Question

Experiment 2- Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector

“Table 1: Fragment Lengths”

“DNA Type”	“Longest Length (in base pairs)”
“Foreign”	720
“Plasmid”	2804

1. What is the expected size of the plasmid plus the cut foreign DNA?

2. What type of ends to the enzymes BamHI and EcoRI produce? How does this type of end facilitate cloning?

3. What enzyme is necessary to permanently link the digested foreign and plasmid DNA together to form the recombinant DNA molecule? How does this enzyme work?

4. How would you clone a gene into a plasmid if there were no common restriction sites between the two DNA sequences?

Answer 1

1.The expected size is- 3524 basepairs.

2. 1st part: BamHI and EcoR1 produce STAGGERED ENDS.

2nd part: in cloning experiment, when a researcher wants to insert a foreign DNA in a plasmid, he/she prefers the use of restriction enzymes that produce staggering ends/sticky ends. This is because when the insert DNA molecule and the foreign DNA molecule both have their 2 ends sticky, in presence of the DNA ligase enzyme, the two DNA molecule just sticks to one another- (refer to the figure). In contrast, when the ends are blunt, there arises issue because the insert DNA molecule can get inserted in either direction and can also attach with its own instead of with the target-creating plasmid that is non-functional.

3. The enzyme that is needed is -DNA ligase. The DNA ligase actually attaches the exposed 5'-Phosphate group present at the end of one DNA molecule with the exposed 3'-OH group of the other DNA molecule and catalyses the formation of a phosphodiester bond between them. This reaction requires the presence of ATP or NAD.

4. If there are no common restriction sites in the two DNA molecule(the plasmid and the insert DNA), then an adaptor molecule can be attached with the two ends of the insert DNA Molecule(flanking in the two ends)- that would have a desired restriction site. Once attached, the desired restriction enzyme can be used