I know some of these might be confusing but your help will really help me learn.
ID: 135723 • Letter: I
Question
I know some of these might be confusing but your help will really help me learn. The teacher requires very very detailed answers and he gives nearly no (and I mean literally nearly zero) partial credit so I'm dying here. Thank you sooooooo much, you have no idea.
Also, I am nearly leaglly blind and I am dyslexic so if you could please write clear it would help so much. I just want to learn like everyone else. SUPER SUPER THANK YOU.
QUESTION 2 (6 PTS) 2A. The last time you were setting up a PCR reaction you accidentally added polymerase from E. coli instead of from T. aquaticus. Why did the reaction fail? What aspects of protein structure were most likely at the root of this failure? (2pt) 2B. To try and overcome this failure, you run the reaction again (still with the E. coli enzyme) but lower all of the temperatures by 15'C. This fails again. Why? (give a different answer than for 2A and be specific about what's going on in the tube) (2pts) 2C. The following is a common way to represent the progression of a PCR reaction. time A common way to detect the amount of DNA in a reaction is with a fluorescent dye that emits light when it binds to double stranded DNA. Would the detection of this signal be most accurate and precise at A, B or C? Why? (2pt)Explanation / Answer
Answer for 2A: Taq polymerase isolated from bacterium Thermus aquaticus is used in PCR reaction as it is an enzyme that can be able to withstand protein denaturing conditions like high temperatures required during PCR.
The reaction has got failed as E.coli DNA polymerase which is used might get denatured and lost its function during PCR reaction.
The DNA polymerase from Thermus aquaticus (Taq polymerase) is homologus to DNA polymerase from E.coli structure (Pol1). But the structure of vestigial editing 3'-5' exonuclease domain of Taq polymerase is dramatically altered but is having ribonuclease H-like motif which confers 5'-3' exonuclease activity that makes it thermostable.
This kind of protein structure can't be seen in E.coli and unable to withstand the higher temperatures during PCR reaction.
Answer for 2B:AS PCR reaction consists of 3 basic steps like initiation, denaturation and anneaing step
Initiation step requires temperature of 94 to 96 degreeC for 1 to 10 mins
Denaturation step requires temperature of 94 to 98 degreeC for 20 to 30 secs
Annealing step requires temperature of 50 to 65 degreeC for 20 to 40 secs
Even though temperature reduced by 15 degreeC, E.coli polymerase can't withstand the temperature and lost its enzymatic function as it will get denatured
A,B and C in PCR graphical reaction represents DNA denaturation,primer annealing and Primer extension steps respectively. During DNA denaturation, primer annealing steps DNA will be in single stranded state but in extension step double stranded DNA is generating. As fluorescent dye binds to double stranded DNA and emits light, the detection of the signal would be more accurate and precise at C compared to And B.