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DNA Manipulation 2. The nucleus. contains DNA. DNA. Restriction Digestion. Restriction enzymes are proteins that cut DNA at specific sequences. Have optimal condition of pH, temperature, ionic strength etc... (enzymes) Scan DNA until they find the sequence. - PowerPoint PPT Presentation
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DNA Manipulation 2
DNA
The nucleus
contains DNA
Restriction Digestion
• Restriction enzymes are proteins that cut DNA at specific sequences.
• Have optimal condition of pH, temperature, ionic strength etc... (enzymes)
• Scan DNA until they find the sequence.
• http://www.youtube.com/watch?v=-sI5vy-cD2g
DNA Hybridisation• DNA denaturation: Two DNA strands can be separated by heat
without breaking phosphodiester bonds
• DNA renaturation = hybridization: Two single strands that are complementary or nearly complementary in sequence can come together to form a different double helix
• Single strands of DNA can also hybridize complementary sequences of RNA
DNA Hybridisation
Gene Cloning
• Inserting new DNA into another piece of DNA.• Most commonly used with bacterial plasmids
which are easily manipulated.• Usually used antibiotic resistance genes to
select for cloned DNA.• Recombination:• http://www.youtube.com/watch?
v=8rXizmLjegI&feature=relmfu
Example of a cloning vector
• What is a vector?• A piece of DNA that can be replicated (usually a plasmid) that cloned
DNA can be inserted into.
Cloning steps
Gene Delivery
• Gene delivery depends on the organism...– Bacteria: Plasmids, electroporation, conjugation
or bacteria phage.– Yeast: Yeast artificial chromosomes– Plants: Agrobacterium or gene gun– Animal: Electroporation, gene gun, transfection
reagents (lipid balls) or viral vectors.
Delivering genes to Plants
The RoundUp Ready Story
• Glyphosate is a broad-spectrum herbicide• Active ingredient in RoundUp herbicide • Kills all plants it come in contact with• Inhibits a key enzyme (EPSP synthase) in an amino acid pathway
• Plants die because they lack the key amino acids
• A resistant EPSP synthase gene allows crops to survive spraying
+ Glyphosate
X
RoundUp Sensitive Plants
X
X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)
Plant EPSP synthase
Aromaticamino acids
Without amino acids, plant dies
X
BacterialEPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate(EPSP)
Aromaticamino acids
RoundUp Resistant Plants
+ Glyphosate
With amino acids, plant lives
RoundUp has no effect;enzyme is resistant to herbicide
Delivering genes to Animals
Lentivirus
Adenovirus
Gene Gun
Liposomes (Lipids)
Transgenic animals
Cloned Genes to make therapiesHuman insulin produced in bacteria....