Transcript Restriction Enzymes

Restriction Enzymes
Discovery
 In
1962, Werner Arber, a Swiss
biochemist, provided the first evidence for
the existence of "molecular scissors" that
could cut DNA.
 He showed that E. coli bacteria have an
enzymatic “immune system” that
recognizes and destroys foreign DNA, and
modifies native DNA to prevent selfdestruction.
Molecular Scissors
 By
the early 1970s these enzymes started
to be identified and purified.
 It was shown that each species of bacteria
had its own population of a SPECIFIC
restriction enzyme.
 Each enzyme recognized its own specific
sequence of DNA bases. It is at this
sequence that the DNA was cut.
Restriction Enzyme Recognition
Sequences
 What
do the following phrases have in
common?:



Dammit, I'm mad!
Doc, note I dissent: a fast never prevents a
fatness. I diet on cod.
Dog DNA and God
 Restriction
enzymes usually recognize
palindromes in the nucleotide sequences
Restriction Enzyme EcoRI
Eco RI recognizes the sequence 5’….GAATTC…..
 A cut is made between the G and the A on each strand.
 This restriction enzyme leaves the nucleotides 5’AATT
overhanging.
 These are known as “sticky ends” because hydrogen
bonds are available to “stick” to a complimentary 3’TTAA
 Note: Restriction enzymes don’t stop with one cut! They
continue to cut at every recognition sequence on a DNA
strand.

Restriction Enzyme
Cut from EcoRI
Videos and Animations
http://www.dnai.org/b/
Click on “Techniques” then “Cutting and Pasting”
and view the 2D animation and 3D Cartoon
Video to see Restriction enzymes in action
Restriction enzymes, DNA, and
Electrophoresis
• DNA normally comes in “Genome sized”
lengths (usually several million bp in length.)
• These are the “elephants” in the race through
the agarose and cant enter the gel matrix when
they are this big.
• Restriction enzymes made possible the cutting
of DNA into smaller fragments together with their
separation and visualization by agarose gel
electrophoresis.
Restriction Sites as “Molecular
Signposts”
• Using two, or more different restriction
enzymes on a DNA fragment enables those
restriction sites to be mapped onto that DNA
fragment.
Eco Digest
Eco cuts to
yield two
DNA
fragments
Eco
Bgl also cuts
to yield two
DNA
fragments.
But where is
the Bgl site in
relation to the
Eco site?
Bgl Digest
Bgl
Eco
Or
Eco
Bgl
A restriction
digest with
both Eco and
Bgl enzymes
provides the
answer.
Eco Bgl Double Digest
Shows it must be:
Eco
Bgl
Your Turn:
• DNA- Off to the Races
• Restriction Enzyme mapping challenge.