Enzymes are very important tools for gene cloning.

3. Introduction
• Restriction Endonucleases are enzymes that produce
internal cuts, called cleavage, In the DNA molecule.
• Restriction Endonuclease (Restriction Enzyme) is a bacterial
enzyme that cuts dsDNA into fragments after recognizing
specific nucleotide sequence known as recognition or
restriction site.
• Restriction Enzymes are believed to be evolved by bacteria to
resist viral attack.
• Restriction Enzymes are also known as molecular scissor.

4. • Restriction Enzymes scan the DNA sequence
• Find a very specific set of nucleotides
• Make a specific cut

5. Palindromes in DNA sequences
Genetic palindromes
are similar to verbal
palindromes. A
palindromic sequence
in DNA is one in which
the 5’ to 3’ base pair
sequence is identical
on both strands.
5’
5’
3’
3’

6. The discovery and function of Restriction Endonucleases
The initial observation that led to the eventual discovery of
restriction endonucleases was made during the early 1950s,
when some strains of bacteria were shown to be immune to
bacteriophage infection, a phenomenon referred to as host-
controlled restriction.
The mechanism of restriction is not very complicated, even
though it took over 20 years to be fully understood. Restriction
occurs because the bacterium produces an enzyme that
degrades the phage DNA before it has time to replicate and
direct the synthesis of new phage particles

10. Type I
• Capable of both restriction and modification
activities
• The co factors S-Adenosyl Methionine(AdoMet),
ATP, and mg+are required for their full activity
• Contain:
two R (restriction) subunits
two M (methylation) subunits
one S (specifity) subunits
• Cleave DNA at random length from recognition
sites

11. Type II
• These are the most commonly available and used
restriction enzymes
• They are composed of only one subunit.
• Their recognition sites are usually undivided and
palindromic and 4-8 nucleotides in length,
• They recognize and cleave DNA at the same site.
• They do not use ATP for their activity
• They usually require only Mg2+ as a cofactor.

12. Type III
• Type III restriction enzymes ) recognize two separate
non-palindromic sequences that are inversely
oriented.
• They cut DNA about 20-30 base pairs after the
recognition site.
• These enzymes contain more than one subunit.
• And require AdoMet and ATP cofactors for their roles
in DNA methylation and restriction

13. Types of Restriction Enzymes
Cleavage
site
Location of
methylase
Requirements for
activity Examples
Type
I
Random,
Around 100-
1000bp away
from recogni-
tion site
Endonuclease
and methylase
located on a
single protein
molecule
co factors S-Adenosyl
Methionine (AdoMet)
, ATP and mg+
EcoK I
EcoA I
CfrA I
Type
II
Specific,
Within the
recognition
site
Endonuclease
and methylase
are separate
entities
Mg2+ EcoR I
BamH I
Hind III
Type
III
Random
20-30 bp
away from
recognition
site
Endonuclease
and methylase
located on a
single protein
molecule
AdoMet and ATP
cofactors
EcoP I
Hinf III
EcoP15 I

14. Restriction fragments can be blunt ended
or sticky ended
5’ G A A T T C 3’ 5’ G A T A T C 3’
3’ C T T A A G 5’ 3’ C T A T A G 5’
Sticky Ends Blunt Ends
Sticky ends or blunt ends can be used to join DNA
fragments.
Sticky ends are more cohesive compared to blunt ends.

15. blunt end
sticky end

17. • Isoschizomers are restriction enzymes that have the same recognition
sequence and the same specificity. For instance, AgeI and BshT1 recognize and
cleave 5′-A↓CCGGT-3′ in the same pattern. Nevertheless, a set of
isoschizomers may differ in site preferences, reaction conditions, methylation
sensitivity, and star activity. (Learn more: Restriction enzyme isoschizomers
and key considerations).
• Neoschizomers recognize the same nucleotide sequence but cleave DNA at
different positions. Examples of neoschizomers are SmaI (5′-CCC↓GGG-3′) and
XmaI (5′-C↓CCGGG-3′), which both recognize 5′-CCCGGG-3′ but cleave them
differently and thus generate different types of ends (in this case, blunt ends
for SmaI and 5′ protruding ends for XmaI).
Isoschizomers and Neochischizomers