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

70% final conc.

Sentrifugasi

“spooling” 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 nucleotides
ssDNA
dU
dC dsDNA

The conjugated p-electron systems of The absorbance of double-stranded

the purine & pyrimidine bases absorb DNA (dsDNA) at 260 nm is less than
strongly in the UV. (That’s why UV that of either single-stranded DNA
light is mutagenic and carcinogenic.) (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
Anode
Cathode (positive)
(negative)
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.

•Agarose was first used in biology

when Robert Koch* used it as a
*Lina Hesse, technician and illustrator for culture medium for Tuberculosis
a colleague of Koch was the first to bacteria in 1882
suggest agar for use in culturing bacteria

Agarose is a linear
PRAKTIKUMpolymer extracted
BIOLOGI MOLEKULER, 2007 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
- +

• Polymerized agarose is porous,

Power 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
- +

Within an agarose gel, linear DNA migrate inversely

Power
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
 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 beta-
galactosidase.
 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!