This document summarizes the key steps in recombinant DNA technology. It describes the required features of plasmids used for cloning, including an origin of replication, selectable marker, screenable marker, and cloning sites. It explains how restriction enzymes are used to cut DNA and how ligases join DNA fragments. It describes transforming bacteria with recombinant plasmids and selecting for transformed colonies. Finally, it provides examples of commercially available proteins and therapeutics produced through recombinant DNA technology, including hormones, enzymes, antibodies, and fusion proteins.

2. Recombinant DNA Technology

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

5. AAGATGCGAATTCGTACA
DNA methylase
AAGATGCGAATTCGTACA
DNA methylase
*
*
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
Restriction endonuclease
Restriction endonuclease
*
*
AAGATGCGAATTCGTACA
AAGATGCG AATTCGTACA

6. 5’ ATGCGAATTCCGGTT 3’
3’ TACGCTTAAGGCCTT 5’
EcoR1
Sticky-end cutter
5’-ATGCG-3’
5’-AATTCCGGTT-3’
3’-TACGCTTAA-5’
3’-GGCCTT-5’
5’ ATGCGATATCCGGTT 3’
3’ TACGCTATAGGCCTT 5’
Blunt-end cutter
EcoRV
5’-ATGCGAT-3’
3’-TACGCTA-5’
5’-ATCCGGTT-3’
3’-TAGGCCTT-5’

7. T4 DNA
Ligase

9. 
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.

10. 
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

12. 0.1 to 1%
of cellular
protein
10 to 70%
of cellular
protein

14. cDNA

15. •Requires that you know
something about the gene
sequence
•Can get sequence information
form purified protein

16. 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 (AntiHepatitis 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

17. 
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

19. Genetically Engineered Salmon
Is Bigger Better?

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