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

Prinsip DNA rekombinan

PEMOTONGAN HpaI

PEMOTONGAN EcoRI

LIGASI

VEKTOR KLONING

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.

Penamaan plasmid

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

pBR328

Pembuatan Plasmid rekombinan

VE

KTO

R P

LAS

MID

pB

r32

2

KLO

NIN

G D

EN

GA

N V

EKT

OR

p

BR

32

2

Dete

ksi ad

an

ya k

lon y

ang

d

iing

inka

n

VE

KTO

R P

LA

SM

ID

puc1

8/1

9

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

Deteksi adanya klon yang diinginkan

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

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

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

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 = ?

TRANSFORMASI

KLONING GENA EUKARIOT

STRATEGI KLONING GENA EUKARIOT

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