Uji kualitatif dan kuantitatif DNA dan RNA
Uji kualitatif dan
kuantitatif DNA dan
RNA
Fatchiyah, PhD
JBUB, fatchiya@yahoo.co.id
Uji kuantitatif DNA dengan spektrofotometri
UV-Vis, DNA murni dapat menyerap cahaya
ultraviolet karena keberadaan basa-basa
purin dan pirimidin. Pita ganda DNA dapat
menyerap cahaya UV pada 260 nm, sedang
kontaminan protein atau phenol akan
menyerap cahaya pada 280 nm.
Sehingga kemurnian DNA dapat dukur
dengan menghitung nilai absorbansi 260
nm dibagi dengan nilai absorbansi 280
(Å260/Å280), dan nilai kemurnian DNA
berkisar antara 1.8-2.0.
Uji Kuantitatif
Serta untuk mengukur konsentrasi DNA
digunakan rumus sebagai berikut:
[DNA] = Å260 x 50 x faktor pengenceran
Å260 = Nilai absorbansi pada 260 nm
50 = larutan dengan nilai absorbansi 1.0
sebanding dengan 50 ug untai ganda DNA
per ml (dsDNA)
[RNA] = Å260 x 40 x faktor pengenceran
40 = 40ug/ml untai tunggal RNA (ssRNA)
Mengukur Konsentrasi DNA/RNA
Metoda standar yang digunakan untuk
memisahkan, mengidentifikasi dan
memurnikan fragmen DNA adalah
elektroforesis gel agorose.
Teknik ini sederhana, cepat terbentuk,
dan mampu memisahkan campuran
potongan DNA sesuai dengan ukurannya
secara akurat, dibanding dengan densitas
gradient sentrifugasi.
Selanjutnya, lokasi DNA dalam gel
tersebut dapat diidentifikasi secara
langsung dengan menggunakan pewarna
berfluorescen.
Uji Kualitatif
11/23/18
fatchiyah, JB-UB
4
Agarose Gel Electrophoresis
Purification for Specific Fragment of DNA
-DNA Electro-elution
-Electrophoresis onto DEAE-cellulose
membranes
Polyacrylamide Gels
Pulse-field Gel Electrophoresis (PFGE)
http://learn.genetics.utah.edu/content/labs/gel/
Electrophoresis for nucleic acid
11/23/18
fatchiyah, JB-UB
5
The equipment and supplies necessary for
conducting agarose gel electrophoresis
are relatively simple and include:
An electrophoresis chamber and power
supply
Gel casting trays, which are available in a
variety of sizes and composed of UVtransparent plastic. The open ends of the trays
are closed with tape while the gel is being
cast, then removed prior to electrophoresis.
Sample combs, around which molten agarose
is poured to form sample wells in the gel.
Preparing and Running Standard
Agarose DNA Gels
11/23/18
fatchiyah, JB-UB
6
Electrophoresis buffer, usually Trisacetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
Loading buffer, which contains
something dense (e.g. glycerol) to allow
the sample to "fall" into the sample wells,
and one or two tracking dyes, which
migrate in the gel and allow visual
monitoring or how far the electrophoresis
has proceeded.
Preparing and Running Standard
Agarose DNA Gels
Ethidium bromide, a fluorescent dye used for
staining nucleic acids. NOTE: Ethidium bromide is
a known mutagen and should be handled as a
hazardous chemical - wear gloves while handling.
Transilluminator (an ultraviolet lightbox), which is
used to visualize ethidium bromide-stained DNA
in gels. NOTE: always wear protective eyewear
when observing DNA on a transilluminator to
prevent damage to the eyes from UV light.
Preparation of Gel
11/23/18
fatchiyah, JB-UB
8
No
1
2
3
4
5
6
7
Konsentrasi Gel
Agarose (%)
0.3
0.6
0.7
0.9
1.2
1.5
2.0
Effisiensi range Pemisahan
pada DNA linier (kb)
60-5
20-1
10-0.8
7-0.5
6-0.4
4-0.2
3-0.1
Tabel 1. konsentrasi gel agarose
dan ukuran molekul DNA
DNA
and RNA molecules are negatively
charged, thus move in the gel matrix
toward the positive pole (+)
Linear DNA molecules are separated
according to size
The mobility of circular DNA molecules is
affected by their topological structures.
The mobility of the same molecular weight
DNA molecule with different shapes is:
supercoiled> linear> nicked or relaxed
Chemistry of nucleic acids
11/23/18
fatchiyah, JB-UB
10
Fragments of linear DNA migrate through
agarose gels with a mobility that is inversely
proportional to the log10 of their molecular
weight.
In other words, if you plot the distance from the
well that DNA fragments have migrated against
the log10 of either their molecular weights or
number of base pairs, a roughly straight line will
appear.
Migration of DNA Fragments in
Agarose
11/23/18
fatchiyah, JB-UB
11
Circular forms of DNA migrate in agarose
distinctly differently from linear DNAs of the
same mass.
Typically, uncut plasmids will appear to
migrate more rapidly than the same plasmid
when linearized. Additionally, most
preparations of uncut plasmid contain at
least two topologically-different forms of
DNA, corresponding to supercoiled forms and
nicked circles.
The image to the right shows an ethidiumstained gel with uncut plasmid in the left lane
and the same plasmid linearized at a single
site in the right lane.
S
large
moderate
M
small
Picture of DNA
separation by gel
11/23/18 electrophoresis
fatchiyah, JB-UB
14
DNA Migration
•
Several additional factors have important effects on
the mobility of DNA fragments in agarose gels, and
can be used to your advantage in optimizing
separation of DNA fragments.
Fig. Agarose Concentration
Chief among these
factors are:
Agarose Concentration
Voltage
Electrophoresis buffer
Effects of Ethidium
Bromide
Factors of DNA Migration
11/23/18
fatchiyah, JB-UB
15
Fig. 13-2, p.331
Fig. 13-1, p.331
In addition to its importance as an analytical tool,
gel electrophoresis is widely used for isolating and
then purifying specific fragments of DNA, usually
in preparation for subcloning
Several techniques can be used to purify DNA
from agarose gels, and choosing between them is,
to some extent, a matter of personal preference.
They all start out by excising the desired "band"
from an ethidium-stained gel viewed with a UV
transilluminator. Because UV light can fragment
DNA, it is best to work expeditiously and keep
exposure time to a minimum.
Purification for Specific
Fragment of DNA
11/23/18
fatchiyah, JB-UB
19
Cut out the desired piece of agarose using a
razor blade or scalpel blade, and try to get as
little extra agarose as possible.
The block of agarose containing DNA is then
subjected to any of the following. The block of
agarose is placed in a piece of dialysis tubing
with a small amount of fresh electrophoresis
buffer, the ends sealed with clamps, and the bag
placed into an electrophoresis chamber.
Application of current will cause the DNA to
migrate out of the agarose, but it will be trapped
within the bag.
DNA Electroelution
11/23/18
fatchiyah, JB-UB
20
Progress can be monitored using a
transilluminator, as shown below. When the DNA
is out of the agarose, the flow of current is
reversed for a few seconds to knock the DNA off
of the side of the tubing.
The buffer containing the DNA is then collected
and the DNA precipitated with ethanol.
Electroelution is more time consuming than
some of the other techniques, but works well
and is probably the best technique for recovery
of large (> 5 kb) fragments of DNA.
DNA Electroelution . .
11/23/18
fatchiyah, JB-UB
21
ectrophoresis
To separate DNA of different
size ranges
Narrow size range of DNA: use
polyacrylamide
Wide size range of DNA: use agarose
gel
Very large DNA(>30-50kb): use
pulsed-field gel electrophoresis
22
At low concentrations of salt,
DNA binds avidly to DEAEcellulose membranes.
Fragments of DNA are
electrophoresed in a standard
agarose gel until they resolve
adequately. One then makes a
slit in the gel slightly ahead
of the fragment(s) of interest
and resumes electrophoresis
until all of that fragment has
migrated and stuck onto the
membrane.
Electrophoresis onto DEAEcellulose membranes
11/23/18
fatchiyah, JB-UB
23
The membrane is then removed, washed free
of agarose in low salt buffer (150 mM NaCl,
50 mM Tris, 10 mM EDTA), then incubated
for about 30 minutes at 65 C in high salt
buffer (1 M NaCl, 50 mM Tris, 10 mM EDTA)
to elute the DNA.
Progress in binding DNA to the membrane
and eluting it can be monitored with UV light
to detect the ethidium bromide bound to
DNA. After elution, DNA is precipitated with
ethanol.
This procedure is simple and provides very
clean DNA. However, fragments larger than
about 5 kb do not elute well from the
membrane.
Electrophoresis onto DEAEcellulose membranes
For some purposes, eg. sequencing by
Maxam-Gilbert procedure. It is
necessary to obtain separated stands of
fragment of DNA. Often this can be
achieved by electrophoresis of
denatured DNA through neutral
agarose.
The strands of DNA fragment less than
1kb in length are separated on
polyacrilamide gel.
Polyacrylamide gel necessary to obtain
separated the each nucleotide of DNA
sequence
Strand-separating Gels
11/23/18
fatchiyah, JB-UB
25
A commonly-used means of recovering DNA
from polyacrylamide gels is by the so-called
"crush and soak" method. The slice of
polyacrylamide containing DNA is crushed in a
microcentrifuge using a plastic pipet tip, and
incubated with constant shaking in elution buffer
(high salt) at 37ºC for several hours. The
polyacrylamide pieces are then eliminated by
centrifugation or by passing the mixture through
a plug of siliconized glass wool. Finally, DNA is
recovered by ethanol precipitation.
DNA can also be recovered from polyacrylamide
by use of certain types of silica gel particles, as
described above for recovery from agarose.
However, small (< 100 bp) fragments of DNA
are very difficult to elute from standard glass
particles.
Polyacrylamide Gels
11/23/18
fatchiyah, JB-UB
27
Ideally, the DNA should separate in straight lanes to
simplify lane-to-lane comparisons.
The original pulsed-field systems used inhomogeneous
electric fields that did not produce straight lanes, making
interpretation of gels difficult (Schwartz and Cantor, 1984).
Again, the simplest approach to straight lanes is FIGE,
which uses parallel electrodes to assure a homogeneous
electric field.
Although extremely useful for separating relatively small
DNA, 4- 1,000 kb (fig. 2),
FIGE's reorientation angle of 180ø results in a separation
range most useful under 2,000 kb. Furthermore, like other
PFGE techniques, FIGE has mobility inversions in which
larger DNA can move ahead of smaller DNA during
electrophoresis.
Pulse-field gel Electrophoresis
(PFGE)
11/23/18
fatchiyah, JB-UB
http://www.protocol-online.org/prot/Molecular_Biology/Electrophoresis/Agarose_Gel_Electrophoresis/index.html
28
ectrophoresis
pulsed-field gel
electrophoresis
Switching between two orientations: the
larger the DNA is, the longer it takes to
reorient
29
PFGE System: Electrode
Configuration
Figure 1:Electrode
configuration of
commonly used
pulsed field gel
electrophpresis
units.
11/23/18
fatchiyah, JB-UB
30
Figure 2. Increased
separation of the 20-50
kb range with field
inversion gel
electrophoresis (FIGE).
Run conditions: 230 V,
7.9 V/cm, 16 hrs., 50
msec. pulse,
forward:reverse pulse
ratio = 2.5:1, 1% GTG
agarose, 0.5X TBE, 10
C.a) 1 kb ladder, 0.5-12
kb; b) Lambda/Hind III,
0.5-23 kb; and c) High
molecular weight
markers, 8.3-48.5 kb.
PFGE Result
11/23/18
fatchiyah, JB-UB
31
kuantitatif DNA dan
RNA
Fatchiyah, PhD
JBUB, fatchiya@yahoo.co.id
Uji kuantitatif DNA dengan spektrofotometri
UV-Vis, DNA murni dapat menyerap cahaya
ultraviolet karena keberadaan basa-basa
purin dan pirimidin. Pita ganda DNA dapat
menyerap cahaya UV pada 260 nm, sedang
kontaminan protein atau phenol akan
menyerap cahaya pada 280 nm.
Sehingga kemurnian DNA dapat dukur
dengan menghitung nilai absorbansi 260
nm dibagi dengan nilai absorbansi 280
(Å260/Å280), dan nilai kemurnian DNA
berkisar antara 1.8-2.0.
Uji Kuantitatif
Serta untuk mengukur konsentrasi DNA
digunakan rumus sebagai berikut:
[DNA] = Å260 x 50 x faktor pengenceran
Å260 = Nilai absorbansi pada 260 nm
50 = larutan dengan nilai absorbansi 1.0
sebanding dengan 50 ug untai ganda DNA
per ml (dsDNA)
[RNA] = Å260 x 40 x faktor pengenceran
40 = 40ug/ml untai tunggal RNA (ssRNA)
Mengukur Konsentrasi DNA/RNA
Metoda standar yang digunakan untuk
memisahkan, mengidentifikasi dan
memurnikan fragmen DNA adalah
elektroforesis gel agorose.
Teknik ini sederhana, cepat terbentuk,
dan mampu memisahkan campuran
potongan DNA sesuai dengan ukurannya
secara akurat, dibanding dengan densitas
gradient sentrifugasi.
Selanjutnya, lokasi DNA dalam gel
tersebut dapat diidentifikasi secara
langsung dengan menggunakan pewarna
berfluorescen.
Uji Kualitatif
11/23/18
fatchiyah, JB-UB
4
Agarose Gel Electrophoresis
Purification for Specific Fragment of DNA
-DNA Electro-elution
-Electrophoresis onto DEAE-cellulose
membranes
Polyacrylamide Gels
Pulse-field Gel Electrophoresis (PFGE)
http://learn.genetics.utah.edu/content/labs/gel/
Electrophoresis for nucleic acid
11/23/18
fatchiyah, JB-UB
5
The equipment and supplies necessary for
conducting agarose gel electrophoresis
are relatively simple and include:
An electrophoresis chamber and power
supply
Gel casting trays, which are available in a
variety of sizes and composed of UVtransparent plastic. The open ends of the trays
are closed with tape while the gel is being
cast, then removed prior to electrophoresis.
Sample combs, around which molten agarose
is poured to form sample wells in the gel.
Preparing and Running Standard
Agarose DNA Gels
11/23/18
fatchiyah, JB-UB
6
Electrophoresis buffer, usually Trisacetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
Loading buffer, which contains
something dense (e.g. glycerol) to allow
the sample to "fall" into the sample wells,
and one or two tracking dyes, which
migrate in the gel and allow visual
monitoring or how far the electrophoresis
has proceeded.
Preparing and Running Standard
Agarose DNA Gels
Ethidium bromide, a fluorescent dye used for
staining nucleic acids. NOTE: Ethidium bromide is
a known mutagen and should be handled as a
hazardous chemical - wear gloves while handling.
Transilluminator (an ultraviolet lightbox), which is
used to visualize ethidium bromide-stained DNA
in gels. NOTE: always wear protective eyewear
when observing DNA on a transilluminator to
prevent damage to the eyes from UV light.
Preparation of Gel
11/23/18
fatchiyah, JB-UB
8
No
1
2
3
4
5
6
7
Konsentrasi Gel
Agarose (%)
0.3
0.6
0.7
0.9
1.2
1.5
2.0
Effisiensi range Pemisahan
pada DNA linier (kb)
60-5
20-1
10-0.8
7-0.5
6-0.4
4-0.2
3-0.1
Tabel 1. konsentrasi gel agarose
dan ukuran molekul DNA
DNA
and RNA molecules are negatively
charged, thus move in the gel matrix
toward the positive pole (+)
Linear DNA molecules are separated
according to size
The mobility of circular DNA molecules is
affected by their topological structures.
The mobility of the same molecular weight
DNA molecule with different shapes is:
supercoiled> linear> nicked or relaxed
Chemistry of nucleic acids
11/23/18
fatchiyah, JB-UB
10
Fragments of linear DNA migrate through
agarose gels with a mobility that is inversely
proportional to the log10 of their molecular
weight.
In other words, if you plot the distance from the
well that DNA fragments have migrated against
the log10 of either their molecular weights or
number of base pairs, a roughly straight line will
appear.
Migration of DNA Fragments in
Agarose
11/23/18
fatchiyah, JB-UB
11
Circular forms of DNA migrate in agarose
distinctly differently from linear DNAs of the
same mass.
Typically, uncut plasmids will appear to
migrate more rapidly than the same plasmid
when linearized. Additionally, most
preparations of uncut plasmid contain at
least two topologically-different forms of
DNA, corresponding to supercoiled forms and
nicked circles.
The image to the right shows an ethidiumstained gel with uncut plasmid in the left lane
and the same plasmid linearized at a single
site in the right lane.
S
large
moderate
M
small
Picture of DNA
separation by gel
11/23/18 electrophoresis
fatchiyah, JB-UB
14
DNA Migration
•
Several additional factors have important effects on
the mobility of DNA fragments in agarose gels, and
can be used to your advantage in optimizing
separation of DNA fragments.
Fig. Agarose Concentration
Chief among these
factors are:
Agarose Concentration
Voltage
Electrophoresis buffer
Effects of Ethidium
Bromide
Factors of DNA Migration
11/23/18
fatchiyah, JB-UB
15
Fig. 13-2, p.331
Fig. 13-1, p.331
In addition to its importance as an analytical tool,
gel electrophoresis is widely used for isolating and
then purifying specific fragments of DNA, usually
in preparation for subcloning
Several techniques can be used to purify DNA
from agarose gels, and choosing between them is,
to some extent, a matter of personal preference.
They all start out by excising the desired "band"
from an ethidium-stained gel viewed with a UV
transilluminator. Because UV light can fragment
DNA, it is best to work expeditiously and keep
exposure time to a minimum.
Purification for Specific
Fragment of DNA
11/23/18
fatchiyah, JB-UB
19
Cut out the desired piece of agarose using a
razor blade or scalpel blade, and try to get as
little extra agarose as possible.
The block of agarose containing DNA is then
subjected to any of the following. The block of
agarose is placed in a piece of dialysis tubing
with a small amount of fresh electrophoresis
buffer, the ends sealed with clamps, and the bag
placed into an electrophoresis chamber.
Application of current will cause the DNA to
migrate out of the agarose, but it will be trapped
within the bag.
DNA Electroelution
11/23/18
fatchiyah, JB-UB
20
Progress can be monitored using a
transilluminator, as shown below. When the DNA
is out of the agarose, the flow of current is
reversed for a few seconds to knock the DNA off
of the side of the tubing.
The buffer containing the DNA is then collected
and the DNA precipitated with ethanol.
Electroelution is more time consuming than
some of the other techniques, but works well
and is probably the best technique for recovery
of large (> 5 kb) fragments of DNA.
DNA Electroelution . .
11/23/18
fatchiyah, JB-UB
21
ectrophoresis
To separate DNA of different
size ranges
Narrow size range of DNA: use
polyacrylamide
Wide size range of DNA: use agarose
gel
Very large DNA(>30-50kb): use
pulsed-field gel electrophoresis
22
At low concentrations of salt,
DNA binds avidly to DEAEcellulose membranes.
Fragments of DNA are
electrophoresed in a standard
agarose gel until they resolve
adequately. One then makes a
slit in the gel slightly ahead
of the fragment(s) of interest
and resumes electrophoresis
until all of that fragment has
migrated and stuck onto the
membrane.
Electrophoresis onto DEAEcellulose membranes
11/23/18
fatchiyah, JB-UB
23
The membrane is then removed, washed free
of agarose in low salt buffer (150 mM NaCl,
50 mM Tris, 10 mM EDTA), then incubated
for about 30 minutes at 65 C in high salt
buffer (1 M NaCl, 50 mM Tris, 10 mM EDTA)
to elute the DNA.
Progress in binding DNA to the membrane
and eluting it can be monitored with UV light
to detect the ethidium bromide bound to
DNA. After elution, DNA is precipitated with
ethanol.
This procedure is simple and provides very
clean DNA. However, fragments larger than
about 5 kb do not elute well from the
membrane.
Electrophoresis onto DEAEcellulose membranes
For some purposes, eg. sequencing by
Maxam-Gilbert procedure. It is
necessary to obtain separated stands of
fragment of DNA. Often this can be
achieved by electrophoresis of
denatured DNA through neutral
agarose.
The strands of DNA fragment less than
1kb in length are separated on
polyacrilamide gel.
Polyacrylamide gel necessary to obtain
separated the each nucleotide of DNA
sequence
Strand-separating Gels
11/23/18
fatchiyah, JB-UB
25
A commonly-used means of recovering DNA
from polyacrylamide gels is by the so-called
"crush and soak" method. The slice of
polyacrylamide containing DNA is crushed in a
microcentrifuge using a plastic pipet tip, and
incubated with constant shaking in elution buffer
(high salt) at 37ºC for several hours. The
polyacrylamide pieces are then eliminated by
centrifugation or by passing the mixture through
a plug of siliconized glass wool. Finally, DNA is
recovered by ethanol precipitation.
DNA can also be recovered from polyacrylamide
by use of certain types of silica gel particles, as
described above for recovery from agarose.
However, small (< 100 bp) fragments of DNA
are very difficult to elute from standard glass
particles.
Polyacrylamide Gels
11/23/18
fatchiyah, JB-UB
27
Ideally, the DNA should separate in straight lanes to
simplify lane-to-lane comparisons.
The original pulsed-field systems used inhomogeneous
electric fields that did not produce straight lanes, making
interpretation of gels difficult (Schwartz and Cantor, 1984).
Again, the simplest approach to straight lanes is FIGE,
which uses parallel electrodes to assure a homogeneous
electric field.
Although extremely useful for separating relatively small
DNA, 4- 1,000 kb (fig. 2),
FIGE's reorientation angle of 180ø results in a separation
range most useful under 2,000 kb. Furthermore, like other
PFGE techniques, FIGE has mobility inversions in which
larger DNA can move ahead of smaller DNA during
electrophoresis.
Pulse-field gel Electrophoresis
(PFGE)
11/23/18
fatchiyah, JB-UB
http://www.protocol-online.org/prot/Molecular_Biology/Electrophoresis/Agarose_Gel_Electrophoresis/index.html
28
ectrophoresis
pulsed-field gel
electrophoresis
Switching between two orientations: the
larger the DNA is, the longer it takes to
reorient
29
PFGE System: Electrode
Configuration
Figure 1:Electrode
configuration of
commonly used
pulsed field gel
electrophpresis
units.
11/23/18
fatchiyah, JB-UB
30
Figure 2. Increased
separation of the 20-50
kb range with field
inversion gel
electrophoresis (FIGE).
Run conditions: 230 V,
7.9 V/cm, 16 hrs., 50
msec. pulse,
forward:reverse pulse
ratio = 2.5:1, 1% GTG
agarose, 0.5X TBE, 10
C.a) 1 kb ladder, 0.5-12
kb; b) Lambda/Hind III,
0.5-23 kb; and c) High
molecular weight
markers, 8.3-48.5 kb.
PFGE Result
11/23/18
fatchiyah, JB-UB
31