Transcript DNA rekombinan

TEKNOLOGI DNA REKOMBINAN
Agustina Setiawati, M.Sc., Apt
Kloning
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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
PEMOTONGAN HpaI
PEMOTONGAN EcoRI
LIGASI
VEKTOR KLONING
Plasmid
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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
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p : plasmid
BR : pembuat Bolivar dan Rodriguez
322 dibuat lebih awal drpd pBR325, pBR328
Pembuatan Plasmid rekombinan
VEKTOR PLASMID pBr322
KLONING DENGAN VEKTOR pBR322
Deteksi adanya klon yang diinginkan
VEKTOR PLASMID puc18/19
Keuntungan pUC
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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
Deteksi adanya klon yang diinginkan
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
KEMUNGKINAN BERHASIL
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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
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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
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Ukuran kromosom 4x109 pb
Ukuran gena isulin 1.700 pb
Ukuran sisipan 10.000 pb
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N = ln(1-0,99)/ln(1-104/4x109)
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Jika sisipan 40.000 pb N = ?
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TRANSFORMASI
KLONING GENA EUKARIOT
STRATEGI KLONING GENA EUKARIOT
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Isolasi total mRNA dari jaringan ttt
Ubah mRNA menjadi cDNA
Sisipkan pada vektor ekspresi
Penapisan klon
Produksi enzim
RNA processing
Produksi Insulin
Proinsulin
Berg JM, et al, 2002
Pembuatan cDNA
DETEKSI PROTEIN PADA SEL HOST
DENGAN PELACAK DNA
DETEKSI PROTEIN PADA SEL HOST
Want to know more?
Just ask!
MATUR NUWUN