CLONING VECTOR

RESTRICTION ENZYME
DNA LIGASE

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WHAT IS CLONE, CLONING?

1970 cloning gen menjadi sebuah kenyataan

clone yang diturunkan dari bahasa Yunani klon ,

artinya potongan yang digunakan untuk memperbanyak
tanaman.
KLON = sekelompok individu yang genetis uniform
berasal dari satu individu, hsl perbanyakan veg/aseksual,
tanpa pembuahan.
Kloning = teknik memperbanyak klon

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Konvensional vs Modern
Konvensional : okulasi, stek
Modern : teknik penggandaan gen yang
menghasilkan turunan yang sama sifat baik dari
segi hereditas maupun penampakannya dengan
induknya

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TUJUAN CLONING GEN

TUJUAN CLONING GEN :
Mendapatkan untuk diekspresikan di
sel inang
Mendapatkan gen sebagai penelusur
Mendapatkan gen untuk dianalisis
urutan nukleotidanya

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KOMPONEN DALAM CLONING GEN

DNA Asal
Cloning Vektor (Pembawa Molekul DNA/gen)
Enzim Restriksi
Enzim Ligase
Sel Inang

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CLONING VECTOR

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Cloning Vectors
Cloning vector - a carrier of foreign DNA into a host cell, replicates
inside a bacterial (or yeast) cell and produces many copies of itself
and the foreign DNA

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Four Features Cloning Vectors

1. can independently replicate themselves and the foreign DNA

segments they carry;

2. contain a number of unique restriction endonuclease
cleavage sites that are present only once in the vector;

3. carry a selectable marker (usually in the form of antibiotic

resistance genes or genes for enzymes missing in the host
cell) to distinguish host cells that carry vectors from host cells
that do not contain a vector; and
4. are relatively easy to recover from the host cell.
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What Makes a Good Cloning Vector?

Size ukuran relatif kecil dibanding pori sel inang

Origin of replication (ori) dapat mereplikasi di sel inang
Multiple cloning site (MCS) sbg tempat penyisipan gen
Selectable marker genes penanda masuk tidaknya
plasmid ke dalam sel inang

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Type of Cloning Vector

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BACTERIAL PLASMID

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PLASMID

Bacterial genome
(5000kb)

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Plasmid DNA
(3kb)

DNA Ekstrakromosom
DNA Utas Ganda Sirkuler Berukuran
Kecil
Dapat Melakukan Replikasi
Secara Genetik Dapat Ditransfer
Secara Stabil

Bagian Plasmid
Titik Replikasi (ORI)
Tempat-tempat Restriksi Untuk Penyisipan Gen
- Multiple Cloning Sites Regions of known
restriction (NdeI, HindIII, EcoRI, etc.) sites that
permit insertion or deletion of the gene of interest
Penanda Ketahanan Genetik

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Plasmid Vector

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Contoh Plasmid dari Bakteri

pUC19
pBR322

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An overview of how bacterial plasmids are used to clone genes

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ENZIM RESTRIKSI

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What is Restriction Enzyme?

Primarily found in bacteria
Cut DNA by cleaving the phosphodiester bond that joins
adjacent nucleotides in a DNA strand
Bind to, recognize, and cut DNA within specific
sequences of bases called a recognition sequence or
restriction site

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How it works?
Cohesive (sticky) ends overhanging single-stranded ends
Blunt ends double-stranded, non-overhanging ends

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Some of Restriction Enzyme

Ezyme

Bacteria

Sequence

EcoRI

E.coli

G AATTC

HindIII

Haemophilus influenzae

A AGCTT

Taq I

Thermus aquaticus

T CGA

SmaI

Serratia marcescens

CCC GGG

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Double Restriction/Digestion
Penggunaan dua enzim untuk memotong DNA

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Mapping Restriction
Proses pencarian informasi struktural pada
potongan2 DNA dengan menggunakan enzim
restriksi
Aim: to determine which restriction enzymes cut the
cloned gene.

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Bam

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ENZIM LIGASE

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DNA ligases catalyze formation of a

phosphodiester bond between the 5-phosphate of a
nucleotide on one fragment of DNA and the 3hydroxyl of another
The DNA ligase most widely used in the lab is
derived from the bacteriophage T4.

Copyright 2009 Pearson Education, Inc.

Copyright 2009 Pearson Education, Inc.