Kloning Gen

Why Clone DNA?

• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
 Kenapa perlu kloning
• Untuk melakukan studi fungsi gen perlu
mendapatkan “molekul” gen di luar sistemnya
(invitro atau mikroorganisme)
• Untuk melakukan modifikasi gen (mutasi,
kontruksi silensing dsb) diperlukan gen di luar
sistemnya
 What is DNA cloning?
• When DNA is
extracted from an
organism, all its
genes are obtained
• In gene (DNA)
cloning a particular
gene is copied
(cloned)
 What is Gene cloning?

• To "clone a gene" is to
make many copies of it
• Act of making many
identical copies of gene
• Gene can be an exact
copy of a natural gene
• Gene can be an altered
version of a natural gene
Whole organisms are cloned too,
but differently
 How is DNA cloned?

Cell-based DNA cloning Cell-free DNA cloning (PCR)

Clone in dividing cells
Clone
in
P
C
R
 Cell-based DNA cloning
The procedure in a gene cloning experiment
is
1. To place a foreign gene into a bacterial cell;
2. To grow a clone of those modified bacteria.

The principle factors for gene cloning

experiment:
 Restriction endonucleases
 Vectors
 Specific probe
 CLONING PROCESS
• Gene of interest is cut
out with RE
• Host plasmid is cut
with same RE
• Gene is inserted into
plasmid and ligated
with ligase
• New plasmid inserted
into bacterium
(transform)
 The Role of Restriction
Endonucleases
• Restriction : restrict the host range of the
virus
• Endonucleases : cut at sites within the
foreign DNA
• How to name: the first 3 letters of the
Latin name of the microorganism + the
strain designation + Roman numeral
 Restriction Endonucleases
--The Molecular Scissors
Host enzymes that prevent the invasion of foreign DNAs
such as viral DNA, by cutting them up.
Restriction
These enzymes cut within the foreign DNAs, rather than
chewing them away from the ends.
Endonucleases
These enzymes recognize a specific DNA sequence (4-12bp)
which is twofold symmetry and cut both DNA strands
GAATTC
Some enzymes make staggered cuts CTTAAG

CCCGGG
Some make even cuts GGGCCC
 Sticky end

Sticky end
 DNA ligase covalently links two DNA strands

5’ 3’
Restriction
enzyme

Ligase

3’ 5’

Restriction
enzyme

Ligase
DIGESTION OF DNA SAMPLE
 Vectors
Vectors serve as carriers to allow
replication of recombinant DNAs.
 Origin of replication
 Multiple cloning site(MCS)
 Selection gene
Plasmids pBR322 pUC
Phages λphage cosmids M13
Phagemids
 Plasmids

• Naturally occurring extrachromosomal DNA

• Plasmids are circular dsDNA
• Plasmids can be cleaved by restriction
enzymes, leaving sticky ends
• Artificial plasmids can be constructed by
linking new DNA fragments to the sticky ends
of plasmid
Plasmids
 Cloning Vectors

Plasmids that can be modified to carry new genes

Plasmids useful as cloning vectors must have :

a replicator (origin of replication)
a selectable marker (antibiotic resistance gene)
a cloning site (site where insertion of foreign
DNA will not disrupt replication or inactivate
essential markers)
 Vectors -- the DNA carriers
Must have a origin of replication
Allow the vector as well as the foreign DNA to amplify in the host cell

1) Plasmids
Origin of replication
2) Phages
Antibiotic-resistant genes
Allow the host to grow on
selective media
Can selectively amplify this
specific vector in the host cell

Multiple cloning sites

Allow insertion of foreign DNA
Figure 4.3 The plasmid pBR322, showing the locations of 11 unique restriction
sites that can be used to insert foreign DNA
The locations of the two antibiotic resistance genes (Ampr =ampicillin resistance; Tetr
=tetracycline resistance) and the origin of replication (ori ) are also shown. Numbers
refer to kilobase pairs (kb) from the EcoRI site.
 Vectors -- the DNA carriers

Plasmid as a vector
Host: E. coli
Vector size: usually about 3kb.
Insert size: up to 20kb. usually below 5 kb.
Insert select: functional inactivation of the ability
to resist an antibiotic
Figure 4.4 Cloning foreign DNA
using the PstI site of pBR322.
We cut both the plasmid and
the insert (yellow) with PstI, then
join them through these sticky
ends with DNA ligase. Next, we
transform bacteria with the
recombinant DNA and screen for
tetracycline-resistant, ampicillin-
sensitive cells. The recombinant
plasmid no longer confers
ampicillin resistance because the
foreign DNA interrupts that
resistance gene (blue).
The first cloning experiment done by Boyer and Cohen
pUC
lacZ’ : coding for the amino
terminalportion of the enzyme β –
galactosidease.
Host E.coli strain carry a gene fragment
that codes the carboxyl potion of β –
galactosidease;
When X-gal cleaved by β –galactosidease, it
releases galactose plus an indigo dye that
stains the bacterial colony blue.
DIGESTION OF PLASMID DNA
LIGATION OF DNA SAMPLE AND
PLASMID DNA
 Figure 4.7 Joining of vector to insert. (a)
Mechanism of DNA ligase.
Step 1: DNA ligase reacts with an AMP
donor—either ATP or NAD(nicotinamide
adenine dinucleotide), depending on the
type of ligase. This produces an activated
enzyme (ligase-AMP). Step 2: The
activated enzyme donates a phosphate to the
free 5’-phosphate at the nick in the lower
strand of the DNA duplex, creating a high-
energy diphosphate group on one side of the
nick. Step 3: With energy provided by
cleavage of the diphosphate, a new
phosphodiester bond is created, sealing the
nick in the DNA. This reaction can also
occur in both DNA strands at once, so two
independent DNAs can be joined together
by DNA ligase.
 Figure 4.7 Joining of vector to insert.
(b)Alkaline phosphatase prevents vector
re-ligation.
Step 1: We cut the vector(blue, top left)
with BamHI. This produces sticky ends
with 5’-phosphates(red). Step 2: We
remove the phosphates with alkaline
phosphatase, making it impossible for the
vector to re-ligate with itself. Step 3: We
also cut the insert(yellow, upper right)
with BamHI, producing sticky ends with
phosphates that we do not remove. Step
4: Finally, we ligate the vector and insert
together. The phosphates on the insert
allow two phosphodiester bonds to
form(red), but leave two unformed
bonds, or nicks, These will be completed
once the DNA is in the transformed
bacterial cell.
DNA can be inserted into a cell by:

• Transformation
• Electroporatio
n
• Protoplast
fusion
 TRANSFORMATION OF
LIGATION PRODUCTS

• The process of transferring exogenous DNA into cells is

call “transformation”
• There are basically two general methods for
transforming bacteria. The first is a chemical method
utilizing CaCl2 and heat shock to promote DNA entry
into cells.
• A second method is called electroporation based on a
short pulse of electric charge to facilitate DNA uptake.
CHEMICAL TRANSFORMATION WITH
CALCIUM CHLORIDE
TRANSFORMATION BY ELECTROPORATION
STEP 5. GROWTH ON AGAR
PLATES
 DNA can be inserted into a cell by:
• Microinjection
• Gene gun
 Screening
• The medium in this petri
dish contains the
antibiotic Kanamycin
• The bacteria on the
right contain Kanr, a
plasmid that is resistant
to Kanamycin, while the
one on the left has no
resistance
• Note the difference in
growth
 Propagation
• Once colonies are
identified, they are
cultured in broth to
increase numbers
and therefore the
amount of DNA
• Samples are also
prepared for storage
at -80 degrees. They
can be kept for many
years this way.
 Figure 4.5 Screening bacteria by
replica plating.
(a) The replica plating process. We
touch a velvet-covered circular tool to
the surface of the first dish containing
colonies of bacteria. Cells from each of
these colonies stick to the velvet and
can be transferred to the replica plate in
the same positions relative to each
other. (b) Screening for inserts in the
pBR322 ampicillin resistance gene by
replica plating. The original plate
contains tetracycline, so all colonies
containing pBR322 will grow. The
replica plate contains ampicillin, so
colonies bearing pBR322 with inserts in
the ampicillin resistance gene will not
grow (these colonies are depicted by
dotted circles). The corresponding
colonies from the original plate can then
be picked.
 Polymerase Chain Reaction (PCR)
(Invitro Cloning)

• Developed in the mid 1980s

• Revolutionized molecular biology
• DNA fragments can be amplified in large amounts
• In vitro technique
• Kary Mullis - Nobel prize (1993)
What do we need for PCR?
• Sequence information
• Oligonucleotide primers
• DNA
• Nucleotides
• Heat-stable DNA polymerase
• Taq (Thermus aquaticus)
 DENATURA
TION
T ≥ 95⁰C
RENATURATION
T <75⁰ C
 PCR technique

ds DNA
Step 1 Denature

Step 2 Anneal

Step 3 Extend
 DENATURA
TION
T ≥ 95⁰C
RENATURATION
T <75⁰ C
 dNTP
dATP
dTTP
dGTP
dCTP

Primer

DNA-polymerase

DNA-template

DENATURATION ANNEALING PRIMER EXTENTION

PCR amplifies DNA (gene)

1st cycle
2nd cycle
3rd cycle

4th cycle

5th cycle

6th cycle
 CYCLE # AMOUNT OF DNA
0 1
1 2
2 4
3 8
1600000000 4 16
1400000000 5 32
AMOUNT OF DNA

1200000000 6 64
7 128
1000000000
8 256
800000000 9 512
600000000 10 1,024
400000000 11 2,048
12 4,096
200000000
13 8,192
0 14 16,384
0 5 10 15 20 25 30 35 15 32,768
PCR CYCLE NUMBER 16 65,536
17 131,072
10000000000 18 262,144
1000000000 19 524,288
20 1,048,576
AMOUNT OF DNA

100000000
10000000
21 2,097,152
22 4,194,304
1000000
23 8,388,608
100000
24 16,777,216
10000
25 33,554,432
1000
26 67,108,864
100
27 134,217,728
10 28 268,435,456
1 29 536,870,912
0 5 10 15 20 25 30 35 30 1,073,741,824
PCR CYCLE NUMBER 31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
 PCR: Theory vs. Reality

• In theory, PCR reactions

amplify exponentially, Theoretical
doubling every cycle increase
and grow forever.

Log Target
Realit
y

DNA
• In reality,

Cycle #