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Cloning Arsalan Sarfraz
Cloning Arsalan Sarfraz
Cloning Arsalan Sarfraz
Technology
• Genetic engineering
– Modification of the genetic material of an
organism by directly
• Changes its nucleic acid genome
Desired
Suitable Restriction Means of Screening
gene/Foreign Host cells
DNA Vector enzymes insertion of clones
Recombinant DNA Technology
• Molecular techniques involved
– Restriction and Digestion analysis
– DNA sequencing
– Polymerase chain reaction
– etc
Foreign DNA
• Genomic DNA
• PCR product
• cDNA
• Synthesize
Vector
• Suitable vector
• Molecular carrier
Vector
Foreign
Not I
Xho I
Xba I
Apa I
Restriction enzymes
• Molecular Scissors
– Bacterial enzymes
• Cut DNA at specific site
Sticky ends
5’ GAATTC 3’
3’ CTTAAG 5’
Sticky ends
5’
G AATTC 3’
3’ CTTAA G 5’
Blunt ends
5’ GAATTC 3’
3’ CTTAAG 5’
Blunt ends
5’ GAA TTC 3’
3’ CTT AAG 5’
Insertion of Foreign DNA into vector
• Ligation of gene of interest into a linearized
vector
• Circular vector
– Digest vector and required gene of interest with
same restriction enzyme
– Ligation reaction
DNA ligase
Recombinant DNA
plasmid
Recombinant Recombinant
DNA plasmid DNA plasmid
Host cells
• Suitable host cells
– Requirement
• Transformation
– Host cells
– Different methods
• Chemical methods
• Physical methods
Screening/selection of positive clones
Bacterial colonies
Plasmid isolation
Confirmation
(PCR, Restriction and digestion
analysis, DNA sequencing)
• After successful cloning
– Expression analysis
• Protein
• RNA
• DNA