Prinsip Dasar Teknik DNA Rekombinan

Gen papain yang Plasmid DNA

diinginkan.
Diseleksi dng PCR
- Promoter kuat/
Kromosom Pustaka Genom
- Multi Kopi
Pepaya
diligasi
Gen
Papain

Plasmid + Gen Papain

E. Coli
Di Transformasi
Di Tumbuhkan

Di induksi dengan penambahan induser

Induser

Papain
Gen Papain diekspresi
Enzim Restriksi
DNA (Gene) Cloning
 Tahapan Teknik Rekayasa Genetika
Dengan Teknik DNA Rekombinan
• Seleksi dan kloning Gen
• Pemilihan Vektor
• Pemotongan dan Penyambungan DNA
• Transformasi (mikroba, tanaman dan hewan beda caranya)
• Seleksi transforman (tergantung marker plasmidnya)
• Ekspresi (gen prokariot di E.coli; gen eukariot di ragi, jika pada
tanaman disebut: molecular farming)

• Isolasi dan pemurnian produk (akan lebih mudah jika

proteinnya diberi `ekor`, mis. (His)6)
 Kloning Gen
• Teknik PCR
• Teknik Pustaka Genom
 Isolasi DNA dari jaringan manusia
Metode Presipitasi

Many different methods and technologies are available for the isolation of genomic DNA.
In general, all methods involve disruption and lysis of the starting material followed by the
removal of proteins and other contaminants and finally recovery of the each patient DNA.
 Bacteriophage

• A virus that infects a bacterium.

Recombinant DNA containing viral DNA and
DNA of interest are packaged into viral
particles in a test tube. Host bacterial cells are
infected with recombinant phage DNA, the
DNA replicates within the host cells, and
progeny phage are produced when the host
cell undergoes lysis.
 Cosmids

• Engineered hybrids of phage DNA and

plasmids that are used for large DNA
fragments.
• Can be used to clone DNA segments of no
more than ~50 kb
 Yeast Artificial Chromosomes
(YACs)
• Are linear DNA molecules useful for eukaryotic
molecular studies and for large DNA
fragments.
 Plant Cloning Vectors

• Commonly used are plant viruses: Tobacco

mosaic virus and Ti plasmid (Agrobacterium
tumefaciens).
 Plant Cloning Vectors

Agrobacterium tumefaciens
Ti plasmid with the new gene
cell’s
+ DNA Transformation

Agrobacterium
Plant cell

The new
gene

Cell division
Transgenic plant
 Mammalian Cell Vectors

• many animal proteins can be produced only in

eukaryotic hosts.

• First eukaryote-infecting virus used for cloning

was Simian virus 40 or SV40, a small circular,
double-stranded DNA tumor virus.
 Mammalian Cell Vectors

• Retroviruses are single-stranded RNA viruses.

They replicate using reverse transcriptase to
make a double-stranded DNA from RNA
template.

• Adenovirus is a double-stranded DNA virus

suited as vectors for gene therapy.
 Mammalian Cell Vectors

• Cloning and expressing eukaryotic genes in

prokaryotic setting is difficult:
– Different promoter and DNA control
sequence
– Eukaryotic genes contain long noncoding
regions (introns)
 Cell Transformation

• introduction of new DNAs into organisms or to

a host for gene cloning.
 Methods of DNA transfer (transfection)

• Electroporation
– protoplasts of host organism are exposed to a
brief electrical pulse, which is thought to
introduce transient openings in the cell membrane
through which DNA molecule enters.
Methods of DNA transfer (transformatiion)

Electroporation
Power supply
Technique
Plant cell
Duracell

Protoplast

DNA containing
the gene of interest DNA inside the The plant cell with
plant cell the new gene
 Methods of DNA transfer (transfection)

• Microprojectile bombardment or
biolistics – very small (4μm) microprojectiles
made of gold or tungsten are coated with
DNA and is shot at high velocity from a
particle gun into cells or tissues.
Methods of DNA transfer (transfection)

“Gene Gun” Technique

DNA coated Cell’s DNA
golden particles

Plant cell
Gene gun

A plant cell with

the new gene

Transgenic plant Cell division

 Methods of DNA transfer (transfection)

• Microinjection
– for multicellular animals, DNA is injected directly
into the nucleus of animal with an extremely fine
pipette. After the DNA is transferred into the cell,
it is integrated into the chromosome, and the
transformed fertilized egg is implanted into an
animal for completion of development.
 Libraries
• Complimentary DNA (cDNA)
-DNA that carries the complete coding sequence for a
gene but no introns.

• Making a cDNA:
– Fully processes mRNA (introns removed) extracted from
eukaryotic cell nucleus.
– Addition of reverse transcriptase to synthesize DNA from RNA.
– DNA polymerase is used to synthesize a second DNA strand.
– Result is cDNA, which carries the complete coding sequence of
the gene but no introns.
 Libraries
• Cloned genes are stored in DNA libraries.
• Thousand of different recombinant plasmids are
produced in cloning, each carrying copies of a particular
segment from the initial genome. These sets of
recombinant plasmid clones are saved in such a library
called Genomic libraries.
– Genomic libraries:
• Plasmid library
• Phage library
• cDNA library
• Screening library
• Expression library
PUSTAKA GENOM
(Koolman, 2005)
 Gel
Electrophoresis
Southern Blotting
 Tahapan Teknik Rekayasa Genetika
Teknik DNA Rekombinan
• Seleksi Gen
• Pemilihan Vektor
• Pemotongan dan Penyambungan DNA
• Transformasi (mikroba, tanaman dan hewan beda caranya)
• Seleksi transforman (tergantung marker plasmidnya)
• Ekspresi (gen prokariot di E.coli; gen eukariot di ragi, jika pada
tanaman disebut: molecular farming)

• Isolasi dan pemurnian produk (akan lebih mudah jika

proteinnya diberi `ekor`, mis. (His)6)
Crown gal disease
 Green Fluorescent Protein (GFP)
(dari Ubur-Ubur )

Struktur 3D GFP

Image: Alexander Brandt, Wikimedia Commons

GFP sendiri terdiri atas 238 asam amino. Bentuknya yang
menyerupai gentong inilah yang menjadi kunci sifat fluoresensi
yang dimiliki GFP
 Kodok hasil rekayasa genetik dengan gen Ubur-
Keturunan marmoset GMO yang dapat berpendar (Image: E.Sasakiubur.
et al. 2009).
 tri

• GFP pada kera (Macaca)

• GFP pada sel ragi

GFP Pada Mencit • GFP pada goresan bakteri dalam

Image: University of Pennsylvania cawan petri
Gambar 3.1. Peta restriksi plasmid pUKC630, Plasmid ini membawa gen SUP45 wild type. Pada ujung
C-terminal terikat enam asam amino histidin. Plasmid pUKC630 diturunkan dari plasmid pT7-7. Fragmen
BglII dan HindIII dari Sup45 diambil dari plasmid pUKC625.