Enzymes used in molecular cloning - L12

Question 1

Molecular cloning is a set of experimental methods used to assemble recombinant DNA. What are the 3 steps to do this?

Answer 1

1. Cut out the specific gene you want and paste into vector (to make recombinant DNA)
2. Transfer recombinant DNA to host.
3. Selection and replication.

Question 2

What is the role of restriction enzymes?

Answer 2

They recognise short, specific DNA sequences.

Question 3

What is the role of DNA ligase?

Answer 3

DNA ligase catalyses the formation of phosphodiester bonds (between phosphate and OH groups).

Question 4

What is the ligation reaction?

Answer 4

1. Complementary “sticky” ends have to interact.

2. DNA ligase catalyses the formation of a new phosphodiester bond.

Question 5

What is the role of endonuclease?

Answer 5

Cleaves (splits) DNA at specific sites.

Question 6

How is a 5’ phosphate group added and why?

Answer 6

T4 Polynucleotide kinase - where there is no phosphate and we need to add one in order for ligation to work

Question 7

How is a 5’ phosphate group removed and why?

Answer 7

Calf Intestinal Phosphatase (CIP) - to stop ligation happening between the wrong molecules as ligation cannot occur when the 5’ phosphate is removed.

Question 8

What is a vector?

Answer 8

A vector – carrier for your gene e.g a plasmid

Question 9

What are essential features common to all vectors?

Answer 9

1. Origin of replication – independent replication inside the host
2. Selectable marker – only survival of host cells that are carrying your plasmid
3. Multiple cloning site – (MCS), unique sites, where to put your gene.

Question 10

In molecular cloning, what is ‘transformation’ and how is it done?

Answer 10

Transformation: Getting plasmid into host = transformation in bacteria. Nearly always using a bacteria called E. coli

1. Electroporation: brief pulse of high-voltage
2. Chemical Transformation: Chemically treated E.coli, Add a heat-shock, Causes cell membrane changes that allow uptake of DNA.