DNA Manipulation 2
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Transcript of DNA Manipulation 2
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....