Transcript Lab # 7 Restriction Enzymes

General Genetics
1.
Introduce the students to digest genomic DNA
by restriction endonucleases.
2.
Observe the results of digestion on agarose
gel
electrophoresis.
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4.
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The activity of restriction enzymes is dependent
upon precise environmental conditions:
PH
Temperature
Salt Concentration
Ions
An Enzymatic Unit (u) is defined as the amount of
enzyme required to digest 1 ug of DNA under optimal
conditions:
3-5 u/ug of genomic DNA
1 u/ug of plasmid DNA
Stocks typically at 10 u/ul
Cohesive Ends
Cohesive Ends
Blunt Ends
(5´ Overhang)
(3´ Overhang)
(No Overhang)
BamH1
KpnI
HaeIII
GGATCC
CCTAGG
GGTACC
CCATGG
GGCC
CCGG
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Hundreds of restriction
enzymes have been identified.
Most recognize and cut
palindromic sequences
Many leave staggered (sticky)
ends by choosing correct
enzymes can cut DNA very
precisely
Important for molecular
biologists because restriction
enzymes create unpaired
"sticky ends" which anneal
with any complementary
sequence
 Bacterial" immune system": destroy any "non-self" DNA
 methylase recognizes same sequence in host DNA and protects it by
methylating it; restriction enzyme destroys unprotected = non-self DNA
(restriction/modification systems)
As an example, consider a 5000 base pair, circular
plasmid DNA containing single recognition sites for
enzymes A, B, and C.
 Any one of these enzymes will cleave the DNA once to
produce a linear molecule of 5000 base pairs.
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Differently paired combinations of enzymes in the
same reaction mixture (double-digests) will produce
the following DNA fragments (sizes in base pairs):
•Arbitrarily placing one of the cleavage sites at the top of a circle. This site
acts as a reference point.
•The closest cleavage site to this point can be placed in a clockwise or
counterclockwise direction.
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Generally, a restriction enzyme map is constructed by
first determining the number of fragments each
individual enzyme produces. The size and number of
fragments is determined by electrophoresis.
If a DNA molecule contains several recognition
sites for a restriction enzyme, then under certain
experimental conditions, it is possible that certain
sites are cleaved but not others.
 These incompletely cleaved fragments of DNA
are called partial digests (partials).
 Partials can arise if an insufficient amount of
enzyme is used or the reaction is stopped after a
short time (Figure 5).
 Reactions containing partials may also contain
some molecules that have been completely
cleaved.
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Two possible maps inferred from the
observations
BamH1 XhoI
4.3 kb
3.7 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
0.7 kb
BamH1
XhoI
PCR and Restriction
enzymes