Uji kualitatif dan kuantitatif DN

A dan RNA
Fatchiyah, PhD
JBUB, fatchiya@yahoo.co.id
 Uji kuantitatif DNA dengan spektrofotometri
UV-Vis, DNA murni dapat menyerap cahaya u
ltraviolet karena keberadaan basa-basa purin
dan pirimidin. Pita ganda DNA dapat menyera
p cahaya UV pada  260 nm, sedang kontami
nan protein atau phenol akan menyerap caha
ya pada  280 nm.
 Sehingga kemurnian DNA dapat dukur denga
n menghitung nilai absorbansi  260 nm diba
gi dengan nilai absorbansi  280 (Å260/Å280
), dan nilai kemurnian DNA berkisar antara 1.
8-2.0.

Uji Kuantitatif
  Serta untuk mengukur konsentrasi DNA di
gunakan 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 me
misahkan, mengidentifikasi dan memurnik
an fragmen DNA adalah elektroforesis gel
agorose.
 Teknik ini sederhana, cepat terbentuk, da
n mampu memisahkan campuran potonga
n DNA sesuai dengan ukurannya secara a
kurat, dibanding dengan densitas gradient
sentrifugasi.
 Selanjutnya, lokasi DNA dalam gel terseb
ut dapat diidentifikasi secara langsung de
ngan menggunakan pewarna berfluoresce
n.

Uji Kualitatif 11/9/2019 fatchiyah, JB-UB 4

 Agarose Gel Electrophoresis
 Purification for Specific Fragment of DNA
-DNA Electro-elution
-Electrophoresis onto DEAE-cellulose me
mbranes
 Polyacrylamide Gels
 Pulse-field Gel Electrophoresis (PFGE)

http://learn.genetics.utah.edu/content/labs/gel/

Electrophoresis for nucleic acid

11/9/2019 fatchiyah, JB-UB 5
 The equipment and supplies necessary for
conducting agarose gel electrophoresis ar
e relatively simple and include:
 An electrophoresis chamber and power su
pply
 Gel casting trays, which are available in a va
riety of sizes and composed of UV-transparent
plastic. The open ends of the trays are closed
with tape while the gel is being cast, then rem
oved prior to electrophoresis.
 Sample combs, around which molten agarose
is poured to form sample wells in the gel.

Preparing and Running Standard A

garose DNA Gels 11/9/2019 fatchiyah, JB-UB 6
 Electrophoresis buffer, usually Tris-acet
ate-EDTA (TAE) or Tris-borate-EDTA (TBE)
.
 Loading buffer, which contains somethin
g dense (e.g. glycerol) to allow the sampl
e to "fall" into the sample wells, and one o
r two tracking dyes, which migrate in the
gel and allow visual monitoring or how far
the electrophoresis has proceeded.

Preparing and Running Standard Agar

ose DNA Gels
 Ethidium bromide, a fluorescent dye used for stai
ning nucleic acids. NOTE: Ethidium bromide is a k
nown mutagen and should be handled as a hazar
dous chemical - wear gloves while handling.
 Transilluminator (an ultraviolet lightbox), which is
used to visualize ethidium bromide-stained DNA i
n gels. NOTE: always wear protective eyewear w
hen observing DNA on a transilluminator to preve
nt damage to the eyes from UV light.

Preparation of Gel 11/9/2019 fatchiyah, JB-UB 8

 No Konsentrasi Gel Agar Effisiensi range Pemisahan
ose (%) pada DNA linier (kb)
1 0.3 60-5
2 0.6 20-1
3 0.7 10-0.8
4 0.9 7-0.5
5 1.2 6-0.4
6 1.5 4-0.2
7 2.0 3-0.1

Tabel 1. konsentrasi gel agarose d

an ukuran molekul DNA
  DNA and RNA molecules are negatively cha
rged, thus move in the gel matrix toward t
he positive pole (+)
 Linear DNA molecules are separated accor
ding to size
 The mobility of circular DNA molecules is a
ffected by their topological structures. Th
e mobility of the same molecular weight DN
A molecule with different shapes is: superc
oiled> linear> nicked or relaxed

Chemistry of nucleic acids11/9/2019 fatchiyah, JB-UB 10

 Fragments of linear DNA migrate through agaros
e gels with a mobility that is inversely proportion
al to the log10 of their molecular weight.
 In other words, if you plot the distance from the
well that DNA fragments have migrated against t
he log10 of either their molecular weights or nu
mber of base pairs, a roughly straight line will ap
pear.

Migration of DNA Fragments in

Agarose
11/9/2019 fatchiyah, JB-UB 11
 Circular forms of DNA migrate in agarose dist
inctly differently from linear DNAs of the sam
e mass.
 Typically, uncut plasmids will appear to migra
te more rapidly than the same plasmid when
linearized. Additionally, most preparations of
uncut plasmid contain at least two topological
ly-different forms of DNA, corresponding to s
upercoiled forms and nicked circles.
 The image to the right shows an ethidium-sta
ined gel with uncut plasmid in the left lane an
d the same plasmid linearized at a single site
in the right lane.
 S M

large moderate small

Picture of DNA se
paration by gel el
11/9/2019 ectrophoresis
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 11/9/2019 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 a
nd then purifying specific fragments of DNA, usu
ally in preparation for subcloning
 Several techniques can be used to purify DNA fro
m agarose gels, and choosing between them is, t
o some extent, a matter of personal preference.
They all start out by excising the desired "band" f
rom an ethidium-stained gel viewed with a UV tr
ansilluminator. Because UV light can fragment D
NA, it is best to work expeditiously and keep exp
osure time to a minimum.

Purification for Specific Fragm

ent of DNA
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 Cut out the desired piece of agarose using a razo
r blade or scalpel blade, and try to get as little ex
tra agarose as possible.

 The block of agarose containing DNA is then subj

ected to any of the following. The block of agaros
e is placed in a piece of dialysis tubing with a sm
all amount of fresh electrophoresis buffer, the en
ds sealed with clamps, and the bag placed into a
n electrophoresis chamber.

 Application of current will cause the DNA to migr

ate out of the agarose, but it will be trapped with
in the bag.

DNA Electroelution
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 Progress can be monitored using a transilluminat
or, 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 t
ubing.
 The buffer containing the DNA is then collected a
nd the DNA precipitated with ethanol.
 Electroelution is more time consuming than som
e of the other techniques, but works well and is
probably the best technique for recovery of large
(> 5 kb) fragments of DNA.

DNA Electroelution . . 11/9/2019 fatchiyah, JB-UB 21

Electrophoresis
To separate DNA of different si
ze ranges

 Narrow size range of DNA: use polyac

rylamide
 Wide size range of DNA: use agarose
gel
 Very large DNA(>30-50kb): use pulse
d-field gel electrophoresis

22
 At low concentrations of salt,
DNA binds avidly to DEAE-c
ellulose membranes. Fragme
nts of DNA are electrophores
ed in a standard agarose gel u
ntil they resolve adequately.
One then makes a slit in the g
el slightly ahead of the fragm
ent(s) of interest and resumes
electrophoresis until all of tha
t fragment has migrated and s
tuck onto the membrane.

Electrophoresis onto DEAE-c

ellulose membranes
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 The membrane is then removed, washed free
of agarose in low salt buffer (150 mM NaCl, 5
0 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 an
d eluting it can be monitored with UV light to
detect the ethidium bromide bound to DNA. A
fter elution, DNA is precipitated with ethanol.
 This procedure is simple and provides very cl
ean DNA. However, fragments larger than ab
out 5 kb do not elute well from the membran
e.

Electrophoresis onto DEAE-cellulos

e membranes
 For some purposes, eg. sequencing by
Maxam-Gilbert procedure. It is necessar
y to obtain separated stands of fragmen
t of DNA. Often this can be achieved by
electrophoresis of denatured DNA throu
gh neutral agarose.

 The strands of DNA fragment less than

1kb in length are separated on polyacril
amide gel.

 Polyacrylamide gel necessary to obtain

separated the each nucleotide of DNA s
equence

Strand-separating Gels
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 A commonly-used means of recovering DNA fro
m polyacrylamide gels is by the so-called "crush
and soak" method. The slice of polyacrylamide c
ontaining DNA is crushed in a microcentrifuge us
ing a plastic pipet tip, and incubated with consta
nt shaking in elution buffer (high salt) at 37ºC fo
r several hours. The polyacrylamide pieces are t
hen eliminated by centrifugation or by passing t
he mixture through a plug of siliconized glass wo
ol. Finally, DNA is recovered by ethanol precipita
tion.
 DNA can also be recovered from polyacrylamide
by use of certain types of silica gel particles, as
described above for recovery from agarose. How
ever, small (< 100 bp) fragments of DNA are ver
y difficult to elute from standard glass particles.

Polyacrylamide Gels 11/9/2019 fatchiyah, JB-UB 27

 Ideally, the DNA should separate in straight lanes to simplif
y lane-to-lane comparisons.
 The original pulsed-field systems used inhomogeneous elec
tric fields that did not produce straight lanes, making interp
retation of gels difficult (Schwartz and Cantor, 1984).
 Again, the simplest approach to straight lanes is FIGE, whic
h uses parallel electrodes to assure a homogeneous electric
field.
 Although extremely useful for separating relatively small D
NA, 4- 1,000 kb (fig. 2),
 FIGE's reorientation angle of 180ø results in a separation r
ange most useful under 2,000 kb. Furthermore, like other
PFGE techniques, FIGE has mobility inversions in which larg
er DNA can move ahead of smaller DNA during electrophor
esis.

Pulse-field gel Electrophoresis

(PFGE)
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http://www.protocol-online.org/prot/Molecular_Biology/Electrophoresis/Agarose_Gel_Electrophoresis/index.html
Electrophoresis

pulsed-field gel ele

ctrophoresis
Switching between two orientations: the l
arger the DNA is, the longer it takes to re
orient

29
PFGE System: Electrode Co
nfiguration

Figure 1:Electrode
configuration of co
mmonly used puls
ed field gel electro
phpresis units.

11/9/2019 fatchiyah, JB-UB 30

 Figure 2. Increased separatio
n of the 20-50 kb range with f
ield inversion gel electrophor
esis (FIGE). Run conditions: 2
30 V, 7.9 V/cm, 16 hrs., 50 ms
ec. pulse, forward:reverse pul
se ratio = 2.5:1, 1% GTG agar
ose, 0.5X TBE, 10 C.a) 1 kb la
dder, 0.5-12 kb; b) Lambda/H
ind III, 0.5-23 kb; and c) High
molecular weight markers, 8.
3-48.5 kb.

PFGE Result
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