Restriction endonuclease
• A restriction enzyme, restriction endonuclease, or
restrictase is an enzyme that cleaves double
standard or single standard DNA into fragments at
specific Recognition nucleotide sequence known
as restriction site.
• Found naturally in a wide variety of prokaryotes
• An important tool for manipulating DNA
APEKSHA KESARWANI.
B.K.BIRLA COLLEGE
T.Y. BSC BIOTECH
Discovery
Arbor and Dussoix in 1962 discovered that certain
bacteria contain Endonucleases which have the ability
to cleave DNA.
In 1970 Smith and colleagues purified and characterized
the cleavage site of a Restriction Enzyme.
Werner Arbor, Hamilton Smith and Daniel Nathans
shared the 1978 Nobel prize for Medicine and
Physiology for their discovery of Restriction Enzymes.
Restrictions enzymes : Molecular scissors
• Most bacteria use Restriction Enzymes as a defence against
bacteriophages.
• Restriction enzymes prevent the replication of the phage by
cleaving its DNA at specific sites.
• The host DNA is protected by Methylases which add methyl
groups to adenine or cytosine bases within the recognition
site thereby modifying the site and protecting the DNA.
• What kind of bonds are broken by R.E.; 1.covalent
phosphodiester bonds between nucleotide (within single
strand)2. Hydrogen bonds ( Between strands)
• They are cut into twoends ;
• 1) Blunt ends :A straight cut, down through the DNA
that results in a flat pair of bases on the ends of
the DNA.
• Sticky End / cohesive : Staggered ends on a DNA
molecule with short, single-stranded overhang.
H
• Nomenclature
Each Enzyme is named after
the bacterium from which
it was isolated using a
naming system based on
bacterial genes, species,
and strain .
Applications
• Restriction enzymes are most widely used in recombinant
DNA technology.
• Isolated restriction enzymes are used to manipulate DNA
for different scientific applications.
• They are used to assist insertion of genesinto plasmid
vectors during gene cloning and protein expression
experiments.
• Restriction enzymes can also be used to distinguish gene
alleles by specifically recognizing single base changes in
DNAknown as single nucleotide polymorphisms (SNPs).
Application
• Restriction Enzymes can be used to generate a restriction map.
• This can provide useful information in characterizing a DNA molecule.
• Restriction Fragment Length Polymorphism – RFLP is a tool to study
variations among individuals & among species.
• Each of these methods depends on the use of agarose gel
electrophoresis for Separation of DNA fragments.
