Transcript Restriction Enzymes - mvhs

Restriction
Enzymes
AP Biology
Unit 2
Images obtained without permission from http://w3.dwm.ks.edu.tw/bio/activelearner/14/images/ch14summary.gif and
http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif and
http://www.symmation.com/gallery/images/restriction-enzyme-ecorV-th.jpg
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 Hydrogen
strands) as a result of the
bond
Covalent bond
strands coming apart
Image taken without permission from http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif
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
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
BamHI
Bacillus amyloliquefaciens
5'AAGCTT 3’
3'TTCGAA 5’
5' GGATCC 3’
3' CCTAGG 5’
AluI
Arthrobacter luteus
5' AGCT 3’
3' TCGA 5’
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’
III I
3’ - - - C T T A A
I I I
G - - - 5’
Sticky ends
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
Recombinant Plasmids
Step 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
Question…
• What kinds of bonds does DNA Ligase
form?
– Covalent bonds (between nucleotides in a
single strand of DNA)
Putting It All Together
Create recombinant plasmid
Transform plasmid into
E. coli
Restriction Analysis
• Using restriction enzymes to find out
information about a piece of DNA
• We can use restriction enzymes to find out
– The size of a plasmid
– If there are any restriction sites for a particular
enzyme on a piece of DNA (ex. EcoRI)
– How many restriction sites for a particular
enzyme
– Where the restriction sites are located