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RESTRICTION ENZYMES
RESTRICTION ENZYMES
classified as endonucleases
(cutting within).
Their biochemical activity is the
hydrolysis ("digestion") of the
phosphodiester backbone at
specific sites in a DNA
sequence.
Specific" means that an enzyme
will only digest a DNA molecule
after locating a particular
sequence.
are
AN EXPERIMENT USING
RESTRICTION ENDONUCLEASE:
BOYER AND COHEN
This early experiment used
EcoRI to cut 2 plasmids, small
circular pieces of DNA
independent of the host
chromosome
Each plasmid had 1 EcoRI site
Cutting converted circular
plasmids into linear DNA with
the same sticky ends
The ends base pair
Some ends re-close
Others joint together
DNA ligase joins 2 pieces with
covalent bonds
RESTRICTION ENZYMES
Restriction endonuclease EcoRI cleaves double-stranded
DNA.
The recognition site for EcoRI is the hexameric sequence
GAATTC:
5' . .NNNNGAATTCNNNN ... 3'
3' . .NNNNCTTAAGNNNN ... 5'
NAMING RESTRICTION
ENDONUCLEASES
Restriction endonucleases are named using the 1st three
letters of their name from the Latin name of their source
microorganism eg Hind III
First letter is from the genus H from Haemophilus
Next two letters are the 1st two letters of the species name
in from influenzae
Sometimes the strain designation is included
d from strain Rd
If microorganism produces only 1 restriction enzyme, end
the name with Roman numeral I Hind I
If more than one restriction enzyme is produced, the others
are numbered sequentially II, III, IV, etc.
NOMENCLATURE OF RE
RE names are based on a species-of-origin.
Taq polymerase (Thermophilus aquaticus, or Thermus
aquaticus).
Eg. EcoR1 (from Escherichia coli)
BamHI (from Bacillus amyloliquifaciens)
Sma I (from Serratia marcescens)
Mlu I (from Micrococcus luteus)
Hpa I (from Haemophilus parainfluenzae)
RE RECOGNITION SEQUENCE
AND TYPE OF ENDS IN PRODUCT
BamHI
G^GATCC
5' overhang
SacI
GAGCT^C
3' overhang
SmaI
CCC^GGG
blunt
meaning
that
they
could
hydrogen bond
to
other
compatible
complementary strands
These
BLUNT ENDS
RESTRICTION ENDONUCLEASE
SPECIFICITY
Restriction endonucleases
recognize a specific DNA
sequence, cutting ONLY
at that sequence
They
They
cut DNA
reproducibly in the
same place
RESTRICTION ENDONUCLEASE
SPECIFICITY
65,000 bp)
The frequency of cuts lessens as
the recognition sequence is
longer
RESTRICTION-MODIFICATION
SYSTEM
What
prevents these
enzymes from cutting up
the host DNA?
They are paired with
methylases
Theses enzymes recognize,
methylate the same site
Together