Restriction Enzymes General Genetics Objectives Introduce the students to digest genomic DNA by restriction endonucleases Observe the results of digestion on agarose gel electrophoresis ID: 389076
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Slide1
Lab # 7Restriction Enzymes
General GeneticsSlide2
Objectives:Introduce the students to digest genomic DNA by restriction endonucleases.
Observe the results of digestion on
agarose
gel electrophoresis.
Slide3Slide4Slide5Slide6
Theoretical Basis Using Restriction EnzymesThe 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/
ulSlide7
Restriction Endonucleases:
Type
II
BamH1
GGATCC
CCTAGG
HaeIII
GGCC
CCGG
Cohesive Ends
(5
´
Overhang)
Cohesive Ends
(3
´
Overhang)
KpnI GGTACCCCATGG
Blunt Ends
(No Overhang)Slide8Slide9Slide10Slide11
Restriction EnzymesHundreds 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 sequenceSlide12Slide13
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)Slide14
Cont.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.
Differently paired combinations of enzymes in the same reaction mixture (double-digests) will produce the following DNA fragments (sizes in base pairs
):Slide15
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.Slide16
The triple digest, A + B + C is a confirmatory testGenerally, 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.Slide17Slide18
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.Slide19Slide20
Restriction Enzyme Mapping
Two possible maps inferred from the observationsSlide21
Restriction Enzyme Mapping
4.3 kb
3.7 kb
2.3 kb
1.9 kb
1.4 kb
1.3 kb
0.7 kb
Bam
H1
Xho
I
Bam
H1
Xho
ISlide22Slide23
PCR and Restriction enzymesSlide24