Chapter 23 (Part 1) Recombinant DNA Technology. Cloning Vector Required features 1.Origin of...
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Transcript of Chapter 23 (Part 1) Recombinant DNA Technology. Cloning Vector Required features 1.Origin of...
![Page 1: Chapter 23 (Part 1) Recombinant DNA Technology. Cloning Vector Required features 1.Origin of replication 2.Selectable marker 3.Screenable marker for recombinant.](https://reader035.fdocuments.net/reader035/viewer/2022062407/56649d605503460f94a4154a/html5/thumbnails/1.jpg)
Chapter 23 (Part 1)
Recombinant DNA Technology
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Cloning Vector
Required features1. Origin of
replication2. Selectable marker3. Screenable marker
for recombinant molecules
4. Cloning sites
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Restriction Modification System
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA* *
AAGATGCGAATTCGTACA* *
DNA methylase
Restriction endonuclease
AAGATGCGAATTCGTACA
AAGATGCGAATTCGTACA
AAGATGCG AATTCGTACA
DNA methylase
Restriction endonuclease
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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
![Page 5: Chapter 23 (Part 1) Recombinant DNA Technology. Cloning Vector Required features 1.Origin of replication 2.Selectable marker 3.Screenable marker for recombinant.](https://reader035.fdocuments.net/reader035/viewer/2022062407/56649d605503460f94a4154a/html5/thumbnails/5.jpg)
T4 DNA Ligase
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![Page 7: Chapter 23 (Part 1) Recombinant DNA Technology. Cloning Vector Required features 1.Origin of replication 2.Selectable marker 3.Screenable marker for recombinant.](https://reader035.fdocuments.net/reader035/viewer/2022062407/56649d605503460f94a4154a/html5/thumbnails/7.jpg)
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
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How to produce a recombinant protein
10 to 70% of cellular protein
0.1 to 1% of
cellular protein
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cDNA
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cDNA Library
cDNA
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DNA hydridization screening for specific gene
•Requires that you know something about the gene sequence•Can get sequence information form purified protein
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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
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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
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Genetically Engineered SalmonIs Bigger Better?
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