Restriction EnzymesRestriction Enzymes Aims:
Must be able to recall the basic functions of restriction enzymes.
Should be able to outline how specific restriction enzymes work.
Could be able to explain the different ends produced by restriction enzymes and their benefits.
Restriction EnzymesRestriction Enzymes RESTRICTION ENZYMES = one of the essential tools of
genetic engineering.
Purified forms of these naturally occurring bacterial enzymes are used as “molecular scalpels”.
Allow genetic engineers to cut up DNA in a controlled way.
Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called RECOGNITION SITES.
By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers are able to isolate and sequence DNA
Restriction EnzymesRestriction Enzymes
Recognition SiteRecognition Site
GAATTC
CTTAAGDNA
CTTAAG
GAATTCcut
cut cut
restriction
enzyme
EcoRI cuts
here
Specific Recognition SequencesSpecific Recognition Sequences
Named according to the bacterial species first isolated from.
Followed by a number to distinguish different enzymes isolated from the same organism.
e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.
A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site:
Enzyme SourceRecognition Sites
EcoRIEscherichia coli
RY13GAATTC
BamHI
Bacillus
amyloliquefaciens
H
GGATCC
HaeIIIHaemophilus
aegyptiusGGCC
HindIIIHaemophilus
influenzae RdAAGCTT
HpalHaemophilus
parainfluenzaeGTTAAC
C T T A A
A A T T C G
G
FragmentRestriction
enzyme: EcoRI
Sticky endRestriction enzyme: EcoRI
DNA from another
source
A restriction enzyme cuts the double-
stranded DNA molecule at its specific
recognition site
The two different fragments cut
by the same restriction
enzyme have identical sticky
ends and are able to join
together
The cuts produce a
DNA fragment with
two “sticky” ends
When two fragments of DNA cut by the same
restriction enzyme come together, they can join by
base-pairing
C T T A A
A A T T C
G
G A A T T C
C T T A AG
G
C T T A A
A A T T C G
G
It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”.
DNA cut in such a way produces ends which may only be joined to other sticky ends with a complementary base sequence.
Sticky EndsSticky Ends
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
Blunt EndsBlunt Ends
Possible to use restriction
enzymes that cut leaving no
overhang; a so-called “blunt
end”.
DNA cut in such a way is
able to be joined to any
other blunt end fragment,
but tends to be non-specific
because there are no sticky
ends as recognition sites.
Restriction enzyme
cuts here
Recognition Site Recognition Site
DNA from another source
The cut by this type of restriction
enzyme leaves no overhang
cutcut
C C C
G G G
G G G
C C C
C C C
G G G
G G G
C C C
G G G
G G G
C C C
C C C
DNA
A special group of
enzymes can join
the pieces together