Cloning

Question 1

What are restriction endonuclease?

Answer 1

Cleave ds DNA
Recognise 4-8bp sequence
Assymetric cuts with 5’ or 3’ sticky ends
But can leave blunt ends
Recognition sites are usually palindromes
Named after bacteria

Question 2

What are restriction enzymes?

Answer 2

Digest DNA- recognise specific sequences in DNA
Natural defence mechanism in bacteria
Hundreds of restriction enzymes
Commonly used in molecular biology

Question 3

What are plasmids?

Answer 3

Double stranded circular DNA
Typically a few kb
Replicate independently of host chromosome (quicker)
Due to their artificial nature, lab plasmids are commonly refered to as vectors or constructs

Question 4

What is the role of DNA ligase?

Answer 4

‘Glue’ can join two DNA molecules with matching ends

Question 5

Where do restriction enzymes bind?

Answer 5

To specific sequences of DNA called restriction sites
Each enzyme recognises only a few restriction sites

Question 6

Is the cut double stranded and equal?

Answer 6

The cut is double stranded but it isn’t always equal, it often leaves ‘overhangs’ called sticky ends

Question 7

Do all restriction enzymes leave sticky ends?

Answer 7

No some do blunt cut SmaI is an example of this

Question 8

Which is easier to stick together blunt or sticky ends?

Answer 8

Sticky, blunt ligation reaction is less efficient and more likely to fail