Restriction enzymes are molecular scissors found in bacteria that cut DNA at specific recognition sequences. They break the covalent bonds within a DNA strand and the hydrogen bonds between the strands. Bacteria use these enzymes to protect themselves from foreign DNA. Restriction enzymes are now used widely in laboratories to cut DNA in recombinant DNA techniques and other applications like DNA fingerprinting. They produce sticky or blunt ends that allow targeted joining of DNA fragments.

2. Restriction Enzymes: Molecular Scissors
Restriction enzymes
(endonuleases) cut
DNA at specific
sequences
What kinds of bonds
are broken when
restriction enzymes
cut?
Covalent bonds (within
a single strand)
Hydrogen bonds
(between strands) as a
result of the strands
Hydrogen
bond
Covalent bond

3. Origins of Restriction Enzymes
Naturally found in different types of bacteria
Bacteria use restriction enzymes to protect themselves
from foreign DNA
Bacteria have mechanisms to protect themselves from the
actions of their own restriction enzymes
Have been isolated and sold for use in lab work

4. Restriction EndonucleasesRestriction Endonucleases
Also called restriction enzymes
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

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

6. Restriction EndonucleasesRestriction Endonucleases
Recognition sites have symmetry (palindromic)
“Able was I I saw Elba”
Bam H1 site:
5’-GGATCC-3’
3’-CCTAGG-5’

7. Restriction EndonucleasesRestriction Endonucleases
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’

8. Sticky Ends vs. Blunt Ends
When restriction enzymes cut, they produce
either
Sticky ends (single stranded sections at the ends)
Blunt ends
5’ - - - G A A T T C - - - 3’
I I I I I I I
3’ - - - C T T A A G - - - 5’
Sticky ends

9. Examples of Restriction Enzymes
Enzyme Organism source Recognized
Sequence
EcoRI Escherichia coli 5' GAATTC 3’
3' CTTAAG 5’
TaqI Thermus aquaticus 5' TCGA 3’
3' AGCT 5’
HindIII Haemophilus influenzae 5'AAGCTT 3’
3'TTCGAA 5’
BamHI Bacillus amyloliquefaciens 5' GGATCC 3’
3' CCTAGG 5’
AluI Arthrobacter luteus 5' AGCT 3’
3' TCGA 5’

11. Restriction EndonucleasesRestriction Endonucleases
Why don’t bacteria destroy their own
DNA with their restriction enzymes?

12. Recombinant DNA
Recombinant DNA is
constructed using
restriction enzymes
Important: In order to
join 2 pieces of DNA
together they have to be cut
by the same restriction
enzyme
Why?
Otherwise, the sticky ends
won’t match– DNA can’t bind
together

13. Recombinant PlasmidsStep 1: Gene of
interest and
plasmid are cut
with the same
restriction
enzyme
Step 2: mix
together
Step 3: Add
DNA Ligase to
seal DNA back
together

14. MethylationMethylation

15. Uses for Restriction EnzymesUses for Restriction Enzymes
RFLP analysis (Restriction Fragment Length Polymorphism)
DNA sequencing
DNA storage – libraries
Transformation
Large scale analysis – gene chips

16. Human DNA cleaved with EcoRI Corn DNA cleaved with EcoRI
5’-C-G-G-T-A-C-T-A-G-OH
3’-G-C-C-A-T-G-A-T-C-T-T-A-A-PO4
PO4
-A-A-T-T-C-A-G-C-T-A-C-G-3’
HO-G-T-C-G-A-T-G-C-5’
+
5’-A-C-G-G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T-G-C-5’
Complementary base pairing
+ DNA Ligase, + rATP
recombinant DNA molecule
5’-A-C-G-G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A-C-G-3’
3’-T-G-C-C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T-G-C-5’
Restriction Enzymes for TransformationRestriction Enzymes for Transformation

17. _
+
DNA is negatively
charged from the
phosphate backbone
Visualize DNA with ethidium
bromide – fluoresces ONLY
when bound to DNA
Restriction Enzymes for RFLPRestriction Enzymes for RFLP