Recombinant DNA

Technology
• Genetic engineering
– Modification of the genetic material of an
organism by directly
• Changes its nucleic acid genome

• Genetic engineering is accomplished by collection of

methods
• Recombinant DNA technology
• Recombinant DNA Technology
– Techniques used in carrying out genetic
engineering
– DNA for a particular phenotype is
• Identification,
• isolation and
• insertion of a gene into a vector
– Recombinant DNA molecule
– Large amount of gene and its products
 Applications
• In the field of
– Medicines
– Industries
– Agriculture
MOLECULAR CLONING
Introduction to Molecular Cloning
 What is Molecular Cloning?
• Identical copies
• Technique
– Insertion of the desired fragment of DNA
– Vector

Property of replicating autonomously

Inside host cells

 Requirements for Cloning

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

Carry required DNA

to host cell
• Plasmids
– Autonomously replicating small circular DNA
– Resistance genes
– ORI
• Bacteriophages
• others
 hindIII
Kpn I
BamH I
DNA

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