In this question, determine the values for the linking number (L), super helical turns (AL), the twisting number (T) and the writhing number (W = supercoiling number) initially and after Steps 1 -4. Each part of your answer will be for the plasmid as a whole. It will be helpful to keep track of whether a nick is present or not.   Below the table, explain your answers. Assumptions: Choose a reference state as explained in class. Once you have chosen this reference state at the outset of the problem, do not change it at any time throughout the problem. (After all, what good is a reference state if it is not constant?) Throughout this problem, assume that all duplex DNA is B-DNA (10 bp/turn), except where it is mentioned otherwise. Initial State: A 5000 bp plasmid is completely in the B-DNA conformation under normal conditions and has 25 underwindings (or 25 negative superhelical turns). Step 1: The plasmid is nicked once in one strand and continues to be B-DNA. Step 2: There is a region of 240 bp in the plasmid that is normally in the B-DNA conformation, butit can be induced to adopt the A-DNA conformation by changing salt concentration. The plasmid DNA is manipulated such that this B-DNA to A-DNA transition occurs in the 240bp region. The rest of the plasmid remains B-DNA. Important Hints: 1.A-DNA was NƠT discussed in class. Assume: right-handed, 12 bp/turn. After step 2, the plasmid is “relaxed", since it remains nicked. HOWEVER, it is not in the same relaxed state as the reference state. In spite of the change in salt concentration, do not change the reference state. Step 3: The plasmid is re-ligated (i.e., the nick is re-sealed). You will have to think through whether DNA Ligase changes DNA topology (is it a topological enzyme?), by thinking about the reaction catalyzed by DNA ligase. Step 4: The plasmid is returned to the original salt concentration, and all of the plasmid regains the original B-DNA conformation. (Note: no nicking occurs in Step 4.)   Initial Step 1 Step 2 Step 3 Step 4 Step 5 L             Delta L             T             W             Nick? No Yes Yes No No You Decide

Curren'S Math For Meds: Dosages & Sol
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Author:CURREN
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Chapter16: Adult And Pediatric Dosages Based On Body Surface Area
Section: Chapter Questions
Problem 7.2P
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In this question, determine the values for the linking number (L), super helical turns (AL), the twisting

number (T) and the writhing number (W = supercoiling number) initially and after Steps 1 -4. Each part of your answer will be for the plasmid as a whole. It will be helpful to keep track of whether a nick is

present or not.

 

Below the table, explain your answers.

Assumptions:

  1. Choose a reference state as explained in class. Once you have chosen this reference state at the

outset of the problem, do not change it at any time throughout the problem. (After all, what good is a

reference state if it is not constant?)

  1. Throughout this problem, assume that all duplex DNA is B-DNA (10 bp/turn), except where it

is mentioned otherwise.

Initial State: A 5000 bp plasmid is completely in the B-DNA conformation under normal conditions and

has 25 underwindings (or 25 negative superhelical turns).

Step 1: The plasmid is nicked once in one strand and continues to be B-DNA.

Step 2: There is a region of 240 bp in the plasmid that is normally in the B-DNA conformation, butit can

be induced to adopt the A-DNA conformation by changing salt concentration. The plasmid DNA is

manipulated such that this B-DNA to A-DNA transition occurs in the 240bp region. The rest of the

plasmid remains B-DNA.

Important Hints:

1.A-DNA was NƠT discussed in class. Assume: right-handed, 12 bp/turn.

  1. After step 2, the plasmid is “relaxed", since it remains nicked. HOWEVER, it is

not in the same relaxed state as the reference state. In spite of the change in salt concentration, do not

change the reference state.

Step 3: The plasmid is re-ligated (i.e., the nick is re-sealed). You will have to think through whether

DNA Ligase changes DNA topology (is it a topological enzyme?), by thinking about the reaction

catalyzed by DNA ligase.

Step 4: The plasmid is returned to the original salt concentration, and all of the plasmid regains the

original B-DNA conformation. (Note: no nicking occurs in Step 4.)

  Initial Step 1 Step 2 Step 3 Step 4 Step 5
L            
Delta L            
T            
W            
Nick? No Yes Yes No No You Decide
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