Teknologi dna rekombinan

Presentasi berjudul: "Teknologi dna rekombinan"— Transcript presentasi:

1 Teknologi dna rekombinan
Agustina Setiawati, M.Sc., Apt

2 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

3 Prinsip DNA rekombinan

4 PEMOTONGAN HpaI

5 PEMOTONGAN EcoRI

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8 LIGASI

9 VEKTOR KLONING

10 Plasmid DNA untai ganda sirkuler, ekstrakromosom
Linier : Streptomyces rochei Ukuran : 2,2 kb – 700 kb Jumlah duplikat: 1-2; 4-8; 20-30,

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14 Penamaan plasmid p : plasmid BR : pembuat Bolivar dan Rodriguez
322 dibuat lebih awal drpd pBR325, pBR328

15 Pembuatan Plasmid rekombinan

16 VEKTOR PLASMID pBr322

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18 KLONING DENGAN VEKTOR pBR322

19 Deteksi adanya klon yang diinginkan

20 VEKTOR PLASMID puc18/19

21 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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23 Deteksi adanya klon yang diinginkan

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25 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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27 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

28 Koloni yang dibutuhkan
Ukuran kromosom kb Ukuran rata-rata sisipan 7,7 kb N=ln(1-99)/ln(1-7,7/4000) = 2390 koloni Manusia 4x106 kb

29 Kloning gena isulin manusia
Ukuran kromosom 4x109 pb Ukuran gena isulin pb Ukuran sisipan pb N = ln(1-0,99)/ln(1-104/4x109) Jika sisipan pb N = ?

30 TRANSFORMASI

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33 KLONING GENA EUKARIOT

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35 STRATEGI KLONING GENA EUKARIOT
Isolasi total mRNA dari jaringan ttt Ubah mRNA menjadi cDNA Sisipkan pada vektor ekspresi Penapisan klon Produksi enzim

36 RNA processing

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38 Produksi Insulin Proinsulin

39 Preparasi cDNA Metode Berg JM, et al, 2002

40 Pembuatan cDNA

41 cDNA

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49 DETEKSI PROTEIN PADA SEL HOST
DENGAN PELACAK DNA

50 DETEKSI PROTEIN PADA SEL HOST

51 Want to know more? Just ask!

52 MATUR NUWUN