4. When the helix axis of a closed circular duplex DNA of 3190 bp is constrained

Question

4. When the helix axis of a closed circular duplex DNA of 3190 bp is constrained to lie in a plane, the DNA has a twist (T) of 290. When released, the B DNA takes up its normal twist. (5 pts; 2.5 pts each) a. Indicate the values of the linking number (L), writhing number (W), and the twist for both the constrained and the unconstrained conformational states of this DNA circle; show your work and explain your answer. b. Also indicate L, W and T for the unconstrained molecule if it is A DNA instead of B DNA; show your work and explain your answer. 5. Describe in detail how a gene of interest (GOI) can be expressed in E.coli using the methods below. Include all relevant steps that must be taken to ensure gene expression inside the host. (10 pts; 5 each) a. Using restriction enzymes b. Using topoisomerases (feel free to copy and paste relevant steps from part a and highlight the differences) 6. In lecture we discussed prokaryotic DNA replication. This replication is primarily done on circular DNA. Given the information you have, discuss how this process becomes problematic for long linear DNA molecules such as those found in Eukaryotic organisms. (5 pts)

Explanation / Answer

DNA replication is the process of producing two identical replicas from one original DNA molecule. This biological process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of two strands and each strand of the original DNA molecule serves as a template for the production of the complementary strand, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication.[1][2]

In a cell, DNA replication begins at specific locations, or origins of replication, in the genome.[3] Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bidirectional from the origin. A number of proteins are associated with the replication fork which helps in terms of the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new DNA by adding complementary nucleotides to the template strand.

DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.

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