Restriction Enzymes and Agarose Gel Electrophoresis
Restriction Enzymes and Vectors-What are they and how do we use them?
Bacteria can produce DNases, which can double cleave the DNA at specific
Restriction endonucleases or restriction enzymes can cleave the DNA at
The 1 endonuclease to be identified is EcoRI isolated from E.Coli
Many other restriction enzymes have been isolated
Restriction enzymes can provide a defense system for DNA against viruses
Restriction enzymes remember different restriction sites
Majority of Restriction enzymes recognize either 4 or 6 base pairs
EX. EcoRI recognizes and cleaves GAATTC, HaeIII recognizes and cleaves
Restriction maps can be used to determine where the enzymes will cleave
Restriction enzymes play a key role in DNA cloning
The Basic Steps required to clone a gene are:
1. The fragment of DNA containing the gene to be cloned is inserted, with
restriction enzymes and ligation enzymes into a vector producing
recombinant DNA. Vector is usually circular but it can also be linear.
2. The vector acts as a vehicle to transport the gene into a host cell, which is
often a bacterium.
3. Host cell multiples producing multiple copies of the vector.
4. The recombinant DNA is replicated.
5. After a large number of cell divisions a colony can be produced
Two naturally occurring DNA
1. Small cir