Restriction enzyme

1. Restriction Enzyme

2. restriction enzymes
,Also called restriction Endonucleses
1962: “molecular scissors” discovered in in bacteria
E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have
unique properties, many are purified and available
commercially

3. •
Molecular scissors that cut double stranded
DNA molecules at specific points.
•
Found naturally in a wide variety of
prokaryotes

4. 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

5. Restriction Enzymes
• Restriction enzymes are endonucleases (Endo (inside),
nuclease(cuts nucleic acid), which catalyze the cleavage
of the phosphodiester bonds within both strands of
DNA.
• They require Mg+2 for activity and generate a 5 prime
(5') phosphate and a 3 prime (3') hydroxyl group at the
point of cleavage.

6. • The distinguishing feature of restriction enzymes is
that they only cut at very specific sequences of bases.
This specific DNA sequence is called recognition
sequence.
Restriction enzymes are traditionally classified according
to the subunit composition, cleavage position,
sequence-specificity and cofactor requirements.

7. • A restriction enzyme requires a specific double stranded
recognition sequence of nucleotides to cut DNA.
• Recognition sites are usually 4 to 8 base pairs in length.
• Cleavage occurs within or near the site.

8. • Restriction enzymes are named according to the
organism from which they are isolated.
• This is done by using the first letter of the genus
followed by the first two letters of the species and
additional letter or number represent the strain or
serotypes.
• Only certain strains or sub-strains of a particular
species may produce restriction enzymes.
Naming of Restriction enzymes

9. Mechanism of Action
•
Restriction Endonuclease scan the length of
the DNA , binds to the DNA molecule when it
recognizes a specific sequence and makes one
cut in each of the sugar phosphate backbones
of the double helix – by hydrolyzing the
phoshphodiester bond. Specifically,the bond
between the 3’ O atom and the P atom is
broken.

10. Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1

11. Example of RE naming:
HindII: Restriction enzyme was isolated from
Haemophilus influenzae serotype d
• There are hundreds of different REs from different
microorganisms.
• Different restriction enzymes have different
recognition sequences.
• This makes it possible to create a wide variety of
different gene fragments.

12. Examples:
Enzyme Organism from which derived
Target sequence
(cut at *)
5' -->3'
Bam HI Bacillus amyloliquefaciens G* G A T C C
Eco RI Escherichia coli RY 13 G* A A T T C
Hind III Haemophilus inflenzae Rd A* A G C T T
Mbo I Moraxella bovis *G A T C
Pst I Providencia stuartii C T G C A * G
Sma I Serratia marcescens C C C * G G G
Taq I Thermophilus aquaticus T * C G A
Xma I Xanthamonas malvacearum C * C C G G G

13. Restriction enzymes recognize specific 4-8 bp sequences
Some enzymes cut in a staggered fashion - “sticky ends”
EcoRI 5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion – “blunt ends”
PvuII 5’…CAGCTG…3’
3’…GTCGAC…5’

17. Many recognition sequences are palindromic. For
example,
5’ GAATTC 3’
3’ CTTAAG 5’
palindromic: read the same in the opposite
direction

18. Sticky and Blunt end cutters
Not all restriction endonucleases cut symmetrically
and leave blunt ends.
Many endonucleases cleave the DNA backbones in
positions that are not directly opposite each other or
can make staggered cuts, which produce single
stranded “sticky-ends”
DNA from different sources can be spliced easily
because of these sticky-end overhangs.

19. Example of RE that produce sticky end cutters:

20. Some restriction enzymes cut DNA at opposite
base.
They leave blunt ended DNA fragments
These are called blunt end cutters
HaeIII

21. Types of Restriction Enzymes
Cleavage
site
Location of
methylase
Examples
Type I Random
Around 1000bp
away from
recognition site
Endonuclease
and methylase
located on a
single protein
molecule
EcoK I
EcoA I
CfrA I
Type II Specific
Within the
recognition site
Endonuclease
and methylase
are separate
entities
EcoR I
BamH I
Hind III
Type III Random
24-26 bp away
from recognition
site
Endonuclease
and methylase
located on a
single protein
molecule
EcoP I
Hinf III
EcoP15 I

22. Isoschizomers and
Neochischizomers
•
Two Restriction enzymes that have the same recognition sequence as well
as the same cleavage site are Isoschizomers.
For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each
other
•
Restriction enzymes that have the same recognition sequence but cleave
the DNA at a different site within that sequence are Neochizomers.
Eg:SmaI and XmaI
C C C G G G
C C C G G G
G G G C C C G G G C C C
Xma I Sma I

23. Restriction Enzyme Uses
1. Recombinant DNA technology
*Discovery of enzymes that cut and paste DNA make
genetic engineering possible.
*Restriction enzyme cuts DNA and generates fragments
*Ligase joins different DNA fragments
*DNA fragments from different species can be ligated
(joined) to create Recombinant DNA

25. 2. Cloning
Replicates a sequence inserted into a host
cell
3. DNA restriction mapping
The location of the restriction enzyme cleavage
sites on the DNA molecule.

27. • Restriction enzyme is part of the cell’s
restriction-modification system in bacteria.
• The phenomenon of restriction modification in
bacteria is a small scale immune system for
protection from infection by foreign DNA.
• Bacteria can protect themselves only after foreign
DNA has entered their cytoplasm.
Biological Function

28. •
Restriction Enzymes can
be used to generate a
restriction map. This
can provide useful
information in
characterizing a DNA
molecule.

29. Restriction Fragment Length
Polymorphism is a tool to study variations
among individuals & among species

30. Why don’t bacteria destroy their own DNA
with their restriction enzymes?

31. Unit Determination Assay
• One unit of restriction endonuclease is defined
as the amount of enzyme required to digest
one microgram of the appropriate substrate
DNA completely in 60 minutes under the
conditions specified for that enzyme.

32. Set up of a restriction enzyme reaction
• A RE reaction contains the DNA to be
analyzed,
• A restriction enzyme,
• A restriction enzyme buffer mix.
–contains a buffering agent to maintain
constant pH,
–and Mg++ (from MgCl2) as a necessary
cofactor for enzyme activity.

33. Digestion by Restriction Enzyme
• Measure the DNA concentration
– Use the Nano-drop spectrophotometer to measure
the concentration of DNA, this is used to determine
the amount of HinfI restriction enzyme to be used.
Digestion of DNA
• Mix the following components in a clean microtube.
• Mix gently and spin down for a few seconds.
• Incubate at 37°C for 16 hours.