This document discusses recombinant DNA technology and its applications. It covers key steps like cloning vectors, restriction modification systems, DNA ligases, transformation, and screening cDNA libraries to identify genes. Examples are given of recombinant proteins used as drugs to treat diseases like growth hormone deficiency, diabetes, and hemophilia. The last section briefly discusses genetic modification of animals and plants, and debates around genetically engineered salmon.

1. Recombinant
DNA Technology
BY- Dr. Armaan Singh

2. Cloning Vector
Required features
1. Origin of replication
2. Selectable marker
3. Screenable marker for
recombinant molecules
4. Cloning sites

3. Restriction Modification System
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
* *
AAGATGCGAATTCGTACA
* *
DNA methylase
Restriction endonuclease
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
AAGATGCG AATTCGTACA
DNA methylase
Restriction endonuclease

4. 5’ ATGCGAATTCCGGTT 3’
3’ TACGCTTAAGGCCTT 5’
5’-ATGCG-3’ 5’-AATTCCGGTT-3’
3’-TACGCTTAA-5’ 3’-GGCCTT-5’
EcoR1
5’ ATGCGATATCCGGTT 3’
3’ TACGCTATAGGCCTT 5’
5’-ATGCGAT-3’ 5’-ATCCGGTT-3’
3’-TACGCTA-5’ 3’-TAGGCCTT-5’
EcoRV
Sticky-end cutter
Blunt-end cutter

5. T4 DNA
Ligase

7. Transformation
• All of the previous steps were
performed in vitro.
• We have generated a very small
amount of a recombinant plasmid
• Need to amplify in bacteria to get
enough to work with.
• Transformation – process to mobilize
DNA into bacterial host
• Select for transformed bacteria on
specific antibiotic that corresponds
to the antibiotic resistance gene
present on the plasmid

8. How to produce a
recombinant protein
10 to 70%
of cellular
protein
0.1 to 1%
of cellular
protein

9. cDNA

10. cDNA
Library
cDNA

11. DNA
hydridization
screening for
specific gene
•Requires that you know
something about the gene
sequence
•Can get sequence
information form purified
protein

12. Product name Protein type Application Company
Adagen (Adenosine
deaminase )
An enzyme Severe combined
immunodeficiency
disease (SCID)
Enzon
Genotropin
(Recombinant
growth hormone)
A hormone Growth hormone
deficiency (GHD) in
children
Pharmacia & Upjohn
Humalog
(Recombinant human
insulin)
A hormone Diabetes Eli Lilly
Nabi-HB (Anti-
Hepatitis B)
An antibody Hepatitis-B Nabi
Novo Seven
(Recombinant
coagulation factor
VIIa)
A modified factor Hemophillia patients
with inhibitors
Novo Nordisk
Ontak (Diphtheria
toxin-interleukin-2)
A fusion protein Cutaneous T-cell
lymphoma (CTCL)
Ligand
Pharmaceuticals
Roferon-A
(Recombinant
interferon alfa-2a)
A modifier Hairy cell leukemia
or AIDS-related
Kaposi's sarcoma
Hoffmann-La Roche

13. Genetic Modification of
Higher Organisms
• Can introduce gene into animals and plants
• These modified organism are powerful research
tools to study the effect of a specific gene
product on metabolism, development etc….
• Has also been used to develop improved
agricultural products

15. Genetically Engineered Salmon
Is Bigger Better?

16. http://www.agwest.sk.ca/sabic_index_tp.shtml