2. Example of Restriction Mapping (7 point)

In the lecture, you have seen an example of how the restriction mapping of a linear DNA can be done with the information from the electrophoretic separation of fragments.

Table 1: DNA fragment sizes after single and double restriction enzymatic digestion of a circular DNA

 

Table 1: DNA fragment sizes after single and double restriction enzymatic digestion of a circular DNA

  

 

EcoRI

 

HindIII

  

EcoRI+HindIII

  

3kb, 7kb

3kb, 7kb

2kb, 3kb

 

(For clear grading purpose, please answer each of the following questions separately, if an

answer to a question(s) requires information/work from a previous section (e.g., answer for ii

requires answer in i) re-write the previous answer/information into the current answer.

Question vi, on the other hand, can be a stand-alone answer. Question vii will be a combination

of answer in vi and ii))

1. Draw a circle and put in the first site of one enzyme arbitrarily (e.g., at 12 o’clock position in your circle). Must label the cut with one of the enzymes of your choice.
2. Put in the second site of that same enzyme based on the fragment size. Must label the cut with the enzymes of your choice.
3. Add up the size of fragments from each of three different restriction digests separately.
4. Compare the sum of fragments size from the single digestions with the double digestions to determine whether there is more than one fragments of the same size. Justify your answer. (Hint: remember that fragment of the same size will migrate the same distance in electrophoresis and are not distinguishable if in the same lane)
5. If enzymatic digestion results in more than one fragment of the same size, find out how many there are. Justify your answer. (Hint: The total size should add up to the size of the circular DNA)
6. Based on your information about the total number of fragments of each size, estimate the cleavage sites of the second enzyme. Must label all the cuts accordingly
7. Put in sites for a second enzyme, and distance it relative to site of first enzyme. Must label all the cuts accordingly 

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