Wright 3 - Recombinant Technology

Question 1

What bond to restriction endonucleases cleave?

Answer 1

Phosphodiester bond

Question 2

What enzyme can be used to covalently join together complementary DNA molecules?

Answer 2

DNA ligase

Question 3

What does a cloning vector do?

Answer 3

Fools the bacterial host, commonly E. coli, into replicating a foreign DNA fragment.

A mini-chromosome called a plasmid, replicates autonomously in the bacterial host. The presence of the plasmid in the bacterium is detected by the bacterium’s resistance to an antibiotic.

Question 4

What is a cloning vector?

Answer 4

a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

Question 5

What type of genes do plasmid cloning vectors frequently carry?

Answer 5

They confer antibiotic resistance on the bacterial host

Question 6

What are restriction endonuclease sites on plasmid cloning vectors used as in cloning?

Answer 6

Employed as cloning (insertion) sites for foreign DNA

Question 7

Plasmid cloning vectors vary in size. When used in cloning, is big or small better?

Answer 7

small. Under 6 Kbp

Question 8

What does ampicillin do?

Answer 8

Inhibits the production of the cell wall around bacteria, causing it to lyse and die when treated with this antibiotic. A plasmid cloning vector can carry a ampicillin resistance gene for the bacteria

Question 9

What does tetracycline do?

Answer 9

Inhibits protein synthesis of bacterial cells by interrupting the function of the bacterial ribosome. A plasmid cloning vector can carry a tetracycline resistance gene for the bacteria.

Question 10

What does rifampicin do?

Answer 10

Inhibits bacterial RNA polymerase, thus transcription of bacterial genes is prevented. A plasmid cloning vector can carry a rifampicin resistance gene for the bacteria.

Question 11

How can cells containing recombinant DNA molecules be selected?

Answer 11

By growth in the presence of antibiotic. The plasmid cloning vector with an antibiotic resistance gene has a foreign DNA fragment inserted, therefore antibiotic resistance will indicate that the plasmid vector with the foreign DNA is incorporated into the host cell’s genome.