DNA Cloning

Pertemuan 10

Imam Hardiman
LET YOUR IMAGINATION FLY
FAMILIAR WITH THIS MOVIE
STORMTROOPER

An Identical Army

An Idea from 1977

YES, WE WILL DISCUSS ABOUT CLONING
 CLONING

“A Number of processes that can be used to

produce genetically identical copies of a biological
entity”

In natural ways we called it twins

 REMEMBER THIS ONE

(2n) (n)
Somatic Cells Reproductive Cells

• Sperm
• Ovum

Somatic cells vs Reproductive cells

AND THIS ONE
AND THIS ONE AS WELL
OK WE CONTINUE TO
GENE CLONING
 MAJOR STEPS IN GENE CLONING

1. Choice of host organism and cloning vector

2. Preparation of vector DNA
3. Preparation of DNA to be cloned
4. Recombination process
5. Transformation of recombinant DNA
6. Selection of organisms containing recombinant
DNA
7. Screening for clones with desired DNA insert and
biological properties
STOP…
 CLONING VECTOR

“A DNA molecule that carries foreign DNA into a

host cell, replicates inside a bacterial (or yeast) cell
and produces many copies of itself and the foreign
DNA”
 VECTOR CHARACTERISTICS

Three functional regions:

1. An origin of replication (ori)
Sequences that permit the propagation of itself in bacteria
(or in yeast for YACs)
2. A drug-resistance gene
Sequence of gene for selecting for bacteria (or yeast for
YACs) containing a vector with foreign DNA
3. A region where DNA can be inserted
Sequence of cloning site to insert foreign DNA
VECTOR CHARACTERISTICS
 TYPES OF CLONING VECTORS (1)

1. Plasmid
An extrachromosomal circular DNA molecule that
autonomously replicates inside the bacterial cell;
cloning limit: 100 to 10,000 base pairs (bp) or 0.1 – 10
kilobases (kb).
2. Phage
Derivatives of bacteriophage lambda; linear DNA
molecules, whose region can be replaced with foreign
DNA without disrupting its life cycle;
cloning limit: 8 – 20 kb.
 TYPES OF CLONING VECTORS (2)

3. Cosmids
An extrachromosomal circular DNA molecule that combines
features of plasmids and phage;
cloning limit: 35 – 50 kb.
4. Bacterial Artificial Chromosomes (BAC)
Based on bacterial mini-F plasmids;
cloning limit: 75 – 300 kb.
5. Yeast Artificial Chromosomes (YAC)
An artificial chromosome that contains telomeres, ORI, a
yeast centromere, and a selectable marker for identification
in yeast cells;
cloning limit: 100 – 1,000 kb
LETS MOVE ON…
 RESTRICTION ENZYMES

“A protein that recognizes a specific, short

nucleotide sequence and cuts the DNA only at that
specific site, which is known as restriction site or
target sequence”
 SOME EXAMPLE OF
RESTRICTION ENZYMES
 HOW WE NAMED IT…

First letter  Genus of organism

Second and third letter  Species of organism
Forth letter  Strain of organism
Number  The order of it been discovered
THE ORIGIN
THE RESULT

Sticky end, 5’ overhang

Sticky end, 3’ overhang

Blunt end
 LET SEE HOW IT WORK

https://www.youtube.com/watch?v=-bTbiafdlqI
LETS MOVE ON…
 DNA LIGATION ENZYMES

“A specific type of enzyme, a ligase, that facilitates

the joining of DNA strands together by catalyzing
the formation of a phosphodiester bond”
 TYPES OF DNA LIGATION

1. E. coli DNA Ligase

Isolated from E. coli and use NAD (nicotinamide adenine
dinucleotide) as cofactor. It does not ligate blunt ends.
2. T4 DNA Ligase
Isolated from bacteriophage T4 and use ATP as cofactor.
Most commonly used in laboratory research.
3. Mammalian Ligases
Use ATP as cofactor.
4. Thermostable Ligases
Isolated from thermophilic bacteria.
 REQUIREMENTS

1. DNA Ligase
A typical reaction for inserting a fragment into a plasmid
vector (subcloning) would utilize about 0.01 (sticky ends) to 1
(blunt ends) units of ligase.
2. Fragments of DNA
Fragments from restriction process that have either blunt or
compatible cohesive (“sticky”) ends.
3. ATP
A donor phosphate in creation of phosphodiester bonds.
LIGATION STEPS
 LET SEE HOW IT WORK

https://www.youtube.com/watch?v=Q3xVGvEGIsg
LETS MOVE ON…
 COMPETENT CELLS

“The cells that have ability to take up extracellular

(“naked”) DNA from its environment”
 TYPES OF COMPETENT CELLS

1. Bacterial
Most commonly used: Escherichia coli.
2. Yeast
Most commonly used: Sacharomyces cerevisiae.
3. Plants
Done by a culture cells.
4. Animals
Done by a culture cells.
 HOW TO MAKE IT COMPETENT

1. Chemical Process/Transformation
Uses a calcium rich environment provided by calcium
chloride to counteract the electrostatic repulsion between
the plasmid DNA and bacterial cellular membrane.

2. Electroporation / Electropermeabilization
A technique which an electrical field is applied to cells in
order to increase the permeability of the cell membrane.
 LET SEE HOW IT WORK

https://www.youtube.com/watch?v=7Ul9RVYG5CM
OK WE ARE READY
 MAJOR STEPS IN GENE CLONING

1. Choice of host organism and cloning vector

2. Preparation of vector DNA
3. Preparation of DNA to be cloned
4. Recombination process
5. Transformation of recombinant DNA
6. Selection of organisms containing recombinant
DNA
7. Screening for clones with desired DNA insert and
biological properties
GENE CLONING
 LET SEE HOW IT WORK

https://www.youtube.com/watch?v=a52sRdYdYJ0
BLUE / WHITE SCREENING
THE APPLICATIONS
THANK YOU
 CERDAS
SPIRITUAL, INTELEKTUAL, EMOSIONAL, SOSIAL