TATAP MUKA 6

STRATEGI
KLONING (1)

CLONESTRATEGY

requires

Method required

DNA fragments

Aligning DNA
fragment to
Vector

Obtained from

Resulted in

Genomics
DNA
through
Mechanica
lly
Restrictio
n enzyme

mRN
A
Synthesise
d to
cDNA

PCR

DNA
Synthesis

Biomolecular
eecombinant

through

Resulted in

Produces
DNA
Fragme
nt for
cloning

Inserted DNA
/Vector
concatameric

Aligned to vector
using
Resulted in

cDNA lybrary
Genomics library

Blunt-end
ligation
Linker
application
Adapter
application
Homopolymer
Can
be
tailing
applied
for of
Ligation
cohesive end Gene
isolatio
n

Learning Outcome (LO)

LO 54: menjelaskan tahapan kloning DNA
LO 55: menjelaskan materi awal kloning
DNA
LO 56: menjelaskan sintesis cDNA
LO 57: menjelaskan kloning cDNA ke dalam
plasmid
LO 58: menjelaskan penggunaan linker,
adaptor, dan homopolymer tailing
LO 59: menjelaskan kloning cDNA ke dalam

Cloning strategies
Four steps of cloning
Cutting DNA strands into fragments
Alignment of DNA fragments
to vector to allow
recombinance
Propagation of DNA
recombinant in the
host cell
Identification and
selection of
targetted DNA
recombinant

LO 54: menjelaskan tahapan kloning DNA

Path ways
Preparat
ion of
cDNA synthesis
DNA
fragmen
t
Blunt end
Ligatio
ligation
n
Inserting
DNA to
the hosts
cell

Cutting of Dna
fragment using
restriction
enzyme

Transformation

Linker
applicatio
n

Mechanical
l cutting

Homopoly
mer tailing

Transfectio
n

LO 54: menjelaskan tahapan kloning DNA

Chemical
synthesis

Cohesive
end ligation

In vitro
packing of
DNA

tial matterial for cloning

Genomic DNA
Representing whole
genome of an organism
If the genome is too
big, it might cause big
problem especially if
the main purpose is to
isolate single gene
Has intron, controlling
area and repeating
sekuens.

mRNA
Representing
genetic
information expressed by
a particular cell
Not all genomic DNA is
represented in an mRNA
population .
Can
only
represent
abundance of particular
mRNA
Contains
sequence
of
gene
codes,
due
to
elimination of intron along
processing of mRNA

LO 55: menjelaskan materi awal kloning

Note: The diversity of mRNAs is indicated by the number of

different mRNA molecules. There is one mRNA that is present in
chick oviduct cells at a very high level (100000 molecules per
cell). This mRNA encodes ovalbumin, the major egg white protein.
Source: After Old & Primrose (1989), Principles of Gene
Manipulation, 4th edition, Blackwell. Mouse data from Young et al.
(1976), Biochemistry 15, 28232828, copyright (1976) American
Chemical Society. Chick data from Axel et al. (1976), Cell 11, 247
254, copyright (1976) Cell Press. Reproduced with permission.
LO 55: menjelaskan materi awal kloning

Sintesis cDNA
short oligo(dT) primer is
annealed to the poly(A) tail
on the mRNA
reverse transcriptase to
begin copying the mRNA

mRNA is
removed
short
double-stranded hairpin loop
second-strand synthesis by
a DNA polymerase

double-stranded cDNA is
trimmed with S1 nuclease
Fig. 6.2. Synthesis of cDNA. Poly(A) RNA (mRNA) is used as the
starting material
LO 56: menjelaskan sintesis cDNA

Producing cDNA from mRNA

 The above statements might cause some

problems as follows:
1) Sinthesis of cDNA might probably not
efficientlly run especially when mRNA is
relatively long.
2) Application of S1 nuclease could relieve
some important bases at 5 end.
The latest method to synthesis cDNA is by
using an Oligo(dG)-primed
secondstrand cDNA synthesis (Figure 6.3).

LO 56: menjelaskan sintesis cDNA

mRNAcDNA hybrid

tailed with C residues

using terminal
transferase

hydrolyses the mRNA

An oligo(dG) primer
is annealed to the C
tails
double-stranded
full-length cDNA
molecule

Fig. 6.3. Oligo(dG)-primed second-strand

cDNA
synthesis
LO
56: menjelaskan
sintesis cDNA

ning of cDNA to the plasmid vector

Plasmid is still, being used as vector especially
when isolation of preffered cDNA including small
numbers of screened-clones.
Ligation of cDNA
Sinthesys of cDNA
fragment to the
vector usually run
in either of the
following
Blunt-end
linker
Homopoly
ligation
mer tailing
methods :
Ligation of
blunt-end
linker
homopolimer
tailing
LO 57: menjelaskan kloning cDNA ke dalam plasmid

 Blunt-end ligation is a ligation process of

DNA molecule to the blunt-end, done by
which is done by DNA ligase
The blunt-end might be obtained from :
Applicatiion of S1 nuclease
Filling the cohesive-end with the DNA
polimerase.
Blunt-ent ligation is inefficent ligation
process since there is no particular relation
between molecules to hold the DNA strand
before the DNA ligase forms fosfodiester
bonds to produce DNA recombinant.
Probability for joining two blunt-ends, the
ligation might be done using high
concentration DNA

 Theoritically, when DNA vector is mixed

with cDNA, it might produced:
Recombinant with one insert.
Self-ligate to make a circular molecule or
insert / vector DNA could form concatemers

Practically, to avoid self ligation vector is

treated by fosfatase (either BAP or CIP).
To solve problems in joining blunt-ends the
following methods are applied :
Linker
Adaptor
Homopolymer tailing

Fig. 6.4. Use of linkers. (a) The 10-mer 5-CCGAATTCGG3 contains the recognition site for EcoRI. (b) The linker is
added to blunt-ended DNA using DNA ligase. (c) The
construct is then digested with EcoRI, which cleaves the
linker to generate protruding 5 termini.
LO 58: menjelaskan penggunaan linker, adaptor, dan homopolymer

Fig. 6.5. Use of adaptors. In this example a BamHI

adaptor (5-GATCCCCGGG-3) is annealed with a singlestranded HpaII linker (3-GGGCCC-5) to generate a double
stranded cohesive-ended molecule, as shown in (a). This is
added to blunt-ended DNA using DNA ligase. The DNA
therefore gains protruding 5 termini without the need for
digestion with a restriction enzyme, as shown in (b). The 5
of the adaptor
can be
dephosphorylated
to prevent
LOterminus
58: menjelaskan
penggunaan
linker,
adaptor, dan homopolymer

Fig. 6.6. Homopolymer

tailing. (a) The vector is
cut with PstI, which
generates protruding 3OH termini. (b) The
vector is then tailed with
dG
residues
using
terminal transferase. The
insert DNA is tailed with
dC residues in a similar
way. (c) The dC and dG
tails are complementary
and
the
insert
can
therefore be annealed
with
the
vector
to
generate a recombinant.
The
PstI
sites
are
regenerated at the ends
the dan
insert
DNA, as
LO 58: menjelaskan penggunaan linker,of
adaptor,
homopolymer

ning cDNA into bacteriophage

(a) The
ds
cDNA
is
treated with EcoRI
methylase,
(b)which methylates any
internal
EcoRI
recognition
sequences.
(c) EcoRI linkers are then
added to the ends of
the methylated cDNA,
and
the
linkers
digested with EcoRI.
(d)The
methylation
prevents digestion at
internal sites, and the
Fig. 6.7 Cloning cDNA in vectors
result is a cDNA with
using linkers
EcoRI
cohesive ends.
LO 59: menjelaskan kloning cDNA ke dalam
bakteriofaga

ng cDNA in phage vectors using link

LO 59: menjelaskan kloning cDNA ke dalam bakteriofaga

MINGGU DEPAN
TATAP MUKA 7