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Question 3: Comment on the advantages and disadvantages of using (a) Agarose and (b) Polyacrylamide gels for the

separation of DNA molecules.


Agarose gel -Gel is easily poured -Gel does not denature the samples -Samples can be recovered


-Gels may melt during electrophoresis, if temperature is too high due to high voltage -Buffer might become exhausted -Different types of genetic material may run in unpredictable ways -Gel is carcinogenic -Can only run nucleic acid fragments of 100 nucleotides to around 10-15kb, not less.

Polyacrylamide gel -Can run fragments as small as 10bp, with resolution of 1bp -High holding capacity of up to 10 mg into each cell -Does not contain many enzymic inhibitors -Pores formed are more controlled and uniform in size -Cannot run fragments above 1kb -mobility of fragments affected by base composition, makes accurate sizing difficult -Bands below 25ng will be hard to visualize because of quenched fluroscence

Question 4: Why is digestion of DNA with enzymes HIND III and Eco RI also known as restriction digest ? Restriction enzymes are enzymes that bind to specific DNA sequences and cleave, or digest the DNA at or next to the binding site. Both HIND III and Eco RI recognize 6 base pair restriction sites, which are symmetrical and inverted sequences. Both produce 5 overhanging ends. They are called restriction enzymes because they cut within the molecule of DNA, at a specific site. The DNA molecule is first scanned for a particular sequence, then the restriction enzyme binds to that location and makes one cut each in each of the two sugar phosphate backbones of the double helix.