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Chapter 23 (Part 1)
Recombinant DNA Technology
Cloning Vector
Required features1. Origin of
replication2. Selectable marker3. Screenable marker
for recombinant molecules
4. Cloning sites
Restriction Modification System
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA* *
AAGATGCGAATTCGTACA* *
DNA methylase
Restriction endonuclease
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
AAGATGCG AATTCGTACA
DNA methylase
Restriction endonuclease
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
T4 DNA Ligase
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
How to produce a recombinant protein
10 to 70% of cellular protein
0.1 to 1% of
cellular protein
cDNA
cDNA Library
cDNA
DNA hydridization screening for specific gene
•Requires that you know something about the gene sequence•Can get sequence information form purified protein
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
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
Genetically Engineered SalmonIs Bigger Better?
