Week 4 Lecture 3, Gene Cloning:

DNA Technology
-Due to our understanding of DNA and gene expression, we can do
almost anything you can imagine with genes.
-Recombine genes from different species.
-Make specific mutations in a gene.
-Insert DNA into bacteria and certain eukaryotic cell to force
expression of an outside gene.
-Make lots of copies of it.
-Sequence it.
-Measure expression of genes in different conditions.
What if you want to
-Research a particular gene or protein.
-Force expression of a gene in another organism.
-Cause crops to be pest resistance or produce vitamins.
-Produce protein for treating disease.
-Insulin
-How can you manipulate a single gene.

Gene Cloning

Our Toolbox
-Restriction Enzymes-the DNA scissors
-Bacterial Plasmids-used to carry our gene of interest
-DNA ligase-the DNA glue
-E.coli bacteria-organism that is easily manipulated in lab
Isolate Gene of Interest

The DNA Scissors

-Restriction Enzymes
-AKA Restriction Endonucleases
-Cut DNA at specific palindrome sequences.
-In nature, used by bacteria to destroy foreign DNA (restrict
replication of
virus).
-In the lab, used to produce useful fragments of DNA.

Plasmids
-The vehicle for the gene of interest.
-Small, circular, self-replicating DNA molecule.
-Separate from bacteriums genome.
-Can replicate itself in the absence of cell division (lots of copies
in one
bacteria).
-The original plasmid used for cloning is a cloning vector.
Cloning Vector
A cloning vector needs to include 3 things
-A cloning site
-A place to insert DNA
-Usually contains multiple restriction sites for versatility
-A selectable marker
-To find which bacteria have the plasmid
-Usually antibiotic resistance (ex. Ampicilin)
-Origin of replication
-So plasmid will be reproduced in bacteria

Plasmid & Protein Factory

Escherichia Coli
-Escherichia coli (E.coli) is the most commonly used bacteria in lab.
-Easy to grow.
-Divides rapidly.
-Non-pathogenic.
Bacterial Transformation
-Some bacteria will import free pieces of DNA floating in their
environment.
-E.coli are induced to transform when
-In a high calcium solution
-And exposed to a heat shock
-Grow bacteria in media with antibiotic
-Antibiotic kills bacteria that were not transformed

Week 4 Lecture 4, Experimental Techniques With DNA:

Gene Cloning Revisited
-Gene cloning is done with large populations of DNA.
-If we know the sequence of the gene of interest, it is easy to get lots
of copies.
Polymerase Chain Reaction
-Used to amplify a particular region of DNA
-Replicate a small region over and over again
-What do we need to replicate DNA?
-Primer
-DNA
-Nucleus
-Heat
-Cool
-Warm
-Repeat