Transcript Molecular Cloning Methods

RECOMBINANT DNA TECHNOLOGY
PART 2
Quiz Essay 2 Topics
Molecular Topics: 35 points
Emphasis: data interpretation, critical thinking with a short answer format
■ Bacterial transformation
■ Plasmid isolation
■ Translation: genetic code table
■ Lac operons: mutational analysis
■ Recombinant DNA technology
Genetics’ Topic 15 points
■ Genetic Fine Structure
Topics
• Cloning & Finding cloned genes
– Using bacteria
– Using viruses
• Genomic libraries
• Transgenic plants
• Transgenic animals
• Timely topics
Technique: Cloning of specific genes or DNA
fragments
= restriction enzyme
+
= ligase
Cut vector
Cut foreign DNA or gene
Paste together
2 different antibiotic resistant genes
Bacteria are now resistant to 2 antibiotics
Bacterial cloning using plasmids
Cut DNA here &
insert foreign DNA
Will your transformed colonies with the insert be white or blue?
Summary of cloning DNA
into a blue/white selection
vector
Cloning and finding desired clone
• Clone into a virus
• Infect bacteria
• Identify infected bacteria containing gene
– Assume you know the protein sequence
Bacterial clones and finding desired clone
Probe design
Genomic library contains all of the organism’s genome
Expression Vectors
Expression vectors
• Sources of proteins
Transgenic Mice
transgen
Non-homologous recombination
Linear DNA
Knockout Mice
G418
gancyclovir
Homozygous
knockout
Phenotype of the knockout mouse gives information on protein function
Applications of DNA technology
• Agriculture
• Drug design
• Genetic testing
• Gene therapy
Genetic Modified Plants: “GM”
Tobacco with luciferase gene from firefly
Transgenic plant
Tumor Inducing Plasmid
Model for making transgenic plants
• Bt toxin genes (from
Bacillus thuringensis)
introduced into corn,
making it resistant to the
European corn borer.
• Millions acres of corn,
cotton, and potatoes
bearing Bt toxin are under
cultivation.
Transgenic Plants
• In 1996: Soybeans resistant to the
weed killer of Round Up.