Since the model organism used undergoes self-induced DNA damage, do you think the expression and activity of DNA repair proteins is contingent on the type of damage experienced (e.g. double vs. single-stranded breaks)?
Yup! That definitely is the case. Different forms of DNA damage are repaired in different ways. For example, double stranded breaks are the most difficult to repair, and it’s the type of DNA break that occurs during conjugation if I’m not mistaken as a part of the mating process.
Hi Josh! Good question!
Short answer: It’s expensive
Long answer: It is a bit more complicated than a regular PCR, so why not do the easier more cost effective first method first.
Yes! We can use Quantitative PCR. It allows us to detect the amplification in real time through fluorescent signals. This detection software is pretty sensitive, so we will probably eventually see some bands at some point after many rounds of amplification.
Hi Sachi! Great question. Quantitative PCR amplifies and visualizes the gene. Gel electrophoresis enables us to visualize the amplification for other forms of PCR that don’t give any visualization of the results. Through gel electrophoresis, we can see both how much we amplified as well as the length of the strand of our DNA. For our current experiment, we did a regular PCR for primer validation and an RT-PCR for the gene expression experiment. Then we did gel electrophoresis on both. Quantitative PCR has an additional bonus: we can see in real time, how much of the Hypr2c gets amplified. This detection software is pretty sensitive, so we will probably eventually see some bands, unlike my experiment. Furthermore, since we have amplified it so many times, the exponential growth will allow us to easily distinguish between brighter and dimmer bands. I should clarify though, we would perform a RT-qPCR. The reverse transcriptase (RT) gets us the cDNA we need for the amplification 🙂
Since the model organism used undergoes self-induced DNA damage, do you think the expression and activity of DNA repair proteins is contingent on the type of damage experienced (e.g. double vs. single-stranded breaks)?
Yup! That definitely is the case. Different forms of DNA damage are repaired in different ways. For example, double stranded breaks are the most difficult to repair, and it’s the type of DNA break that occurs during conjugation if I’m not mistaken as a part of the mating process.
what are the downsides to doing a quantitative pcr experiment?
Hi Josh! Good question!
Short answer: It’s expensive
Long answer: It is a bit more complicated than a regular PCR, so why not do the easier more cost effective first method first.
is there any way to detect lower levels of expression to further determine the results?
Yes! We can use Quantitative PCR. It allows us to detect the amplification in real time through fluorescent signals. This detection software is pretty sensitive, so we will probably eventually see some bands at some point after many rounds of amplification.
How is a quantitive PCR test different from the gel electrophoresis used in the experiment ?
Hi Sachi! Great question. Quantitative PCR amplifies and visualizes the gene. Gel electrophoresis enables us to visualize the amplification for other forms of PCR that don’t give any visualization of the results. Through gel electrophoresis, we can see both how much we amplified as well as the length of the strand of our DNA. For our current experiment, we did a regular PCR for primer validation and an RT-PCR for the gene expression experiment. Then we did gel electrophoresis on both. Quantitative PCR has an additional bonus: we can see in real time, how much of the Hypr2c gets amplified. This detection software is pretty sensitive, so we will probably eventually see some bands, unlike my experiment. Furthermore, since we have amplified it so many times, the exponential growth will allow us to easily distinguish between brighter and dimmer bands. I should clarify though, we would perform a RT-qPCR. The reverse transcriptase (RT) gets us the cDNA we need for the amplification 🙂