9.1 Manipulating DNA
KEY CONCEPT
Biotechnology relies on cutting DNA at specific
places.
9.1 Manipulating DNA
WHAT ARE RESTRICTION
ENZYMES?
Restriction Enyzmes – molecular scissors
able to cut DNA
9.1 Manipulating DNA
HOW DO RESTRICTION
ENZYMES WORK?
Usually cut DNA at a “palindrome” such as GAATTC.
Palindrome – word or phrase when spelled backwords,
spells the same word or phrase
Ex. BOB
MADAM I’M ADAM
A Toyota! Race fast, safe car. A Toyota
5’
3’
GAATTC
| | | | | |
3’ CTTAAG 5’
“Restriction site”
or
“Recognition Sequence”
9.1 Manipulating DNA
HOW DO RESTRICTION
ENZYMES WORK?
RE’s cut DNA’s
phosphodiester bonds
and hydrogen bonds.
9.1 Manipulating DNA
HOW DO RESTRICTION
ENZYMES WORK?
- RE’s generate two
different types of
“cuts”
- Sticky ends
- Blunt cuts.
9.1 Manipulating DNA
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WHERE DO RE’S COME
FROM?
Bacteria
“Immune system” to
protect against
bacteriophages
(bacteria-infecting
viruses like Lambda).
9.1 Manipulating DNA
HOW ARE RE’S NAMED?
After bacteria which produces them.
EcoRI
HindIII
Genus
Escherichia
Haemophilus Bacillus
Species
coli
influenzae
amylo.
Strain
R
d
H
Order Isolated
I
III
I
Recognition Site
G^AATTC
A^AGCTT
BamHI
G^GATGC
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HOW DO RESTRICTION
ENZYMES WORK?
Must provide correct temperature and buffer (salt, pH)
for enzyme to work.
Mimics cellular conditions of bacteria they come from.
9.1 Manipulating DNA
Why cut up DNA?
• If different alleles can be cut up and identified (using
GE), we can identify unknown persons (e.g., criminals,
biological relatives), and document unknown genotypes
(e.g., Huntington’s Disease)
• http://www.youtube.com/watch?v=KVnFBCzg8Ms
9.1 Manipulating DNA
Gel Electrophoresis
• You have a vial of undescribed DNA fragments—now
what?
• Samples pipetted into wells on one end of a gel (e.g.,
agarose)
• Electricity is added to the gel
• DNA fragments move through the gel at different rates,
away from the negative and toward the positive end
– Smaller fragments move easier and further from well,
larger fragments more cumbersome and move
shorter distances from well
• The resulting “fingerprint” of fragments can be matched
to a known sample or investigated for specific alleles
9.1 Manipulating DNA
A B C D E F G
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Eco Digest
Eco cuts
to yield
two DNA
fragments
Eco
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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
9.1 Manipulating DNA
Eco Bgl Double Digest
A
restriction
digest with
both Eco
and Bgl
enzymes
provides
the
answer.
Shows it must be:
Eco
Bgl
9.1 Manipulating DNA
Review
• Restriction enzymes cut up DNA into pieces unique to
your DNA
– DNA pieces are a function of your genetics and the
restriction enzyme used
• DNA fragment soup placed in Gel well and distributes
based on fragment/sequence length
• Resulting gel is unique—like a fingerprint
9.1 Manipulating DNA
Your Turn:
• DNA- Off to the Races
• Restriction Enzyme mapping challenge.
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WHAT ARE RE’S USED FOR?
Genetic engineering –
pasting together DNA
from two different
organisms.
9.1 Manipulating DNA
HOW DO RESTRICTION
ENZYMES WORK?
Which are more useful in genetic
engineering? RE’s that generate sticky ends
or ones that produce blunt cuts?
STICKY ENDS
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HOW IS DNA PASTED
TOGETHER?
Ligase – another enzyme which reconnects
phosphodiester bonds.
RE Video
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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
9.1 Manipulating DNA
WHAT ELSE ARE RE’S USED
FOR?
Forensics – DNA Fingerprinting for crime scene
investigation and paternity testing.
Everyone’s DNA has a different sequence – even though
only 0.1% different.
How frequently would EcoRI cut DNA?
6
4 = once every 4096 bp
Lambda (48,514 bp) would expect about 12 EcoRI sites
9.1 Manipulating DNA
THOUGHT QUESTION
Bacteria are prokaryotes.
Prokaryotes do not have a nucleus.
Both DNA and RE’s are in cytoplasm.
Why isn’t bacterial DNA cut by RE’s?
9.1 Manipulating DNA
Methylation
See board
In humans, methyl groups are used to tag genes to turn
them on or off. Stay tuned.
9.1 Manipulating DNA
Scientists use several techniques to manipulate DNA.
• Chemicals, computers, and bacteria are used to work
with DNA.
• Scientists use these tools in genetics research and
biotechnology.
9.1 Manipulating DNA
Restriction enzymes cut DNA.
• Restriction enzymes act as “molecular scissors.”
– come from various types of bacteria
– allow scientists to more easily study and manipulate
genes
– cut DNA at a specific nucleotide sequence called a
restriction site
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• Different restriction enzymes cut DNA in different
ways.
– each enzyme has a different restriction site
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– some cut straight across and leave “blunt ends”
– some make staggered cuts and leave “sticky ends”
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Restriction maps show the lengths of DNA fragments.
• Gel electrophoresis is used to separate DNA fragments
by size.
– A DNA sample is cut with restriction enzymes.
– Electrical current pulls DNA fragments through a gel.
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– Smaller fragments move faster and travel farther
than larger fragments.
– Fragments of different
sizes appear as bands
on the gel.
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• A restriction map shows the lengths of DNA fragments
between restriction sites.
– only indicate size, not
DNA sequence
– useful in genetic
engineering
– used to study
mutations