Download Biotech I Restriction Enzymes File

Biotechnology I
POINT > Define what restriction enzymes are
POINT > Describe how restriction enzymes cut DNA
POINT > Show how restriction enzymes facilitate
recombinant DNA technology
POINT > Describe the basics of gel electrophoresis
POINT > Define restriction enzyme
In 1962 a molecular “DNA scissors” was discovered
in bacteria cells
E. coli and other bacteria have an enzymatic immune
system that recognizes and destroys foreign DNA (cuts
it into pieces)
3,000 restriction enzymes have been identified,
around 200 have unique properties, many are purified
and available commercially
Use of restriction enzymes has made cloning and other
DNA technologies possible
POINT > Define restriction enzyme
Restriction enzymes are named for the bacterial
genus, species, strain, and type from which they
were isolated
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
WB CHECK:
What do restriction enzymes do?
Where do restriction enzymes come from?
Bacteria use restriction enzymes as a defense
against what?
POINT > Describe how restriction enzymes cut DNA
Restriction enzymes recognize specific DNA base
sequences (usually 5-6 base pairs long) and cut the
DNA at those sites
Cut sites are called restriction sites
These cuts result in pieces of DNA called restriction
fragments (usually a few hundred or thousand bases
long)
DNA fragments can be separated by size using gel
electrophoresis
POINT > Describe how restriction enzymes cut DNA
Restriction sites usually have symmetry (palindromic)
“Step on no pets”
BamH1 cut site:
5’-GGATCC-3’
3’-CCTAGG-5’
POINT > Describe how restriction enzymes cut DNA
Many restriction enzymes cut in a staggered fashion,
which results in fragments with “sticky ends”
EcoRI cut site:
5’……GAATTC…….3’
3’……CTTAAG……5’
Sticky ends:
5’…….G
3’…….CTTAA
AATTC…….3’
G…….5’
POINT > Describe how restriction enzymes cut DNA
Other restriction enzymes cut in a direct fashion,
leaving “blunt ends”
PvuII cut site:
Blunt ends:
5’……CAGCTG……3’
3’……GTCGAC……5’
5’……CAG
3’……GTC
CTG…….3’
GAC……5’
WB CHECK:
How many base pairs long are most “restriction sites”?
How long are the DNA fragments that result from
being cut by restriction enzymes?
What are the pieces of DNA that have been cut by
restriction enzymes called?
Restriction enzymes cut at specific places in DNA.
What do we call these places?
POINT > Show how restriction enzymes facilitate
recombinant DNA technology
Recombinant DNA is used for:
DNA fingerprinting
DNA sequencing to compare individuals and species
Recombinant DNA for genetically modified organisms
and production of proteins like insulin in bacteria
Large scale analysis – gene chips to screen for
potential diseases
POINT > Show how restriction enzymes facilitate
recombinant DNA technology
Human DNA cut with EcoRI
5’G-T-A-C-T-A-G
3’C-A-T-G-A-T-C-T-T-A-A
Baboon DNA cut with EcoRI
+
A-A-T-T-C-A-G-C-T-A3’
G-T-C-G-A-T5’
Complementary base pairing
5’G-T-A-C-T-A-G A-A-T-T-C-A-G-C-T-A3’
3’C-A-T-G-A-T-C-T-T-A-A G-T-C-G-A-T5’
+ DNA Ligase
5’G-T-A-C-T-A-G-A-A-T-T-C-A-G-C-T-A3’
3’C-A-T-G-A-T-C-T-T-A-A-G-T-C-G-A-T5’
Recombinant DNA molecule
POINT > Show how restriction enzymes facilitate
recombinant DNA technology
Any two pieces of DNA that are cut with the
same restriction enzyme can be combined, if
they have sticky ends!
WB CHECK: Which restriction enzyme would be
no good for recombinant DNA technology?
POINT > Describe the basics of gel electrophoresis
Gel electrophoresis is a technique that uses an electric
current to separate DNA restriction fragments by size
DNA is (-) charged and always moves to the (+)
Smaller fragments move more quickly through the gel
POINT > Describe the basics of gel electrophoresis
POINT > Describe the basics of gel electrophoresis
POINT > Describe the basics of gel electrophoresis
POINT > Describe the basics of gel electrophoresis
_
+
DNA is negatively
charged (due to the
phosphate backbone)
Visualize DNA with
ethidium bromide –
fluoresces ONLY
when bound to DNA
POINT > Describe the basics of gel electrophoresis
WB CHECK:
Why do DNA fragments move toward the positively
charged end of an electrophoresis gel?
Which DNA fragments move slowest?
What are three ways scientists use DNA technology?
If two restriction fragments were cut with the same
restriction enzyme, and the sticky ends matched up,
what enzyme is needed to finish putting them
together?
https://www.youtube.com/watch?v=aA5fyWJh5S0
Homework:
Read your textbook pages 403-405
Workbook pages 247-250