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 VectorA 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