Genes, Genomes

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Genes, Genomes Genetic Engineering
Part 2

• Dan Graur
• University of Houston

http//nsm.uh.edu/dgraur/
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Genetic Engineering A collection of techniques
used to manipulate genes, genomes, and other
pieces of DNA, as well as their products and
expression.
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1. Isolating DNA
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2. Reading DNA
sequencing
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3. Cutting DNA
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Restriction Endonucleases
Protect bacteria from bacteriophages.
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Restriction Endonucleases
Palindromes Madam, Im Adam. RADAR A dog. A
panic in a pagoda. Campus motto Bottoms up, Mac.
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Molecular Palindrome
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4. Ligating DNA
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ligase
Fragments that have been produced by a
restriction enzyme can be ligated to other
fragments produced by the same enzyme.
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5. Separation by size
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6. Identification
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7. Transportation
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Texas-style molecular biology
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8. Selection
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Plasmid pBR322
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Ampicillin
Tetracycline
1 2 3 4
1 2 3 4
Streak individual colonies with sterile toothpick.
5 6 7 8
5 6 7 8
1 2 3 4
1 2 3 4
Incubate overnight.
5 6 7 8
5 6 7 8
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Q Which colony is resistant to ampicillin and
sensitive to tetracycline. A 6
Ampicillin
Tetracycline
1 2 3 4
1 2 3 4
5 6 7 8
5 6 7 8
1 2 3 4
1 2 3 4
5 6 7 8
5 6 7 8
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9. Reverse transcription
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10. Copy
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Cycle 0 1 2 3 4 5 n Number 1 2 4 8 16 64 2n of
copies
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11. Genomic library
Library
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27 February 1997
12. Cloning
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Dolly
Incubator
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Another Dolly
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13 Many Present Techniques I Know Nothing About
Many Future Techniques
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Engineering is artificial. There is nothing
artificial about genetic engineering.
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Oocyte from Drosophila infected with Wolbachia.
Note concentration of bacteria in the anterior
region of the cell.
Drosophila ananassae
D. ananassae is a natural transgenic
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What is genetic engineering good for?
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1. Diagnostics of genetic traits (e.g.,
disorders)
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2. Protein Synthesis
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Coagulation Factors
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(1) Vessel damage (2) Vascular spasm (3)
Platelet plug forms. (4) Coagulation.
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Hemophilias
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Treating hemophilia A and hemophilia B Factors
VIII and IX are purified from the pooled blood of
several thousands donors. Injections of factors
VIII and IX have allowed hemophiliacs to live
relatively normal lives.
1/2
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Treating hemophilia A and hemophilia B In the
1980s, HIV-contaminated blood was unknowingly
used to manufacture factors VIII and IX. In
some countries (e.g., France), gt 90 of the
hemophiliacs became infected by HIV.
2/2
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Possibility of HIV contamination
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Human Growth Hormone The first GE product.
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Is Jintropin (recombinant human growth hormone)
the new national past time?
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Insulin The best selling GE product.
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3. Transgenics
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Photinus pyralis A source of luciferase
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Insert luciferase gene into tobacco cell culture,
cause differentiation to occur, water plants with
ATP solution, and get a glowing-in-the-dark
transgenic tobacco plant.
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Green fluorescent protein (GFP)
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Attach to Opsin promoter Expression in Eyes
Attach to Lysin promoter Expression in Sperm
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4. Personal identification (Fingerprinting)
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People are different from one another
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Paternity determination
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Paternity determination
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How to distinguish between a relatively cheap
Trebbiano and a much more expensive Verdelho?
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expensive
cheap
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5. Somatic gene therapy
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Injection of Transformed Cells
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Severe combined immunodeficiency (SCID) due to
adenosine deaminase (ADA) deficiency