Please help me, I am deeply confused in this class and trying so hard to underst

Question

Please help me, I am deeply confused in this class and trying so hard to understand. Please give detailed answers. 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.

Explanation / Answer

2A) PCR or polymerase chain reaction is a common molecular biology technique used to amplify DNA. One of the important features of PCR is that it is carried out in high temperature. Thus it is important that the DNA polymerase enzyme used should be able to withstand this temperature. This is why polymerase from T. aquaticus is added as it can withstand the high temperature used in PCR ie 95 degree celcius. The reaction fails here because the polymerase from E. Coli cannot withstand this temperature and it gets denatured thereby not able to perform its function that is polymerisation. The polymerase from E.Coli is protein in nature, so any protein when subjected to a temperature higher than its optimal temperature tend to denature because of the disturbance in its qaurternary structure mostly breakage of bonds that makes it stable.

2B) PCR is a technique based on thermal cycling ie., the reactants are exposed to different temperature repeatedly. This is because three reactions should take place in PCR:

1. DENATURATION- In this step the double stranded DNA is converted to single strand which requires high temperature upto 95 degree celcius.

2. RENATURATION/ ANNEALING- In this step temperature drops to 55 degree celcius for the primers to bind to single stranded DNA.

3. SYNTHESIS- In this Taq polymerase carry out the synthesis of DNA by polymerisation reaction. The temperature is raised to 75 deegree celcius. And these steps are repeated.

From the above description I hope it is clear for you that PCR depends greatly on the temperature and time. Thus if one carries out a reaction at lower temperatures say for example if you start the reaction at 5o degree celcius the DNA denaturation won't take place properly thereby leaving behind double stranded DNA resulting in the failure of primer binding and subsequent steps.

2C) In the graphical representation of PCR :

A peak indicates denaturation ie., the dna undergoes melting and the hydrogen bonds are broken resulting in single strands.

B indicates renaturation or annealing where the primer binds to the single stranded DNA.

C indicates synthesis phase where the taq polymerase synthesis new DNA strand .

Comparing the three the detection using the fluorescent dye will be more accurate at C as there the DNA exist in double stranded state.

Related Questions

Navigate

• Browse (All)
• Browse P
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.