W11 Gene Cloning

Question 1

Restriction endonucleases:

Answer 1

-RE hydrolyse ‘cleave’ phosphodiester bonds between adjadcent nucleotides
-They recognise a specific sequence in the DNA called a recognition site
-3 types of RE: Type 1, 2, 3
-Type 1 and 3 both have restriction and modification activity, requiring ATP, and cut away from recognition site

Question 2

Type 2 RE:

Answer 2

-Used in molecular biology
-Have restriction activity but no modification ability
-Cut in predictable manner at recognition sequence
-Require only Mg2+ as cofactor, ATP not required

Question 3

Naming Type 2 RE’s:

Answer 3

-Hind3, EcoR1, BamH1
-Name refers to genus, species, strain and isolation order of bacterial enzyme

Example:
EcoRI:
Genus and species: Escherichia coli
Strain: R
Order isolated: 1

Question 4

Recognition sequences:

Answer 4

-Type 2 RE’s typically 4-8 bp long
-Cleaving these sequences can be carried out in several ways producing Blunt ends, 5’ or 3’ overhanging (sticky ends)

Question 5

Plasmids:

Answer 5

-Bacteria contains small circular DNA molecules involved in bacterial gene transfer
-Contain an origin of replication & antibiotic resistance gene
-Have a mutliple cloning site

Question 6

Multiple cloning site:

Answer 6

-Also called polylinkers
-Collection of RE sites occurring next to one another
-These sites are unique (cut only once) and occur nowhere else in the plasmid

Question 7

Gel electrophoresis:

Answer 7

-DNA samples added into wells formed in the gel, gel submerged in buffer
-Gel subjected to electric field
-DNA has net negative charge so migrates towards +ve pole of electric field
-DNA molecules become separated by size (distance moved is inversely proportional to size
-DNA is visualised as ‘bands’ on the gel by staining
-Restriction digestion of a recombinant molecule releases the plasmid vector (of constant size) and the insert