Pulsed-Field Gel Electrophoresis: Separating Large DNA Molecules
H
i
s
t
o
r
i
c
a
l
b
a
c
k
g
r
o
u
n
d
o
f
P
G
F
E
Stand
a
rd
gel e
l
ectrop
h
oresis
techn
i
qu
e
s
w
a
s un
a
b
l
e
to
se
p
arate very
l
a
rge
mol
e
cu
l
es
of
D
N
A
e
f
fectiv
e
l
y
.
D
N
A
molecu
l
es
l
a
rger
than 1
5
-20kb
migrating
throu
g
h
a
gel w
i
ll
essent
i
a
l
ly
move
together in
a size-i
n
de
p
en
d
e
nt
manne
r
.
At
C
o
l
u
mbia
U
n
iv
e
rsity
in
1
9
8
4
,
D
a
vid
C. Schwartz
an
d
Charles
C
antor
dev
e
l
o
ped
a variation
on the
standard protocol by introduci
n
g
a
n
a
l
ternati
n
g
voltage grad
i
ent
to
i
mprove
the
reso
l
uti
o
n
of
l
a
rger
mol
e
cu
l
es.
Th
i
s techn
i
que
bec
a
me
kno
w
n
as
pu
l
se
d
-field
gel e
l
ectrop
h
oresis
(PFG
E
).
2
W
H
A
T
I
S
“
P
u
l
s
e
d
F
i
e
l
d
G
e
l
e
l
e
c
t
r
o
p
h
o
r
e
s
i
s
”
…
?
?
?
F
i
g
.
-
A
m
i
c
r
o
b
i
o
l
o
g
i
s
t
r
u
n
s
a
p
u
l
s
e
d
-
f
i
e
l
d
g
e
l
e
l
e
c
t
r
o
p
h
o
r
e
s
i
s
t
e
s
t
u
s
e
d
i
n
b
a
c
t
e
3
r
i
a
l
t
y
p
i
n
g
.
•
Pulsed
field
g
e
l
el
e
ctro
p
ho
re
sis
is
a
t
e
c
hnique
used
f
or
the
sepa
r
a
ti
o
n
o
f la
r
g
e
de
o
x
y
ribo
n
ucleic
acid
(DNA)
mole
c
ules
b
y appl
y
ing
t
o
a
g
el
m
a
trix
an
el
e
ct
r
ic
field th
a
t
pe
r
iodi
c
ally chan
g
es di
r
e
c
tion.
•
P
F
GE
is a
t
e
c
hnique
used
by sci
e
n
ti
s
ts
t
o
g
en
e
r
a
t
e
a
DNA
•
fin
g
erpri
n
t
f
or
a bac
t
erial
is
o
l
at
e.
•
P
u
lsed
Field
-
a
n
y
ele
c
t
r
oph
o
r
esis
p
r
ocess
th
a
t uses
m
o
r
e
than
one
ele
c
tric
field al
t
e
r
n
a
ting.
H
o
w
d
o
e
s
P
F
G
E
w
o
r
k
?
4
•PFGE
u
ses mo
l
ecu
l
ar
sciss
o
rs,
c
a
l
l
e
d
restriction
enzymes, to
cut
b
acterial
D
N
A
at c
e
r
t
a
i
n locat
i
ons
kno
w
n
as restriction
sites.
•Th
e
se mo
l
ecu
l
ar
sciss
o
rs
are
sel
e
cted
to
g
en
e
rate
a
small
number of
D
NA
p
i
eces
that
can be sep
a
rated
bas
e
d
on siz
e
.
•Usu
a
l
l
y
these D
N
A
p
i
eces,
or restriction
f
ragments,
are
l
a
rge
and
ne
e
d
to
b
e spec
i
a
l
ly
t
r
eated
and
separated
to
ge
n
erate
a
D
N
A
fingerprint.
•F
i
rst
the
b
acteria
are load
e
d
i
n
to an
ag
a
rose
suspe
n
si
o
n, simi
l
ar
to
g
e
l
atin,
then the
bacterial
cell is
ope
n
ed
to rele
a
se
the DN
A
.
•
Once
the D
N
A
is re
l
e
a
sed
then the
a
g
arose
a
n
d
D
N
A
•The
t
reated
p
l
u
gs
are
then loa
d
ed
onto an
ag
a
rose
g
e
l
a
n
d the restrict
i
on
fragme
n
ts
a
re
se
p
arated
b
a
sed
on size
usi
n
g
an e
l
ectric
fiel
d
.
•What makes PFGE
d
i
f
ferent
f
r
om
how scie
n
tists
usua
l
ly
separate
D
N
A
is beca
u
se
PFGE
c
an sep
a
rate
sev
e
ral
l
a
rge
restriction
fragme
n
ts.
T
o do th
i
s an
e
l
ectric
field that
constantly chan
g
es
d
i
recti
o
n
to the
gel is used to
g
en
e
rate
a DNA
fingerprint.
5
C
o
n
t
i
n
u
e
d
…
The
scientist
takes
bacterial
c
e
l
l
s
from
an
agar
plate.
i
l
Pulsed-field
Gel
E
l
ectrophoresis
(PFGE)
Cut
DNA
with
R
e
s
triction
Enzyme
e
The
bacterial
cel
l
s
are
broken
open
with
blochemlcaJs,
or
lysed,
so
that
the
DNA
is
free
in
the
agarose
plugs.
Plug
Mold
DODD
e
The
scie
n
t
ist
m
i
xes
bacterial
cells
with
melted
agarose
and
pours
i
nto
a
plug
m
o
l
d
.
Data
Analy
s
i
s
(
BioNumerics
)
e
The
gel
is
stained
so
that
DNA
can
be
seen
under
ultraviolet
(
UV
)
light
A
d
i
gital
camera
takes
a
ph
o
t
ograph
of
the
g-el
and
stores
the
picture
in
the
compute
r
.
0
The
s
c
i
en
t
i
st
loads
the
DNA
g
e
l
a
t
i
n
plug
i
n
t
o
agel.
and
places
i11n
an
electric
field
that
separates
DNA
fragments
according
to
th
e
i
r
size.
A
P
P
L
I
C
A
T
I
O
N
•
P
F
GE
may
be
used
for
g
e
n
o
typi
n
g
o
r
g
e
n
e
tic
fingerprinti
n
g
.
•It
is common
l
y consid
e
red
a go
l
d
stan
d
ard
i
n
e
p
i
d
emi
o
l
o
gical
stud
i
es
of
p
a
thog
e
n
i
c
organ
i
sms.
•S
u
btyping
has made it e
a
si
e
r
to
d
iscrimi
n
ate
among
st
r
a
i
ns of
L
i
steria
monocytogen
e
s
and
thus to
l
i
nk
env
i
ronmental
or food is
o
l
a
tes
w
i
th
cl
i
n
i
cal
i
n
fecti
o
ns.
7
Ad
v
a
n
tag
e
s
of
P
F
GE
8
PFGE
sub
t
yping
has
b
ee
n
success
f
u
l
l
y
app
l
i
e
d
to
the
sub
t
yping
of
m
a
n
y
pathog
e
nic
bact
e
r
i
a
a
nd
has
high
conco
r
dance
with
e
pi
d
e
m
i
o
l
og
i
cal
r
e
l
a
tedn
e
ss.
PFGE
has
b
ee
n
r
e
p
e
at
e
dly
shown
to
be
m
o
re
disc
r
i
m
in
a
ting
t
h
an
m
e
thods
such
as
r
i
bo
t
yping
or
mu
l
t
i
-
l
o
cus
s
e
quence
t
yping
for
m
a
n
y bacte
r
i
a
.
PF
G
E
in
the
s
am
e
b
a
s
i
c fo
rm
at
c
a
n
be
a
p
p
l
i
e
d
as
a
uni
v
e
rs
a
l
g
e
ne
r
ic
m
e
thod
for
sub
t
yping
of
bact
e
r
i
a.
(On
l
y the
cho
i
ce
of
the
r
e
str
i
cti
o
n
e
n
z
yme
and
cond
i
ti
o
ns
for
e
l
e
ctro
p
hor
e
sis
ne
e
d
to
be
opt
i
m
i
ze
d
for
e
ach
sp
e
ci
e
s.)
DNA
r
e
str
i
cti
o
n
p
a
tte
r
ns
g
e
ne
r
at
e
d
by
PF
G
E
a
r
e
sta
b
le
and
r
e
pr
o
ducib
l
e
.
Limit
a
tions
of
P
F
GE
9
•
T
ime consumi
n
g.
•
R
e
q
u
ires
a t
r
a
i
n
e
d
a
n
d sk
i
l
l
ed
tech
n
ic
i
a
n
.
•D
o
es
not
d
i
scriminate
betw
e
en
a
l
l
unre
l
ated
is
o
l
a
tes.
•P
a
t
t
ern
results vary
sl
i
ghtly
betw
e
en
techn
i
ci
a
ns.
•C
a
n
’
t
optimize
separation
in
every
part of the
gel at the
same time.
•D
o
n
’
t
real
l
y
know if
ba
n
ds
of s
a
me size
are
same
pieces of
D
N
A
.
•B
a
nds
are not i
n
de
p
en
d
ent.
•
C
h
a
n
ge
in
o
n
e restrict
i
on
site can mean
more
than o
n
e
ba
n
d
chan
g
e.
•“Re
l
atedness”
shou
l
d
be used
as
a gui
d
e,
not
t
r
ue phy
l
og
e
netic
measure.
•
S
o
me st
r
a
i
ns ca
n
n
o
t
be
typ
e
d by P
F
GE.
Video Link:
•
https://www.youtube.com/watch?v=__QhCX12h8I
Reference Book:
•
Wilson and Walker's Principles and Techniques of Biochemistry and
Molecular Biology
Dec
em
b
e
r
17
pankaj
gao
nk
a
r
1
2
9
93@g
m
a
i
l
.c
o
m
10
Pulsed-Field Gel Electrophoresis (PFGE) is a technique developed to effectively separate large DNA molecules through the application of an electric field that periodically changes direction. This method, introduced by David C. Schwartz and Charles C. Cantor in 1984, revolutionized the resolution of DNA fragments larger than 15-20kb. PFGE uses restriction enzymes to cut bacterial DNA at specific sites, generating fragments that can be separated based on size. The process involves loading treated DNA plugs onto an agarose gel and applying an electric field to separate the restriction fragments. Unlike traditional DNA separation methods, PFGE can effectively separate multiple large DNA fragments, allowing scientists to create DNA fingerprints for bacterial isolates.
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December 17 pankajgaonkar12993@gmail.com 1
Historical background of PGFE Standard gel electrophoresis techniques was unable to separate very large molecules of DNAeffectively. DNAmolecules larger than 15-20kb migrating through a gel will essentially move together in a size-independent manner. At Columbia University in 1984, David C. Schwartz and Charles Cantor developed a variation on the standard protocol by introducing an alternating voltage gradient to improve the resolution of larger molecules. This technique became known as pulsed-field gel electrophoresis (PFGE). 2
WHAT IS Pulsed Field Gel electrophoresis ??? Pulsed field gel electrophoresis is a technique used for the separation of large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric field that periodically changes direction. PFGE is a technique used by scientists to generate a DNA fingerprint for a bacterial isolate. Pulsed Field - any electrophoresis process that uses more than one electric field alternating. Fig.-A microbiologist runs a pulsed-field gel electrophoresis test used in bacte3 rial typing.
How does PFGE work? PFGE uses molecular scissors, called restriction enzymes, to cut bacterial DNAat certain locations known as restriction sites. These molecular scissors are selected to generate a small number of DNApieces that can be separated based on size. Usually these DNA pieces, or restriction fragments, are large and need to be specially treated and separated to generate a DNAfingerprint. First the bacteria are loaded into an agarose suspension, similar to gelatin, then the bacterial cell is opened to release the DNA. Once the DNAis released then the agarose and DNA 4
Continued The treated plugs are then loaded onto an agarose gel and the restriction fragments are separated based on size using an electric field. What makes PFGE different from how scientists usually separate DNAis because PFGE can separate several large restriction fragments. T o do this an electric field that constantly changes direction to the gel is used to generate a DNAfingerprint. 5
The Pulsed-feld Ge i l Electrophores is Process Pulsed-field Gel E l ectrophoresis (PFGE) Cut DNA with Restriction Enzyme e The bacterial cells are broken open with blochemlcaJs, or lysed, so that the DNA is free in the agarose plugs. The scientist takes bacterial cells from an agarplate. 0 The scientist loads the DNA gelatin plug into agel. and places i11nan electric field that separates DNA fragments according to theirsize. DODD Plug Mold Data Analysi s (BioNumerics)e The gel isstained sothat DNA can beseen under ultraviolet (UV) light Adigital camera takes a photograph of the g-el and stores the picture in the computer. e The scientist mixes bacterial cells with melted agarose and pours into a plug mold.
APPLICATION PFGE may be used for genotyping or genetic fingerprinting. It is commonly considered a gold standard in epidemiological studies of pathogenic organisms. Subtyping has made it easier to discriminate among strains of Listeria monocytogenes and thus to link environmental or food isolates with clinical infections. 7
Advantages of PFGE PFGE subtyping has been successfully applied to the subtyping of many pathogenic bacteria and has high concordance with epidemiological relatedness. PFGE has been repeatedly shown to be more discriminating than methods such as ribotyping or multi- locus sequence typing for many bacteria. PFGE in the same basic format can be applied as a universal generic method for subtyping of bacteria. (Only the choice of the restriction enzyme and conditions for electrophoresis need to be optimized for each species.) DNA restriction patterns generated by PFGE are stable and reproducible. 8
Limitations of PFGE Time consuming. Requires a trained and skilled technician. Does not discriminate between all unrelated isolates. Pattern results vary slightly between technicians. Can t optimize separation in every part of the gel at the same time. Don t really know if bands of same size are same pieces of DNA. Bands are not independent. Change in one restriction site can mean more than one band change. Relatedness should be used as a guide, not true phylogenetic measure. Some strains cannot be typed by PFGE. 9
Video Link: https://www.youtube.com/watch?v=__QhCX12h8I Reference Book: Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology
December 17 pankajgaonkar12993@gmail.com 10
