Restriction Endonuclease as

Molecular Scissors
Presented By:-

Avni Jethva
M.Sc Microbiology
(Sem2)
 Content
• Introduction
• History
• Restriction enzymes
• Nomenclature
• Different recognition sequences
• Classification
• Applications
 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.
 History
• 1962-Arber & Dussoix :
E.coli can restrict lambda
phage DNA
• 1965-Arber: E.coli
methylate their DNA
• 1968-1st restriction
endonuclease in E.coli
discovered
• 1970-Smith, Wilcox &
Kelly: Discovered 1st DNA-
specific restriction
endonuclease in
H.influenzae
 Restriction enzymes

• Specific endonucleases
• Recognize specific short sequences of DNA and
cleave the DNA at or near the recognition
sequence
• Recognition sequences: usually 4 or 6 bases but
there are some that are 5, 8, or longer
• Recognition sequences are palindromes
• Palindrome: sequence of DNA that is the same
when one strand is read from left to right or the
other strand is read from right to left– consists of
adjacent inverted repeats
Blunt & Sticky ends
 Nomenclature
• Restriction enzymes are named based on the bacteria
in which they are isolated in the following manner:

Derive names from the bacteria

Genus- first letter capitalized
Species- second and third letters (small case)
Additional letters from “strains”
Roman numeral designates different enzymes
from the same bacterial strain, in numerical order
of discovery
Example: EcoRI
E Escherichia
co coli
R R strain
I first enzyme discovered from Escherichia
coli R
Different Recognition Sequences
 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)
3)TypeIII(rare) - asymmetric recognition
sites, nicks at measured distances from
recognition sequences. Not used for gene
manipulation.
eg: EcoRI
 The EcoRV-DNA complex

• Only the DNA from the structure is

shown.
• The backbone of the segments
separated by cleavage are shown in
different shades of blue.
• The orange atoms are magnesium ions
bound to the DNA that play a role in the
cleavage reaction.
• These ions are located at the cleavage
site; blunt ends are created.

PDB code: 1RVC

The EcoRV-DNA complex

• The EcoRV
endonuclease
has two sub-
units (shown in
blue and gray).
• The DNA is
highlydistorted
…
 Applications
• rDNA Technology
• Plasmid Insertion
• Southern Blotting
• DNA Sequencing
• PCR
DNA Sequencing.
PCR