EKSTRAKSI, PURIFIKASI
DAN PRINSIP KLONING DNA
Agustina Setiawati, M.Sc., Apt
EKSTRAKSI DNA
Why we do it
What do we need DNA for?
 Forensik/DNA profiling
 Kloning
 Diagnosis penyakit
 Sekuensing DNA
 Genetically modified organism (GMO)
 Pengujian lingkungan
Sistematika ekstraksi dan purifikasi DNA
Penumbuhan sel
Panen sel dan lisis
Pemekatan DNA
Purifikasi DNA
1. Ekstraksi sel
Butuh reagen LYSIS
• detergen
• Buffer
• enzim protease
• panas
“cell extract”
DETERGEN
Melarutkan lipid pada membran
sel
CTAB
(hexadecyltrimethylammonium
bromide) – sel tumbuhan
Laurylsarcosine—bakteri gram
negative
DETERGEN

Soap molecules and grease molecules are made
of two parts:


Heads, which like water
Tails, which hate water.
DETERGEN

Sel mempunyai membran lipid double layer
dan protein.
DETERGEN

When detergent comes close to the cell, it
captures the lipids and proteins.
Enzim protease—Proteinase K



Menghilangkan nuclear protein, enzim
Memecah ikatan peptida
Bisa ditambahkan atau tidak (optional)
Panas

Paling sering 40-60ºC
Buffer

Tris HCl pH 8 untuk menjaga stabilitas DNA
2. Penghilangan protein & RNA
a. Ekstrak sel dicampur dengan fenol, perlahan!
b. Tambahkan Rnase pada lapisan aqueous
3. Pemekatan konsentrasi DNA
Sentrifugasi
“spooling”
70% final conc.
Ethanol precipitation
ANALISIS KUANTITATIF DNA



Absorbansi/Optical density (OD): c x b x (extinction coefficient, E).
Asam nukleat mengabsorbsi sinar UV pada 260 nm
1 A260 O.D. unit for dsDNA = 50 µg/ml

1 A260 O.D. unit for ssDNA = 33 or 50 µg/ml

1 A260 O.D. unit for RNA = 40 µg/ml
Spectrophotometric analysis of DNA
Double-stranded and single-stranded DNA differ in their optical
absorption at 260 nm
dA
dG
dU
dC
The conjugated p-electron systems of
the purine & pyrimidine bases absorb
strongly in the UV. (That’s why UV
light is mutagenic and carcinogenic.)
nucleotides
ssDNA
dsDNA
The absorbance of double-stranded
DNA (dsDNA) at 260 nm is less than
that of either single-stranded DNA
(ssDNA) or the free bases. This is
called “hypochromism.”
Kemurnian DNA


The purity of the DNA is reflected in the
OD260:OD 280 ratio and must be between 1.6
and 2.00.
Decreased 260:280 ratio means that
contaminating protein is still present.
ANALISIS KUALTITAIF

Metode: Elektroforesis Gel Agarose
buffer 

wells
Cathode
(negative)



Anode
(positive)
Add enough electrophoresis buffer to cover the gel to a depth of
at least 1 mm. Make sure each well is filled with buffer.
Agarose
D-galactose
3,6-anhydro
L-galactose
•Sweetened agarose gels have
been eaten in the Far East since
the 17th century.
*Lina Hesse, technician and illustrator for
a colleague of Koch was the first to
suggest agar for use in culturing bacteria
•Agarose was first used in biology
when Robert Koch* used it as a
culture medium for Tuberculosis
bacteria in 1882
PRAKTIKUMpolymer
BIOLOGI MOLEKULER,
2007
Agarose is a linear
extracted
from seaweed.
• DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
• An agarose gel is used to slow the movement of DNA and separate by size.
H

O2

DNA
-
Power
+
• Polymerized agarose is porous,
allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (1×1 µm)

How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size
DNA
small
large
-
Power
+
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
VISUALISASI DNA
TEKNOLOGI DNA REKOMBINAN
Agustina Setiawati, M.Sc., Apt
Kloning


DNA Cloning – the act of making many
identical copies of a particular piece of DNA
(often a gene)
As you know, the first stop often involves joining
a piece of DNA of interest to a cloning vector
using DNA ligase
Prinsip DNA rekombinan


Penyisipan DNA target
ke dalam vektor
Memasukkan DNA
rekombinan dalam
bakteri
TAHAP KLONING





Pemilihan vektor
Pemotongan vektor dan DNA target dengan enzim
retriksi
Penyambungan DNA pada vektor
Transformasi
Seleksi hasil transformasi
VEKTOR KLONING
Pemilihan Vektor
Syarat vektor:
1.
Kecil
2.
Mempunyai gen spesifik untuk penanda
3.
Punya retriksi untuk beberapa enzim retriksi
4.
Origin of Replication (ORI)
Plasmid




DNA untai ganda sirkuler, ekstrakromosom
Linier : Streptomyces rochei
Ukuran : 2,2 kb – 700 kb
Jumlah duplikat: 1-2; 4-8; 20-30, 700-1000.
Penamaan plasmid



p : plasmid
BR : pembuat Bolivar dan Rodriguez
322 dibuat lebih awal drpd pBR325, pBR328
VEKTOR PLASMID pBr322
VEKTOR PLASMID
puc18/19
Keuntungan pUC





Jumlah duplikat 500 – 700 plasmid/sel
Mudah mendeteksi % plasmid rekombinan
Adanya polycloning sites
pUC18 = pUC19, polycloning sitesnya
berlawanan
Membawa promoter lacUV dan ribosome
binding site
Pemotongan DNA & vektor
PEMOTONGAN HpaI
PEMOTONGAN EcoRI
Ligasi
LIGASI
Transformasi
TRANSFORMASI
Electroporation


Prinsip: pembukaan membran pembentukan pori sel
tanaman dengan muatan listrik
DNA in the surrounding solution can enter the cell
through these pores and become incorporated into the
cell’s nuclear genome through illegitimate recombination
Biolistic transformation – “Gene
gun”
DNA is precipitated on the
surface of heavy metal (gold;
tungsten) particles
 Loaded particles are driven
into plant cells by high velocity
gas propulsion (originally
gunpowder; now helium)
 Distance between discharge
nozzle and tissue can be
optimized, as can particle
velocity
 Target tissue must be
regenerable

Seleksi Transformasi
pUC18/19
Deteksi adanya klon yang diinginkan pada pUC
Seleksi pUC18/19


Sel yang membawa plasmid (non rekombinan dan
rekombinan) resisten terhadap ampisilin.
Sel yang punya plasmid non rekombinan menghasilkan
warna biru pada medium yang mengandung laktosa
Plasmid Polylinkers and Marker Genes for Blue-White
screening




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A vector usually contains a sequence (polylinker) which
can recognize several restriction enzymes so that the
vector can be used for cloning a variety of DNA
samples.
Colonies with recombinant plasmids are white, and
colonies with nonrecombinant plasmids are blue.
Example: pUC19
Resistant to ampicillin, has (ampr gene)
Contains portion of the lac operon which codes for betagalactosidase.
X-gal is a substrate of beta-galactosidase and turns blue
in the presence of functional beta-galactosidase is
added to the medium.
Insertion of foreign DNA into the polylinker disrupts the
lac operon, beta-galactosidase becomes non-functional
and the colonies fail to turn blue, but appear white.
DNA rekombinan pada plasmid
pBR322
KLONING DENGAN VEKTOR
pBR322
KEMUNGKINAN BERHASIL


PERSAMAA CLARCK DAN CARBON
N = ln(1 – P)/ln(1 – F)
P
: probalitas 0,99
 F : ukuran sisipan/ukuran kromosom
 N : jumlah koloni dg plasmid rekombinan
Koloni yang dibutuhkan




Ukuran kromosom
4.000 kb
Ukuran rata-rata sisipan 7,7 kb
N=ln(1-99)/ln(1-7,7/4000) = 2390 koloni
Manusia 4x106 kb
Kloning gena isulin manusia

Ukuran kromosom 4x109 pb
Ukuran gena isulin 1.700 pb
Ukuran sisipan 10.000 pb

N = ln(1-0,99)/ln(1-104/4x109)

Jika sisipan 40.000 pb N = ?


Want to know more?
Just ask!