Question

3. Assume that a bacterial culture is grown on medium containing radioactive thymine until essentially all of the thymine in the DNA is radioactive. Then the bacteria with labeled T were washed and transferred to a medium containing nonradioactive T for one round of replication. Given the four double stranded sequences below, which one is likely to represent the replicated double stranded structure? Note: A,C,T,G represent typical nucleotides, and 3T represents labeled T.)

	1	2	3	4
One strand	A G 3T C	A G 3T C	A G 3T C	A G 3T C
One strand	A G T C	T C A G	U C 3A G	3T C A G

In PCR reactions, the only enzyme that is needed is a polymerase. Provide reasoning as to why the following essential replication enzymes/components are not needed:

Helicase

Topoisomerase

Ligase

Primase

Replication clamp

Answer 1

HELICASE- We want to go through melting and melding the DNA. We can't do this with helicase because once we add it, it will unzip all the strands and keep continuing so. Using heat, we can increase and decrease the temp to allow for multiple reactions to occur.

TOPOISOMERASE- Topoisomerase is not needed to unwind and release tension in DNA, because their workl is done by a combination of high temperature and the short length of the DNA being amplified.

LIGASE- Equivalenta of DNA polymerase I and DNA ligase are also unnecessary because of the absence of RNA primers and Okazaki fragments during the process of PCR.

PRIMASE- RNA primase is not necessary because a sequence-specific pair of DNA primers produced synthetically are added to the reaction.

REPLICATION CLAMP- It is a component of  DNA polymerase III holoenzyme, clamp protein binds DNA polymerase and prevents this enzyme from dissociating from the template DNA strand.