Uji kualitatif dan kuantitatif DNA dan RNA

(1)

Uji kualitatif dan

kuantitatif DNA dan

RNA

(2)

Uji Kuantitatif



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.

(3)

Mengukur Konsentrasi DNA/RNA



Serta untuk mengukur konsentrasi DNA

digunakan rumus sebagai berikut:

[DNA] = Å260 x 50 x faktor pengenceran



Å

₂₆₀

= 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

(4)

Uji Kualitatif



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.

(5)

Electrophoresis for nucleic acid



Agarose Gel Electrophoresis



Purification for Specific Fragment of DNA

-DNA Electro-elution

-Electrophoresis onto DEAE-cellulose

membranes



Polyacrylamide Gels



Pulse-field Gel Electrophoresis (PFGE)

11/23/18 fatchiyah, JB-UB 5

(6)

Preparing and Running Standard

Agarose DNA Gels

 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

UV-transparent 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.

(7)

Preparing and Running Standard

Agarose DNA Gels



Electrophoresis buffer, usually

Tris-acetate-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

(8)

Preparation of Gel

_11/23/18 _{fatchiyah, JB-UB} ₈

 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

(9)

Tabel 1. konsentrasi gel agarose

dan ukuran molekul DNA

No Konsentrasi Gel

Agarose (%) Effisiensi range Pemisahan 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

(10)



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)

Migration of DNA Fragments in

Agarose

 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.

(12)

(13)



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

(14)

DNA Migration

large moderate small

Picture of DNA separation by gel electrophoresis

M

S

(15)

Factors of 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.

Chief among these factors are:

 Agarose Concentration  Voltage

 Electrophoresis buffer  Effects of Ethidium

Bromide

(16)

(17)

(18)

(19)

Purification for Specific

Fragment of DNA

 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.

(20)

DNA Electroelution

 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.

(21)

DNA Electroelution . .

 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

(22)

22

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

(23)

Electrophoresis onto

DEAE-cellulose membranes

cellulose membranes



_At

_{low concentrations of salt}

_,

DNA binds avidly to

DEAE-cellulose 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.

(24)

Electrophoresis onto

DEAE-cellulose membranes

cellulose membranes



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

(25)

Strand-separating Gels

 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

(26)

(27)

Polyacrylamide Gels

 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.

(28)

Pulse-field gel Electrophoresis

(PFGE)

 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.

(29)

29

pulsed-field gel

electrophoresis

Switching between two orientations: the

larger the DNA is, the longer it takes to

reorient

(30)

PFGE System: Electrode

Configuration

(31)

PFGE Result

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.