teknologi dna rekombinan
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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TEKNOLOGI DNA REKOMBINAN
Agustina Setiawati, M.Sc., Apt
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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
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Prinsip DNA rekombinan
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PEMOTONGAN HpaI
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PEMOTONGAN EcoRI
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LIGASI
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VEKTOR KLONING
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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.
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Penamaan plasmid
p : plasmid BR : pembuat Bolivar dan Rodriguez 322 dibuat lebih awal drpd pBR325,
pBR328
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Pembuatan Plasmid rekombinan
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VE
KTO
R P
LAS
MID
pB
r32
2
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KLO
NIN
G D
EN
GA
N V
EKT
OR
p
BR
32
2
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Dete
ksi ad
an
ya k
lon y
ang
d
iing
inka
n
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VE
KTO
R P
LA
SM
ID
puc1
8/1
9
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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
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Deteksi adanya klon yang diinginkan
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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
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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
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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
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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 = ?
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TRANSFORMASI
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KLONING GENA EUKARIOT
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STRATEGI KLONING GENA EUKARIOT
Isolasi total mRNA dari jaringan ttt Ubah mRNA menjadi cDNA Sisipkan pada vektor ekspresi Penapisan klon Produksi enzim
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RNA processing
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Produksi Insulin
Proinsulin
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Berg JM, et al, 2002
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Pembuatan cDNA
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DETEKSI PROTEIN PADA SEL HOST
DENGAN PELACAK DNA
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DETEKSI PROTEIN PADA SEL HOST
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Want to know more? Just ask!
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MATUR NUWUN