Download Document

9.1 Manipulating DNA
KEY CONCEPT
Biotechnology relies on cutting DNA at specific
places.
WHAT ARE RESTRICTION
ENZYMES?
Restriction Enyzmes – molecular scissors
able to cut 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”
HOW DO RESTRICTION
ENZYMES WORK?
RE’s cut DNA’s
phosphodiester bonds
and hydrogen bonds.
HOW DO RESTRICTION
ENZYMES WORK?
- RE’s generate two
different types of
“cuts”
- Sticky ends
- Blunt cuts.
WHERE DO RE’S COME FROM?
Bacteria
“Immune system” to
protect against
bacteriophages
(bacteria-infecting
viruses like Lambda).
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
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.
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.
WHAT ARE RE’S USED FOR?
Genetic engineering –
pasting together DNA
from two different
organisms.
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
HOW IS DNA PASTED
TOGETHER?
Ligase – another enzyme which
reconnects phosphodiester bonds.
RE Video
restriction enzymes.exe
Bill Nye on Restriction Enzymes
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
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
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?
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
9.1 Manipulating DNA
• Different restriction enzymes cut DNA in different
ways.
– each enzyme has a different restriction site
9.1 Manipulating DNA
– some cut straight across and leave “blunt ends”
– some make staggered cuts and leave “sticky ends”
9.1 Manipulating DNA
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.
9.1 Manipulating DNA
– Smaller fragments move faster and travel farther
than larger fragments.
– Fragments of different
sizes appear as bands
on the gel.
9.1 Manipulating DNA
• 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