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Molecular Cloning
Cloning
Gene
Expression
Applications
on
ati
Mutational
Analysis
Blunt ends? Sticky ends?
LIGATION can happen in 5 minutes!
Trans
for TRANSFORMATION
mation
for
1) Genetic simplicity – ~4400 genes.
2) Growth Rate – doubles every 20 minutes!
3) Safety – naturally occurs in the
1PLASMID in1E. coli cell
in 24 hours yields
4.7224 × 1021 clones
Cloning F I R S T S
1973 FIRST
recombinant
piece of DNA
was made.
animal gene
was cloned.
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Sele
human digestive tract and harmless
if handled with care.
4) Completely Sequenced
Genome – easier analysis.
5) Its ability to host foreign DNA.
on
cti
Transformants per μg DNA
Genetically
Modified
Organisms
5 Reasons E. coli is used
1972 FIRST
are biochemically characterized. 621
of these are commercially available.
2) The first three letters of the restriction enzyme acronym come from the bacteria from which it was isolated; e.g., EcoRI is isolated from E. coli.
3) Werner Arber, Hamilton Smith and
Dan Nathans jointly won the Nobel Prize
in 1978 for the discovery and innovative
work on restriction enzymes.
1.00E+07
Sticky ends
1.00E+06
Blunt ends
1.00E+05
1.00E+04
1.00E+03
0
5
10
15
20
Time, mins
25
30
2 Ways TO IDENTIFY Positive Clones
1) Blue/White screening using
X-Gal and IPTG.
2) Positive selection via disruption of
a lethal E.coli gene.
DID YOU
Recombinant
Vaccines
1) Currently 4091 restriction enzymes
A
DN
Drug
Production
Lig
Antisense
Technology
RESTRICTION ENZYMES
n
tio
Disease
Therapy
Production
of Human
Proteins
3 fun FACTS about
gment Gene
ra
Fra
OW?
KN
Molecular Cloning
is NOT the same as
Animal Cloning. Dolly
the Sheep was the
result of Somatic Cell
Nuclear Transfer.