PCR Flashcards
What is the normal temperature for DNA denaturation
92-94°C
What are full/incomplete separations and which one si used in PCR?
Incomplete separation - some regions (c-G rich) remain bound, which significantly speeds up reannealing process.
Full separation - when entire sequence is separated into single DNA strands. This separation is used in PCR, because the strands take much longer to reanneal together, which allows the primers to anneal first (as they are shorter).
What factors influence the efficiency of denaturation?
- ionic strength -> the higher the more efficient
- BP composition -> AT-rich fragments more efficiently than C-G-rich
- pH -> basic conditions increase the efficiency
What are the constraints of the denaturation process?
Buffer solution cant be changed through PCR. The buffer needs to be able to stabilize the enzyme even in denaturating temperatures.
How does the length of the sample DNA influence primer design?
Length of the primer is proportional to the length of the DNA sample.
What happens to DNA at the Tm?
50% of DNA is annealed
What are 2 rules for determining the annealing temperature? What is the approximate temp value?
Wallace rule: 2°C(A-T) + 4°C(C-G) –> works for 14-30 oligomers
Bolton and McCarthy rule –> works for 14-70 oligomers
avg temp ~65°C
How long does the annealing phase last and why?
Annealing lasts 10-30s because the primers are supplied in excess, which increases the efficiency of the reaction.
What is the undesired side reaction in the process of annealing and how it can be minimised?
Self-annealing of primers can be prevented by technique called “hot start”.
What is the avg temp for the extension of primers phase of PCR and why?
It is ~72°C, it is set close to the optimal temp for the DNA polymerase used.
How long does the extension phase last and why?
It should last long enough for the whole sequence to be synthesised. E.g. 1min/kbp for Taq polymerase
What are the required factors for the proper activity of theDNA polymerase?
Sufficient supply of dNTPs, a divalent cation (mg2+) and some monovalent ions.
What happens when the annealing temp is much lower than extension temp?
The annealed primers separate from the sample strands.
What causes high error rate of DNA polymerase and what can be done to prevent it.
2 major causes are insufficient dNTP and Mg2+ concentration. Apart from manipulating these, alternative DNA polymerases or their mixtures may be used.
What are Klenow enzyme and Taq polymerase? What are the advantages of their use?
Klenow enzyme is a polymerase that doesn’t have 5’-3’ exonuclease activity(nick translation and DNA repair), but it is not heat resistant and needs to be supplemented after each denaturation process.
Taq is a heat resistant polymerase isolated from the thermophilic bacterium.
