Genetic Engineering
Restriction Endonucleases
EcoRI was the first E.Coli restriction endonuclease discovered. It was discovered when studying
how bacteria protect themselves from bacteriophages as they contain enzymes (restriction
endonucleases) which bind and cut short DNA sequences in the bacteriophage genome. EcoRI
binds the DNA double helix as a homodimer and uses Mg2+ to catalyse the cleavage of DNA.
Restriction endonucleases recognise palindromic sequences which are sequences of DNA which
are the same when read backwards and forwards.
DNA Ligase
Catalyses the formation of phosphodiester bonds in double-stranded DNA molecules. It requires
nearby 5’-phosphate and 3’-hydroxyl groups to undergo a condensation reaction. ATP is the
cofactor.
Plasmids
They have unique cloning sites where DNA can be inserted, drug resistant gene, and an origin of
replication.
1. Vector for Gene Insertion: Plasmids can be engineered to contain specific sequences that
facilitate the insertion of foreign DNA fragments. These sequences typically include
restriction enzyme recognition sites, which allow the plasmid to be cut open at specific
points.
2. DNA Manipulation: Researchers can cut both the plasmid DNA and the DNA fragment
they want to insert using the same restriction enzymes. This creates compatible ends on both
the plasmid and the foreign DNA fragment, which can then be joined together using DNA
ligase.
3. Transformation: Once the plasmid has been engineered to contain the desired foreign
DNA, it is introduced into a host organism through a process called transformation. Bacteria
are commonly used as host organisms for recombinant DNA experiments. During
transformation, the bacteria take up the engineered plasmids.
4. Replication and Expression: Inside the host organism, the recombinant plasmid replicates
along with the host DNA during cell division. The foreign DNA carried by the plasmid can
Genetic Engineering 1
Restriction Endonucleases
EcoRI was the first E.Coli restriction endonuclease discovered. It was discovered when studying
how bacteria protect themselves from bacteriophages as they contain enzymes (restriction
endonucleases) which bind and cut short DNA sequences in the bacteriophage genome. EcoRI
binds the DNA double helix as a homodimer and uses Mg2+ to catalyse the cleavage of DNA.
Restriction endonucleases recognise palindromic sequences which are sequences of DNA which
are the same when read backwards and forwards.
DNA Ligase
Catalyses the formation of phosphodiester bonds in double-stranded DNA molecules. It requires
nearby 5’-phosphate and 3’-hydroxyl groups to undergo a condensation reaction. ATP is the
cofactor.
Plasmids
They have unique cloning sites where DNA can be inserted, drug resistant gene, and an origin of
replication.
1. Vector for Gene Insertion: Plasmids can be engineered to contain specific sequences that
facilitate the insertion of foreign DNA fragments. These sequences typically include
restriction enzyme recognition sites, which allow the plasmid to be cut open at specific
points.
2. DNA Manipulation: Researchers can cut both the plasmid DNA and the DNA fragment
they want to insert using the same restriction enzymes. This creates compatible ends on both
the plasmid and the foreign DNA fragment, which can then be joined together using DNA
ligase.
3. Transformation: Once the plasmid has been engineered to contain the desired foreign
DNA, it is introduced into a host organism through a process called transformation. Bacteria
are commonly used as host organisms for recombinant DNA experiments. During
transformation, the bacteria take up the engineered plasmids.
4. Replication and Expression: Inside the host organism, the recombinant plasmid replicates
along with the host DNA during cell division. The foreign DNA carried by the plasmid can
Genetic Engineering 1
