Fergus - Restriction Enzymes Flashcards
What do restriction enzymes do?
They recognize and make a cut within specific palindromic sequences, known as restriction sites in DNA
Enzymes which recognise and cut restriction sites in DNA
What are restriction sites?
Specific palindromic sequences in DNA that can be recognised by restriction enzymes
How big are restriction sites?
Between 4 and 8 base pairs long
The frequency of a restriction site in DNA relates to the number of base pairs in the sequence, what does this mean?
Restriction sites that are shorter e.g. 4 bps occur on average more frequently than longer restriction sites
How are restriction enzymes used in cell based cloning
The DNA and bacterial plasmid must be cut using the same restriction enzyme
How do bacteria use restriction enzymes?
(3)
Bacteria defend against viral infections by means of restriction-modification complexes
Restriction enzymes recognise and cleave viral DNA
Modification enzymes keep host DNA methylated
Give the different type of restriction endonucleases
Type 1
Type II
Type III
Write a note on type II endonuclease
(5)
Single subunit
Most common
Cleave DNA within recognition sequence
Over 3000 of this type of endonuclease
250 available commercially
How does type II endonucleases work?
(3)
Consist of a single subunit
Cut both strands at a specific palindromic recognition site of (4-8) bps long
Endonuclease and methylase components are separate, single subunit enzymes
Give an example of a type II restriction enzyme
Hae III
How does Hae III work?
Searches DNA until it finds either a (5’->3’ GGCC) or (3’ <– 5’ CCGG)
It then cleaves at this site -> split between the Gs and Cs
Produces blunt ends
Give an example of a restriction enzyme that produces sticky ends
EcoR1
Where do we get restriction enzymes/
They are enzymes found in bacteria
Explain how BamH1 is used to create recombinant DNA
(3)
BamH1 works on ‘DNA molecule A’ and ‘DNA molecule B’
This produces sticky ends from both A and B
Mix these ends in the presence of DNA ligase and recombinant DNA will form
