RNA and not from any genomic DNA contamination that may be present in the cDNA sample.
Interesting question. I am not entirely sure what you’re asking… Twi7 can be expressed in nature as it serves a specific function for these yeast cells. We are still unsure about what triggers this expression (the catalyst like you said), but researchers like us are working to figure that out. Hope this helps! If you have any other questions I’d be happy to answer.
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Hello! I know that you mentioned in your future directions that you would like to see how Twi-7 is expressed in other types of breaks – based on your research this semester, would you expect Twi-7 to have low expression in single stranded DNA breaks as well or has there been research that Twi-7 acts differently with different breaks? Thank you!
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Great question! There is not much literature currently present on this specific gene which is why we included it in our research. It is difficult to say whether or not we would get more expression in the case of a single-strand break, but it is definitely possible!
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Why exactly is gel electrophoreses too insensitive a method to detect genes like twi7 that are not highly expressed?
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That’s a good question. Gel electrophoresis is known as a qualitative method, meaning that it provides information about the presence or absence of DNA fragments, but it does not provide quantitative information about the amount of DNA present. Gel electrophoresis also has a certain threshold for this qualitative information, so when a low amount of DNA is present, it may not be visible on the gel, or it may appear as a faint band that is difficult to distinguish from background noise. This can be particularly problematic for genes that are not highly expressed, as the amount of DNA they produce may be very small.
I mentioned qPCR as a potential alternative experiment because qPCR is a quantitative experiment that can be much more sensitive to relative levels of DNA expression compared to gel electrophoresis.
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how will your procedure change if you test Twi7 in single stranded breaks? is there any modification of the gene that need to be preformed?
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Great question! The procedure we conducted to find double strand breaks used Hydroxyurea as our DNA damaging reagent. For a single strand break, we would need to use a different reagent that would only damage DNA on one strand. Hope that helps!
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What was the reason you used g and c primers? Would you test to see if the gene is just not highly expressed?
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Great question! We used the same primers for both the gDNA and cDNA. We had 2 reasons to test on both of these strands. First, testing the primer on gDNA helps to confirm its specificity to the target gene and ensures that it does not amplify any other regions of the genome. Secondly, testing the primer on cDNA helps to ensure that it can amplify the target gene from the transcribed RNA and not from any genomic DNA contamination that may be present in the cDNA sample.
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Can twi-7 be catalyzed in nature like you did in the LAB?
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Interesting question. I am not entirely sure what you’re asking… Twi7 can be expressed in nature as it serves a specific function for these yeast cells. We are still unsure about what triggers this expression (the catalyst like you said), but researchers like us are working to figure that out. Hope this helps! If you have any other questions I’d be happy to answer.
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How and why were specific primers used in the experiment for the gels.
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