3. Observe the diagram given for the length of the DNA strand degraded by two re

Question

3. Observe the diagram given for the length of the DNA strand degraded by two restriction enzymes X and Y The arrows indicate the regions that are attacked by the enzymes X and Y Enzyme Enzyme Enzyme Enzyme DNA Segment, Length (base pairs) 400 500 1,200 1,300 1,500 a) A mutation event takes place in the DNA strand. This results in 3 DNA fragments, when the strand is treated with enzyme Y. Interpret this and comment on the effect of mutation event on the restriction sites. (1 mark) ) Determine the approximate length of fragments generated when both the restriction enzymes are utilized together to digest the given DNA strand. (1 mark) c) If a short stretch of DNA is to be inserted in the given DNA strand using the enzyme X, design the appropriate protocol for stable insertion of the short DNA stretch in to the given strand at the site between 1300 and 1500 bp. (2 mark)

Explanation / Answer

a) For the above strand there is only one restriction site for enzyme y. So a digestion with enzyme y will yield two bands – one of size 900bp (400+500) and another sized 4000 (1200+1300+1500). The mutation event would have resulted in formation of one more restriction site for enzyme y. Two restriction sites for enzyme y in the DNA strand thus will yield three DNA fragments.

b) For the above strand a digestion using both the enzymes –y and x will yield DNA strands of lengths

400bp, 500bp, 1200bp, 1300bp and 1500bp

c) A digestion with enzyme x will yield 4 bands of 400, 1700 (500+1200), 1300 and 1500 bps. In order to cut only between 1300 and 1500 one should standardize a partial digestion protocol. Partial digestion is an incomplete digestion where the enzyme concentration and time of digestion is manipulated so that you will get a cut only at the desired position.   So set up a series of digestion of the given strand with enzyme x , with varying time and enzyme concentration to find out at what point and enzyme concentration one can yield only 2 bands with enzyme x that is, 3400bp (400+ 500+ 1200 +1300) and 1500 bp , as a result of cut between 1300 and 1500.

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