Tek DNA Rek-Pgtr Awal-Pbio-01
Tek DNA Rek-Pgtr Awal-Pbio-01
Tek DNA Rek-Pgtr Awal-Pbio-01
Technology
(An Introduction)
Bacterial Plasmid
chromosome Gene of
Recombinant interest DNA of
DNA (plasmid) chromosome
2 Plasmid put into
Gambaran bacterial cell (“foreign” DNA)
gene Recombinant
cloning dan bacterium
4 Basic research
Basic and various Basic
research applications research
on gene on protein
Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth
Cloning into
Enzim restriksi
Plasmids
(contoh lebih spesifik)
Enzim restriksi Enzim ligase
Prinsip dasar
Seleksi putih biru.
Syarat utama plasmid dpt dijadikan
vektor kloning :
Berukuran kecil (mudah dimanipulasi,
biasanya bersifat multiple copy)
Mempunyai sifat yg dpt digunakan
untuk seleksi
Mempunyai situs spesifik utk enzim
restriksi.
Bakteriophage sebagai vector
Bagaimana cara memodifikasi bakteriophage sehingga
dapat dijadikan sebagai vektor?
Ujung tumpul
Hasil pemotongan molekul DNA dengan enzim restriksi endonuklease berbeda.
(A) ujung blunt dan ujung sticky. (B) Tipe ujung sticky berbeda.
(C) Tipe sticky sama dihasilkan oleh dua enzim restriksi berbeda.
B. 5` and
3` overhangs
Belum tersambung
DNA dipotong dgn EcoRI
LIGASI
• Penyambungan potongan DNA target
dengan DNA plasmid membentuk ikatan
fosfodiester oleh enzim DNA Ligase
• Sticky ends can bond with complementary sticky
ends of other fragments
• DNA ligase is an enzyme that seals the bonds
between restriction fragments
Restriction site
5 3
GAATTC
DNA CTTAAG
3 5
1 Restriction enzyme
cuts sugar-phosphate
Penggunaan backbones.
3 5
5 3
Enzim restriksi
dan DNA ligase 3
5 Sticky 3
5
untuk membuat end
5
DNA rekombinan 3
5 3 5 3 5 3
G AATT C G AATT C
C TTAA G C TTAA G
3 53 5 3 5
One possible combination
3 DNA ligase
seals strands
5 3
Sel pembawa
plasmid TUMBUH Sel yg tdk membawa
pada cawan yg plasmid MATI pada
mengandung cawan yg
antibiotik ttt mengandung
antibiotik ttt
Teknik INSERTIONAL INACTIVATION
Transformasi host
Seleksi (penapisan)
Diperoleh koloni E.
coli yang membawa Mengapa demikian?...
DNA rekombinan
Plasmid pBR322 untuk cloning DNA dgn
teknik insertional inactivation
Salah satu
unique site
Teknik Komplementasi α
Alpha complementation
β-Galactosidase activity can be used as an indicator
of the presence of a foreign DNA insert.
Blue/White Screening for β-galactosidase
(polylinker)
Pada plasmid
(Kloning berhasil)
pUC18 adalah salah satu contoh plasmid vektor utk kloning
dengan teknik seleksi WHITE-BLUE Colony
Such as : insulin
REKAYASA GENETIKA
ORGANISMA
• MIKROBA
• TANAMAN
• HEWAN
MIKROBA
Production of Human Insulin
(disederhanakan)
1) Obtaining the human insulin gene
Human insulin gene can be obtained by making a
complementary DNA (cDNA) copy of the
messenger RNA (mRNA) for human insulin.
2)Joining the human insulin gene
into a plasmid vector
The bacterial plasmids and the cDNA are mixed
together. The human insulin gene (cDNA) is
inserted into the plasmid through complementary
base pairing at sticky ends.
3)Introducing the recombinant DNA
plasmids into bacteria
Further manipulation :
Production and
purification of human
insulin from large scale
culture sistem
( bioreactor)
Gambaran
REKAYASA GENETIKA
TANAMAN melalui
Agrobacterium
PRODUKSI TANAMAN TRANSGENIK
(dimediasi Agrobacterium)
Tikus transgenik dengan GFP