Molecular Cloning & Restriction Enzymes

Question 1

What are Restriction Enzymes?

Answer 1

Biological defence systems against foreign DNA invasion (e.g. phage attack)

Question 2

How can a restriction site on DNA be modified to protect against restriction enzymes?

Answer 2

Methylation
(adds a Methyl group to A base)

Question 3

What size fragments move through agarose gel faster?

Answer 3

Small DNA fragments

Question 4

What is Gel Electrophoresis used for?

Answer 4

To separate DNA fragments based on size

Question 5

What is Southern Blotting used for?

Answer 5

Identification of desired DNA fragments using a labelled probe

Question 6

Which type of blotting uses RNA?

Answer 6

Northern blotting

Question 7

Which type of blotting involves protein-antibody interactions?

Answer 7

Western blotting

Covid test is based on Western blotting

Question 8

How is DNA denatured into a single strand in Southern blotting?

Answer 8

Incubation with NaOH

Question 9

What is the purpose of Cloning?

Answer 9

Isolate large quantities of a specific gene

Question 10

What is the process of Cloning?

Answer 10

1. Isolation & Fragmentation of the source DNA (restriction enzymes/PCR)
2. Joining the desired DNA fragment to a cloning vector (DNA ligase)
3. Introduction into a suitable host organism (transformation)

Question 11

Why do Plasmids make good cloning vectors?

Answer 11

• Capable of autonomous replication
• Contain suitable, unique restriction sites to insert foreign DNA into
• Exist in low or high copy number
• Small size (2-6kb)
• Selectable markers
• Easy to introduce into bacteria by transformation
• Easy to isolate from bacterial host
• Easy to examine by restriction analysis

Question 12

What plasmid was the original cloning vector?

Answer 12

pBR322