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Dolly the sheep (1997-2003)
1. Animal and human cloning
2. Gene cloning
1. Animal and human cloning
To "clone a gene" is to make multiple copies of it in vivo
What is Gene cloning?
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 bacteriaGene encoding insulin
Ligation
RE1
RE2
RE1RE2
RE1
RE1
RE2RE2
Insulin overexpression in bacteria
Transformation into E. coli cells
Insulin
What do we need for gene cloning?
•Insert
Plasmid vector
ligation
transformation
Target DNA
•Ligation of the palsmid+Insert
•Plasmid Vector
•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.
All plasmids contain:
1. Origin of replication (ori)
2. Selection marker (Ampicillin)
3. Multiple Cloning Site (MCS)
Plasmid vectors
Circular DNA molecules capable of autonomous replication in living cells
A Plasmid (vector) is a DNA molecule used for transferring foreign DNA fragments (genes) into host cells
MCS
Insert – Target DNA
2 .Restriction Enzymes
1 .PCR product
RE1RE2
RE1
RE1
RE2RE2
T
T
Ligation
REs will produce ends that enable the gene to be spliced into a plasmid
Ligation
REs and DNA ligase
DNA ligase
11
1 1
Ligation of the insert to the plasmid cut with only one enzyme
RE1 RE2
Ligation of the insert to the plasmid cut with only two enzyme
ligation
There is two possible outcomes
Transformation
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 10-20kV/cm. Plasmid DNA can enter the cell through these holes.
Use of bacterial cells to amplify the DNA of interest
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)?
Plasmid + insert
Ampicillin resistant
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)?
Plasmid + insert
Ampicillin resistant
Cloning procedure
+ IPTG
+ X-Gal
transformation
Lac Z gene
LacZ genepromotorRNA pol.
Gene expression dogma
DNA
LacZ mRNARibosome
β-galactosidase
RNA
Protein
X-gal BLUE coloniesBLUE colonies
WHITE WHITE coloniescoloniesX-gal
LacZpromotor operator
Repressor
Lac Z gene
LacZpromotorRNA pol.
RNA pol.
IPTG
IPTG
IPTG
LacZpromotor operator
IPTGIPTG
RNA pol.
IPTG
X-galΒ-galactosidase
X + galactoseCells which produce ß-galactosidase form BLUE coloniesBLUE colonies. Cells without ß-galactosidase production form WHITE WHITE coloniescolonies.
X
XX
X
X
Plasmid without Insert
Plasmid +Insert
without plasmid
Screening
LacZ
pGEM
Insert
WHITE coloniesWHITE colonies BLUE BLUE coloniescolonies
promotor operatorT
T
A plasmid DNA will be purified from the bacteria cells.
Insert
Vector
Confirmation by digestion with restriction enzyme and separation of the digestion
products on agarose gel
EcoRI
EcoRI
Plasmid DNA will be digested with EcoRI, and analyzed by gel electrophoresis for identification of the clone containing insert.
pGEM
