Question 3:

Comment on the advantages and disadvantages of using (a) Agarose and (b) Polyacrylamide gels for the separation of DNA molecules.

Agarose gel Polyacrylamide gel

Advantages

-Gel is easily poured-Gel does not denature the

samples-Samples can be recovered

-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

Disadvantages

-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.

-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.