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....