Download Chapter 23 (Part 1)

Chapter 23 (Part 1)
Recombinant DNA Technology
Cloning Vector
Required features
1. Origin of replication
2. Selectable marker
3. Screenable marker
for recombinant
molecules
4. Cloning sites
Restriction Modification System
AAGATGCGAATTCGTACA
DNA methylase
AAGATGCGAATTCGTACA
DNA methylase
*
*
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
Restriction endonuclease
Restriction endonuclease
*
*
AAGATGCGAATTCGTACA
AAGATGCG
AATTCGTACA
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’
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
0.1 to 1%
of cellular
protein
10 to 70%
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 (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
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 Salmon
Is Bigger Better?
http://www.agwest.sk.ca/sabic_index_tp.shtml