Deck 15: The Action of Restriction of Endonucleases on Plasmid or Viral DNA

Question 1

How does the EDTA quench buffer stop the restriction enzyme reaction?

Answer 1

In this problem, we are asked the way in which the EDTA buffer stops the restriction enzyme reaction.
EDTA, an abbreviation for ethylene diamine tetra acetic acid, binds metal ions very effectively; it works as a chelating compound.
Restriction enzymes need magnesium ions to function properly. EDTA chelates these metal ions out of solution, making them unavailable to support the activity of the enzyme, thus stopping the process.

Question 2

Why are glycerol and bromophenol blue dye added to the gel-loading buffer?

Answer 2

In this problem, we are asked why we would use glycerol and bromo phenol blue in our buffer for our polyacrylamide gel.
Glycerol is a viscous sugar-alcohol; it makes the sample thicker and stickier, thus easing loading onto the gel lanes.
Bromo phenol blue is a tracking dye; most substances loaded onto a gel cannot be seen with the naked eye as they move through the gel. The bromo phenol blue can be seen during operations and thus we can see how the gel is progressing.

Question 3

Describe the pattern obtained from electrophoresis of a standard Eco RI digest of ? phage DNA oN₂% agarose gel.

Answer 3

In this problem, we are asked to describe the polyacrylamide gel (PAGE) that would result from the digestion of ? phasge using the restriction endonuclease Eco RI.
The phage in question is linear and double-stranded and has more than 48,000 base-pairs. Eco RI is a nuclease derived from E. coli , and it cleaves this phage into six fragments. Therefore, a PAGE run separating these fragments would have six bands.

Question 4

Why is polyacrylamide electrophoresis not suitable for analysis of re­striction enzyme fragments of most DNA?

Question 5

How many DNA fragments result from the action of the restriction en­zyme Hae II on the plasmid pBR322?

Question 6

Assume the reaction digest from Problem 5 is analyzed by agarose elec­trophoresis. Draw an electrophoresis gel and show the approximate location of each DNA fragment.

Question 7

Explain the action of ethidium bromide in locating the DNA fragments after electrophoresis.

Question 8

Ethidium bromide is a mutagen. Explain all precautions you took dur­ing this experiment in order to avoid contact with the chemical.

Question 9

What assumptions must be made about the relative mobility of bromophenol blue dye and DNA fragments during electrophoresis?

Question 10

Which of the following base sequences are probably not recognition sites for cleavage by restriction endonucleases? Why not?
(a) 5′GAATTC3′
(b) 5′CATTAG3′
(c) 5′CATATG3′
(d) 5′CAATTG3′