DNA

Clonning
DONE BY: ENID HII LIN WEI & CHONG
HUI JUN
DEFINITION:
DNA a
CLONIN molecul
G ar
biology
techniq
ue that
makes
many
identica
l copies
 OVERVIEW PROCESS OF DNA
CLONING
the gene
replicate
or other
the
DNA
recombina plasmid
fragment produces a
“cut and nt plasmid and pass it
of interest molecule
paste” is on to their
is inserted of
DNA using introduced offspring,
into a recombina
enzymes into making
circular nt DNA
bacteria copies of
piece of
the DNA it
DNA called
contains
a plasmid.
 Click icon to add picture

THE GENE OF INTEREST IS INTRODUCED INTO

THE PLASMID TO BECOME A RECOMBINANT DNA
plasmi
d and
DNA cloning can be used to synthesize
"paste
Grow
a
"protein
in (such as human insulin) up
in bacteria.
the Use
lots of
gene. antibi
plasmi
This otic Harve
d-
proces selecti st the
Insert carryi
s on to protei
the ng
relies identif n from
plasmi bacter
on y the the
d into ia and
restric bacter bacter
bacter use
tion ia that ia and
ia them
enzym took purify
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es up the it.
make
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the
1. Cutting and pasting DNA
• restriction enzymes and DNA ligase
i. restriction enzyme
• a DNA-cutting enzyme that
recognizes a specific target
sequence and cuts DNA into two
pieces at or near that site.
• produce cut ends with short,
single-stranded overhangs
• two molecules have matching
overhangs-base-pair and stick
together
ii. DNA ligase
• two molecules have
matching overhangs
joined and combined
by DNA ligase combine
to form an unbroken
DNA molecule , which
seals gaps in the DNA
backbone.
 The gene a
combine
plasmid, fragment, recombina
the
which has which has nt plasmid
fragments
a single a cut site containing
with DNA
cut site is near each the gene
ligase
digested. end is is made
digested
2. Bacterial
transformation and
selection
i. transformation
• Plasmids and other
DNA introduced into
bacteria, such as the
harmless E. coli
ii. Selection
• A plasmid typically contains an antibiotic
resistance gene, which allows bacteria to
survive in the presence of a specific
antibiotic.
• bacteria that took up the plasmid can be
selected on nutrient plates containing the
antibiotic.
• Bacteria without a plasmid - die
• Bacteria carrying a plasmid - live and
reproduce
• Each surviving bacterium will give rise to a
colony of identical bacteria that all carry
the same plasmid.
ligation, DNA fragments don’t
always get “pasted” in exactly
the way intend.
- restriction enzyme digestion
and PCR are commonly used to
check the plasmids.
 Give the
a bacterial grow a large bacteria a
colony culture of chemical
with the plasmid- signal that
right instructs them
bearing
to make the
plasmid bacteria target protein
For instance, if our
target protein is plasmid contained
purified, or the human insulin
separated from the bacterial gene, the bacteria
would start
the other cells can be split
transcribing the
contents of the open to release gene and translating
cells by it the mRNA to
biochemical produce many
molecules of human
techniques
insulin protein.
https://www.you
tube.com/watch
LET’S HAVE A VIDEO!
?v=MIfDx417SD
Uses of DNA cloning
Biopharmaceuticals
• make human proteins with biomedical applications, eg.
insulin
• Other examples of recombinant proteins include
human growth hormone - given to patients who are
unable to synthesize the hormone
• Tissue plasminogen activator (tPA) - used to treat
strokes and prevent blood clots. Recombinant proteins
like these are often made in bacteria.
GENE THERAPY
• genetic disorders - patients lack the functional form
of a particular gene
• Gene therapy provide a normal copy of the gene to
the cells of a patient’s body
• Eg. DNA cloning was used to build plasmids
containing a normal version of the gene that's non-
functional in cystic fibrosis. When the plasmids were
delivered to the lungs of cystic fibrosis patients, lung
function deteriorated less quickly.
 GENE ANALYSIS
• In labs, biologists often use DNA cloning to build artificial,
recombinant versions of genes that help them understand how
normal genes in an organism function. 
• Eg, researchers studying neurone in fruit flies use DNA cloning
to assemble a reporter construct for a neural gene. In this
construct, the regulatory region (promoter) of the gene might
be pasted in front of a gene encoding a fluorescent protein.
When transferred into a fly, the "reporter gene" would be
expressed in the same neurons as the neural gene itself,
causing those neurons to glow (fluoresce) under UV light.