EKSTRAKSI, purifikasi dan PRINSIP KLONING DNA

Presentasi berjudul: "EKSTRAKSI, purifikasi dan PRINSIP KLONING DNA"— Transcript presentasi:

1 EKSTRAKSI, purifikasi dan PRINSIP KLONING DNA
Agustina Setiawati, M.Sc., Apt

2 EKSTRAKSI DNA Why we do it

3 What do we need DNA for? Forensik/DNA profiling Kloning
Diagnosis penyakit Sekuensing DNA Genetically modified organism (GMO) Pengujian lingkungan

4 Sistematika ekstraksi dan purifikasi DNA
Penumbuhan sel Panen sel dan lisis Pemekatan DNA Purifikasi DNA

5 1. Ekstraksi sel Butuh reagen LYSIS detergen Buffer enzim protease
panas “cell extract”

6 DETERGEN Melarutkan lipid pada membran sel
CTAB (hexadecyltrimethylammonium bromide) – sel tumbuhan Laurylsarcosine—bakteri gram negative

7 DETERGEN Soap molecules and grease molecules are made of two parts:
Heads, which like water Tails, which hate water.

8 DETERGEN Sel mempunyai membran lipid double layer dan protein.

9 DETERGEN When detergent comes close to the cell, it captures the lipids and proteins.

10 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

11 2. Penghilangan protein & RNA
a. Ekstrak sel dicampur dengan fenol, perlahan! b. Tambahkan Rnase pada lapisan aqueous

12 3. Pemekatan konsentrasi DNA
Sentrifugasi 70% final conc. “spooling” Ethanol precipitation

13 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

14 Spectrophotometric analysis of DNA

15 Double-stranded and single-stranded DNA differ in their optical absorption at 260 nm
dC dG dU dsDNA ssDNA nucleotides The conjugated p-electron systems of the purine & pyrimidine bases absorb strongly in the UV. (That’s why UV light is mutagenic and carcinogenic.) The absorbance of double-stranded DNA (dsDNA) at 260 nm is less than that of either single-stranded DNA (ssDNA) or the free bases. This is called “hypochromism.”

16 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.

17 ANALISIS KUALTITAIF Metode: Elektroforesis Gel Agarose buffer  
Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer. Cathode (negative) Anode (positive)  wells

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19 Agarose is a linear polymer extracted from seaweed.
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 culture medium for Tuberculosis bacteria in 1882 *Lina Hesse, technician and illustrator for a colleague of Koch was the first to suggest agar for use in culturing bacteria Agarose is a linear polymer extracted from seaweed. PRAKTIKUM BIOLOGI MOLEKULER, 2007

20 + - DNA H  O2  Power • DNA is negatively charged.
• When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2 • An agarose gel is used to slow the movement of DNA and separate by size. + - Power Scanning Electron Micrograph of Agarose Gel (1×1 µm)  • Polymerized agarose is porous, allowing for the movement of DNA

21 + - How fast will the DNA migrate? DNA small large Power
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 + - Power small large Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.

22 VISUALISASI DNA

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

26 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

27 Prinsip DNA rekombinan
Penyisipan DNA target ke dalam vektor Memasukkan DNA rekombinan dalam bakteri

28 TAHAP KLONING Pemilihan vektor
Pemotongan vektor dan DNA target dengan enzim retriksi Penyambungan DNA pada vektor Transformasi Seleksi hasil transformasi

29 VEKTOR KLONING

30 Pemilihan Vektor Syarat vektor: Kecil
Mempunyai gen spesifik untuk penanda Punya retriksi untuk beberapa enzim retriksi Origin of Replication (ORI)

31 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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34 Penamaan plasmid p : plasmid BR : pembuat Bolivar dan Rodriguez
322 dibuat lebih awal drpd pBR325, pBR328

35 VEKTOR PLASMID pBr322

36 VEKTOR PLASMID puc18/19

37 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

38 Pemotongan DNA & vektor

39 PEMOTONGAN HpaI

40 PEMOTONGAN EcoRI

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42 Ligasi

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44 LIGASI

45 Transformasi

46 TRANSFORMASI

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48 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

49 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

50 Seleksi Transformasi

51 pUC18/19

52 Deteksi adanya klon yang diinginkan pada pUC

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54 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

55 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

56 pBR322

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58 KLONING DENGAN VEKTOR pBR322

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61 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

62 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

63 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 = ?

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65 Want to know more? Just ask!