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Restriction Endonuclease as
Molecular ScissorsPresented By:-
Avni JethvaM.Sc Microbiology
(Sem2)
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Content
Introduction
History
Restriction enzymes Nomenclature
Different recognition sequences
Classification Applications
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Introduction
Bacteria possess restriction systems as
protection mechanism against incoming
DNA.
This system employs restriction enzymes
like endo- & exo- nucleases.
Nucleases cleave phosphodiester bonds
between nucleotides.
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History
1962-Arber & Dussoix :E.coli can restrict lambdaphage DNA
1965-Arber: E.colimethylate their DNA
1968-1st restrictionendonuclease in E.colidiscovered
1970-Smith, Wilcox &Kelly: Discovered 1st DNA-specific restrictionendonuclease inH.influenzae
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Restriction enzymes
Specific endonucleases
Recognize specific short sequences of DNA andcleave the DNA at or near the recognitionsequence
Recognition sequences: usually 4 or 6 bases butthere are some that are 5, 8, or longer
Recognition sequences are palindromes
Palindrome: sequence of DNA that is the samewhen one strand is read from left to right or theother strand is read from right to left consists ofadjacent inverted repeats
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Blunt & Sticky ends
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Nomenclature
Restriction enzymes are named based on the bacteriain which they are isolated in the following manner:
Derive names from the bacteria
Genus- first letter capitalizedSpecies- second and third letters (small case)
Additional letters from strains
Roman numeral designates different enzymes
from the same bacterial strain, in numerical order
of discoveryExample: EcoRI
E Escherichia
co coli
R R strain
I first enzyme discovered fromEscherichia
coliR
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Different Recognition Sequences
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Classification
3 Basic types:-1)TypeI - cleavage occurs at random
site, so not used for gene manipulation.
2)TypeII recognize a particular
target in duplex DNA, so give fragments of
defined length.
eg: SmaI (blunt ends)
BamHI (sticky ends)
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3)TypeIII(rare) - asymmetric recognition
sites, nicks at measured distances from
recognition sequences. Not used for gene
manipulation.
eg: EcoRI
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The EcoRV-DNA complex
PDB code: 1RVC
Only the DNA from the structure isshown.
The backbone of the segmentsseparated by cleavage are shown indifferent shades of blue.
The orange atoms are magnesium ionsbound to the DNA that play a role in thecleavage reaction.
These ions are located at the cleavagesite; blunt ends are created.
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The EcoRV-DNA complex
The EcoRVendonucleasehas two sub-units (shown in
blue and gray). The DNA is
highlydistorted
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Applications
rDNA Technology
Plasmid Insertion
Southern Blotting DNA Sequencing
PCR
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DNA Sequencing.
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PCR