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1. Animal and
human cloning
Dolly the sheep (1997-2003)
2. Gene cloning
1. Animal and human cloning
What is Gene cloning?
To "clone a gene" is to
make multiple copies of it
in vivo
Why Clone DNA?
• A particular gene can be isolated and its nucleotide
sequence determined
• 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
Insulin overexpression in bacteria
Gene encoding insulin
RE1
RE2
RE1
RE2
RE1
RE1
RE2
RE2
Ligation
Insulin overexpression in bacteria
Transformation into
E. coli cells
Insulin
What do we need for gene cloning?
•Insert
•Plasmid Vector
•Ligation of the palsmid+Insert
•transformation
Target DNA
Plasmid vector
ligation
transformation
Plasmids are Used to Replicate a Recombinant DNA
Plasmids are small circles of DNA found in bacteria.
Plasmids replicate independently of the bacterial chromosome.
Pieces of foreign DNA can be added within a plasmid to
create a recombinant plasmid.
Plasmid vectors
A Plasmid (vector) is a DNA molecule used for transferring foreign DNA
fragments (genes) into host cells
Circular DNA molecules capable of autonomous replication in living cells
All plasmids contain:
1. Origin of replication (ori)
2. Selection marker (Ampicillin)
3. Multiple Cloning Site (MCS)
MCS
Insert – Target DNA
1. PCR product
2. Restriction Enzymes
RE1
RE2
RE1
RE1
RE2
RE2
Ligation
REs will produce ends that enable the gene to be spliced into a plasmid
Ligation
REs and DNA ligase
Ligation of the insert to the plasmid cut with only one enzyme
1
1
1
1
DNA ligase
Ligation of the insert to the plasmid cut with only two enzyme
RE1
RE2
ligation
There is two possible outcomes
Transformation
Use of bacterial cells to amplify
the DNA of interest
Two main methods:
1. Chemical transformation – Chilling cells in the presence
of Ca2+ prepares the cell walls to become permeable
to plasmid DNA. Cells are briefly heat shocked which
causes the DNA to enter the cell
2. Electoporation- making holes in bacterial cells, by
briefly shocking them with an electric field of 1020kV/cm. Plasmid DNA can enter the cell through
these holes.
Possible products of the transformation:
Plasmid + insert
Ampicillin resistant
Plasmid without insert
Ampicillin resistant
No plasmid
No ampicillin resistance
How can we differentiate between the bacteria
containing plasmid+insert and the ones with the self
ligated plasmid (no insert)?
Our lab experimemet
Insert that was amplified by PCR
pGEM Vector
ligation
Transformation
Screening
Possible products of the transformation:
Plasmid + insert
Ampicillin resistant
Plasmid without insert
Ampicillin resistant
No plasmid
No ampicillin resistance
How can we differentiate between the bacteria
containing plasmid+insert and the ones with the self
ligated plasmid (no insert)?
Cloning procedure
transformation
+ IPTG
+ X-Gal
Lac Z gene
Gene expression dogma
DNA
promotor
RNA
pol.
RNA
Ribosome
LacZ gene
LacZ mRNA
β-galactosidase
Protein
X-gal
BLUE colonies
X-gal
WHITE colonies
Lac Z gene
promotor
LacZ
RNA
pol.
promotor
operator
RNA
pol.
LacZ
Repressor
IPTG
IPTG
promotor
operator
RNA
pol.
IPTG
LacZ
IPTG
IPTG
X-gal
Β-galactosidase
X + galactose
Cells which produce ß-galactosidase form BLUE colonies.
Cells without ß-galactosidase production form WHITE colonies.
Screening
Insert
promotor operator
T
LacZ
T
pGEM
X X
X without plasmid
X
X
WHITE colonies
BLUE colonies
Plasmid without Insert
Plasmid +Insert
Confirmation by digestion with restriction
enzyme and separation of the digestion
products on agarose gel
A plasmid DNA will be purified from the bacteria cells.
Plasmid DNA will be digested with EcoRI, and analyzed by gel
electrophoresis for identification of the clone containing insert.
EcoRI
pGEM
Vector
EcoRI
Insert