Biological Sciences Laboratory Course
DNA
electrophoresis
Migration
of
DNA
fragments
in
agarose
-‐ When
charged
molecules
are
placed
in
an
electric
field,
they
migrated
towards
either
the
–ve
pole
or
+ve
pole
according
to
their
charge
-‐ DNA:
Negative
charged
due
to
the
phosphate
backbone
à
DNA
migrates
from
–ve
pole
to
+ve
pole
-‐ Fragments
of
linear
DNA
migrate
through
agarose
gel
with
a
mobility
that
is
inversely
proportional
to
the
log10
of
their
molecular
size
Molecular
Size
(bp)
100000
10000
1000
100
1
2
3
4
5
1
Distance
(cm)
Biological Sciences Laboratory Course
Migration
of
DNA
fragments
in
agarose
-‐ Migration
is
also
determined
by
the
configuration
of
the
DNA
-‐ In
most
preparations
of
uncut
DNA
plasmid,
two
topological
forms
including
supercoiled
and
nicked
forms
are
present
-‐ Supercoiled
one
migrate
faster
than
linearized
DNA
(same
molecular
mass)
DNA
of
the
same
molecular
mass
in
different
configurations
Supercoiled
Linearized
Relaxed,
closed
circular
Ethidium-‐stained
gel
Open,
circular
(Nicked)
2
Biological Sciences Laboratory Course
Reagents:
Agarose
gel
Electrophoresis
buffer
Loading
dye
Ethidium
bromide
(or
other
DNA
staining
dye)
Power
pack
Transilluminator
(UV
box)
3
Biological Sciences Laboratory Course
Agarose
concentration
and
efficient
separation
of
linear
DNA
Agarose
-‐
Sulfonated
polysaccharides
isolated
from
algae
-‐
Forms
complex
tertiary
structure
and
acts
as
a
molecular
sieve
Agarose
concentration
-‐
Higher
concentrations
of
agarose
allow
separation
of
small
DNA
-‐
Lower
concentrations
of
agarose
facilitate
resolution
of
larger
DNA
5000
bp
fragment
5000
bp
fragment
1000
bp
fragment
200
bp
fragment
1000
bp
fragment
Larger
fragments
(e.g.
5
kb)
are
much
better
resolved
in
the
0.7%
gel,
while
the
small
fragments
(e.g.
200
bp)
separated
best
in
1.5%
agarose
4
Biological Sciences Laboratory Course
Limitation
of
agarose
gel
electrophoresis
-‐
Effective
separation
of
DNA
fragments
of
size
ranging
from
200
bp
to
50
kb
-‐
Not
good
in
separating
fragments
of
size
from
1
bp
to
200
bp
-‐
Solution:
polyacrylamide
gel
electrophoresis
(PAGE)
à
allows
high
resolution
of
fragments
from
1
bp
to
500
bp
(Prof.
Billy
Chow
will
cover
the
knowledge
on
PAGE
in
his
lecture)
Agarose
gel
electrophoresis
Electrophoresis
buffer
-‐
Commonly
used
are
TAE
(Tris-‐acetate-‐EDTA)
and
TBE
(Tris-‐borate-‐EDTA)
-‐
Establish
pH
-‐
Provide
ions
to
support
conductivity
Loading
dye/buffer
-‐
Contains
glycerol
to
increase
the
density
of
the
loading
DNA
sample
-‐
Contains
one
or
two
tracking
dyes
(such
as
bromophenol
blue
and
xylene
cyanol)
to
allow
visual
monitoring
how
far
the
electrophoresis
has
processed
5
Biological Sciences Laboratory Course
Agarose
gel
electrophoresis
Ethidium
bromide
(EtBr)
and
transilluminator
(UV
box)
-‐ EtBr
intercalates
into
the
DNA
molecule
and
emits
orange
fluorescent
when
excited
by
ultraviolet
light
-‐ Wear
protective
eyewear
when
observing
DNA
on
a
transilluminator
to
prevent
damage
to
the
eyes
from
UV
light
-‐
Ethidium
bromide
is
carcinogenic
-‐
Other
staining
dyes
can
be
used
such
as
methylene
blue
6
Biological Sciences Laboratory Course
Restriction
enzyme
and
its
application
What
are
restriction
enzymes/restriction
endonucleases?
-‐
Found
naturally
in
bacteria
-‐
Bacteria
produce
these
enzymes
to
restrict
the
proliferation
of
invading
virus
by
cleaving
the
viral
DNA
-‐
Different
restriction
enzymes
recognize
and
cut
at
specific
nucleotide
sequences
(known
as
restriction
site
or
recognition
site)
in
the
DNA
DNA
helix
is
shown
in
pink.
The
two
symmetrical
domains
of
the
restriction
enzyme,
EcoRI,
are
shown
in
blue
and
yellow.
7
Biological Sciences Laboratory Course
How
can
the
bacterial
DNA
be
protected
from
cleavage?
-‐
Bacteria
has
another
enzyme
present
that
modifies
the
nucleotides
on
their
own
DNA
-‐
Addition
of
methyl
(-‐CH3)
groups
to
adenine
(A)
or
cytosine
(C)
bases
Example:
-‐
[Link]
makes
the
restriction
enzyme
EcoRI
and
the
methylating
enzyme
EcoRI
methylase
-‐
EcoRI
methylase
modifies
EcoRI
sites
in
the
bacteria's
own
genome
to
prevent
it
from
being
digested
CH3
5’-‐ GAATTC -‐
3’
5’-‐
GAATTC -‐
3’
3’-‐ CTTAAG -‐
5’
3’-‐
CTTAAG -‐
5’
CH3
“Normal”
EcoRI
site
recognized
by
EcoRI
Methylated
EcoRI
site
8
Biological Sciences Laboratory Course
Restriction
site/Recognition
site
-‐
Most
of
recognition
sequences
vary
between
4
and
8
nucleotides
-‐
Many
of
them
are
inverted
repeat
(reversed
complementary)
palindromic
Inverted
repeat
palindromic
Palindromic
e.g.
EcoRI
site
5’-‐
GATTAG
-‐3’
5’-‐
GAATTC
-‐
3’
-‐
Restriction
enzyme
digestion
produces
either
blunt
ends
or
sticky
ends
5’-‐
-‐3’
5’-‐
-‐3’
3’-‐
-‐5’
3’-‐
-‐5’
SmaI
digesEon
EcoRI
digesEon
produces
blunt
ends
produces
sEcky
ends
9
Biological Sciences Laboratory Course
Restriction
enzymes
Neoschizomers
Isoschizomers
-‐
Restriction
enzymes
recognize
the
same
-‐
Restriction
enzymes
recognize
the
same
nucleotide
sequence
but
cleave
at
a
nucleotide
sequence
and
cleave
at
the
different
site
same
locales
of
the
sequence
-‐
Isolated
from
different
strains
of
bacteria
5’-‐
GGGCCC
-‐
3’
5’-‐
CGTACG
-‐
3’
3’-‐
CCCGGG
-‐
5’
3’-‐
GCATGC
-‐
5’
SmaI
digestion
SphI
digestion
5’-‐
CGTACG
-‐
3’
5’-‐
GGGCCC
-‐
3’
3’-‐
GCATGC
-‐
5’
3’-‐
CCCGGG
-‐
5’
BbuI
digestion
XmaI
digestion
[Link]
10
Biological Sciences Laboratory Course
Nomenclature
of
the
restriction
enzymes
-‐
Reflect
their
origin
-‐
Naming
is
based
on
bacterial
genus,
species
and
strain
-‐
The
number
indicates
the
order
of
identification
in
the
bacterium
Example:
EcoRI
E co R I
Escherichia coli RY13 First identified
Genus Species Strain Order of identification in the bacterium
There
are
>100
known
restriction
enzymes
that
recognize
specific
sequence….
How
to
find
out
the
restriction
sites
within
a
DNA
sequence?
Use
web
resource
[Link]
[Link]
11
The linked image cannot be displayed. The file may have been moved, renamed, or deleted. Verify that the link points to the correct file and location.
Biological Sciences Laboratory Course
Restriction
enzyme
digestion
-‐
Restriction
enzymes
are
isolated
from
different
bacteria
-‐
Different
restriction
enzymes
require
different
conditions
to
facilitate
the
digestion
-‐
More
than
one
restriction
enzymes
can
be
used
per
reaction
Attention:
Condition
(e.g.
reaction
buffer,
temperature
for
incubation)
should
fit
for
all
restriction
enzymes
being
used
per
digestion
This
table
showing
some
restriction
enzymes
available
from
a
Biotech
company
12
Biological Sciences Laboratory Course
Restriction
enzyme
digestion
-‐
Different
ion
compositions
in
different
reaction
buffers
-‐
Reaction
buffers
provided
are
normally
at
higher
concentration
(e.g.
10X)
Working
concentration
Restriction
enzyme
digestion
DNA
x
µl
10x
RE
buffer
1
µl
Restriction
enzyme
y
µl
ddH2O
9-‐x-‐y
µl
Total
10
µl
-‐
Incubate
at
specified
temperature
for
digestion
-‐
Heat
inactivate
the
restriction
enzymes
after
digestion
13
Biological Sciences Laboratory Course
Applications
1.
Gene
cloning
-‐
Produces
recombinant
DNA
Steps:
-‐
RE
digestion
of
plasmid
-‐
RE
digestion
of
DNA
insert
DNA Insert
-‐
Generate
compatible
sticky
ends
-‐
Purified
RE-‐digested
plasmid
and
insert
-‐
Ligate
them
by
DNA
ligase
3’
overhang
of
plasmid
5’
overhang
of
insert
14
Biological Sciences Laboratory Course
Applications
2.
Restriction
Mapping
-‐
Find
out
the
relative
locations
of
restriction
sites
with
respect
to
one
another
in
DNA
molecules
Example:
You
have
a
circular
DNA
with
7666bp,
named
pTut,
you
would
like
to
know
whether
BglII
and
EcoRV
sites
are
present
in
the
circular
DNA
and
their
relative
locations
Step
1:
Cut
the
DNA
with
each
enzyme
singly
Circular
DNA
Step
2:
Cut
the
DNA
with
both
enzymes
Step
3:
Gel
electrophoresis
with
DNA
marker
-‐
Analyze
the
samples
of
the
restriction
pTut
7666
bp
digests
Step
4:
Visualizing
the
bands
Step
5:
Determine
the
length
of
the
DNA
fragments
Without
knowing
the
sequence
Step
6:
Generate
the
restriction
map
1.
Are
BglII
and
EcoRV
sites
there?
2.
Where
do
they
locate
(relative
to
each
other)?
15
Biological Sciences Laboratory Course
Step
1:
Cut
the
DNA
with
each
enzyme
singly
Step
2:
Cut
the
DNA
with
both
enzymes
Step
3:
Gel
electrophoresis
with
DNA
marker
-‐
Analyze
the
samples
of
the
restriction
digests
Step
4:
Visualizing
the
bands
Step
5:
Determine
the
length
of
the
DNA
fragments
Step
6:
Generate
the
restriction
map
3
1
2
pTut
pTut
pTut
X
4
X
X
BglII +
DNA
Results
after
performing
steps
1-‐4
BglII EcoRV EcoRV
marker
16
Biological Sciences Laboratory Course
Step
5:
Determine
the
length
of
the
DNA
fragments
using
DNA
marker
as
the
standard
3
-‐
Construct
a
calibration
curve
using
the
DNA
1
2
pTut
pTut
pTut
X
4
marker
data
X
X
BglII +
DNA
-‐
Measure
the
migration
distances
for
bands
in
BglII EcoRV EcoRV
marker
the
experimental
lanes
-‐
Use
the
calibration
curve
to
calculate
DNA
fragment
sizes
Results
shown
in
the
following
table
1 2 3
RE
BglII EcoRV BglII+EcoRV
Band
size
7666 4729 4729
2937 1992
945
17
Biological Sciences Laboratory Course
Step
6:
Generate
the
restriction
map
Results
shown
in
the
following
table
Digestion 1 2 3
RE BglII EcoRV BglII+EcoRV
Band size 7666 4729 4729
2937 1992
945
Both
BglII
and
EcoRV
maps
are
not
No. of 1 2
restriction site putting
the
sites
at
absolute
locations
Comparing
digestions
2
and
3,
the
BglII
site
must
be
in
2937
bp
EcoRV
fragment
The
BglII
cuts
the
2937
bp
EcoRV
fragment
into
two,
1992
bp
and
945
bp
respectively
1,992
BglII 2937
bp
945
The
relative
locations
of
BglII
and
EcoRV
in
the
pTut
plasmid
18
Biological Sciences Laboratory Course
Practical
2
Restriction
mapping
Two
to
three
students
in
a
group
Four
restriction
enzyme
(RE)
digestion
reactions
will
be
provided
Tube
1:
Lambda
DNA
cut
by
HindIII
(as
reference)
Tube
2:
Lambda
DNA
cut
by
unknown
restriction
enzyme
B
Tube
3:
Lambda
DNA
cut
by
unknown
restriction
enzyme
C
Tube
4:
Lambda
DNA
cut
by
unknown
restriction
enzymes
C
and
D
Information
Provided:
A
complete
restriction
enzyme
map
of
Lambda
DNA
will
be
provided
What
you
need
to
do:
1.
Agarose
gel
electrophoresis
2.
Determine
the
restriction
enzymes
being
used
in
your
experiment
19
