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Teknologi dna rekombinan

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.

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Teknologi dna rekombinan

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  1. Teknologidnarekombinan AgustinaSetiawati, 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 interestto a cloning vector using DNA ligase

  3. PrinsipDNA rekombinan

  4. PEMOTONGAN HpaI

  5. PEMOTONGANEcoRI

  6. LIGASI

  7. VEKTOR KLONING

  8. 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.

  9. Penamaan plasmid • p : plasmid • BR : pembuat Bolivar dan Rodriguez • 322 dibuatlebihawaldrpd pBR325, pBR328

  10. Pembuatan Plasmid rekombinan

  11. VEKTOR PLASMID pBr322

  12. KLONING DENGAN VEKTOR pBR322

  13. Deteksiadanyaklon yang diinginkan

  14. VEKTOR PLASMID puc18/19

  15. 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

  16. Deteksiadanyaklon yang diinginkan

  17. 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. DNArekombinanpada plasmid

  18. KEMUNGKINAN BERHASIL • PERSAMAA CLARCK DAN CARBON • N = ln(1 – P)/ln(1 – F) • P : probalitas 0,99 • F : ukuransisipan/ukurankromosom • N : jumlahkoloni dg plasmid rekombinan

  19. Koloni yang dibutuhkan • Ukurankromosom 4.000 kb • Ukuran rata-rata sisipan 7,7 kb • N=ln(1-99)/ln(1-7,7/4000) = 2390 koloni • Manusia4x106 kb

  20. 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 = ?

  21. TRANSFORMASI

  22. KLONING GENA EUKARIOT

  23. STRATEGI KLONING GENA EUKARIOT • Isolasi total mRNA darijaringanttt • Ubah mRNA menjadicDNA • Sisipkanpadavektorekspresi • Penapisanklon • Produksienzim

  24. RNA processing

  25. Produksi Insulin Proinsulin

  26. PreparasicDNA Metode Berg JM, et al, 2002

  27. Pembuatan cDNA

  28. cDNA

  29. DETEKSI PROTEIN PADA SEL HOST DENGAN PELACAK DNA

  30. DETEKSI PROTEIN PADA SEL HOST

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