Download SBI4U MG Restriction Enzymes

2017-04-04
Restriction Endonucleases!
Mr.Masri
SBI4U
Humberside C. I.
Restriction Endonucleases (RE)!
!  also called restriction enzymes!
!  naturally found in bacteria!
!  used as a defense mechanism
against bacteriophages by degrading
their DNA!
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2017-04-04
Restriction Endonucleases (RE)!
!  The DNA of a bacterial cell is protected
from the cell’s own RE by the addition
of methyl groups to adenines or
cytosines within the sequences
recognized by the enzymes.!
Restriction Enzymes (RE)!
! molecular scissors which bind to DNA at
a specific base sequence (recognition
site) and then cuts (or hydrolyzes) the
phosphodiester bonds on both strands
of DNA
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Restriction Enzymes (RE)!
!  once the cuts have been made, the
weakness of the hydrogen bonds
holding the DNA fragments together
allow the DNA fragments to separate
Restriction Enzymes (RE)!
!  Cuts are defined and reproducible
(very important)!
!  Each RE has its own unique
recognition site (4-8 nucleotides
long)!
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Palindromes!
!  All recognition sites are palindromes,
(read the same way forward and
backward) !
Palindromes in English!
! Never odd or even!
! Tips spill, lips spit!
! Devil never even lived!
! Go hang a salami, I’m a lasagna hog!
! So many dynamos!
! Sit on a potato pan, Otis!
! Mr. Owl ate my metal worm!
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Palindromes in Molecular Biology!
!  a DNA sequence can be read the
same in the 5I à 3I direction on
COMPLEMENTARY strands:
5 GCAATTGC 3
3 CGTTAACG 5
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The Cutting…!
When restriction enzymes cut
phosphodiester bonds directly
across each strand, blunt ends are
generated.!
GATC
CTAG
GA
CT
+! T C
! AG
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The Cutting…!
When restriction enzymes cut
phosphodiester bonds in a
staggered (or offset) pattern across
from each strand, sticky ends are
generated.!
G A A T T C!
C T T A A G!
!
A A T T C!
G!
+!! !
G!
C T T A! A! !
!
Sticky Ends!
!  Sticky ends are very useful because if two
different pieces of DNA are cut with the
same restriction enzyme, the overhanging
sticky ends will complementarily base
pair, creating a recombinant DNA
molecule.!
!  DNA ligase joins the fragments by
producing the phosphodiester bonds
between nucleotides!
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Significance of Sticky Ends
Two different
fragments cut by
the same
restriction
enzyme have
identical
sticky ends
and are able to
join together
How are RE named?!
! RE are named after the bacteria from
which they were isolated.!
! Example: EcoRI
E! !Escherichia (genus)!
co !coli (species)!
R! !RY13 (strain)!
I ! !first endonuclease isolated!
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Examples!
Applications of RE!
(1)  Forensics!
•  Inherited variations in base sequence lead to differences
in places where DNA is cut
•  RFLPs (Restriction Fragment Length Polymorphisms)
DNA from different individuals give different fragment
lengths
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Applications of RE!
Differences detected
by cutting the DNA
with specific RE’s
and running the DNA
fragments through
gel electrophoresis
Significance of RE!
(2) Recombinant DNA Technology!
•  DNA from different sources are cut with the same RE
and are then joined together!
•  Commonly used in used in amplifying DNA, GM foods,
medicine and creating new DNA strands.!
•  E.g. human insulin inserted into a bacteria’s plasmid
DNA using RE!
!!
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Homework!
!  Read pg. 266-267!
!  Pg. 267 #1-5!
!  Read pg. 278-284 !
!  pg. 281 #1, 2 and 5!
!  pg. 282 #6, 9!
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