Mullis's 1998 autobiography Dancing Naked in the Mind Field, gives his account of the commercial development of PCR, as well as providing insights into his opinions and experiences. In the book, Mullis chronicles his romantic relationships, use of LSD, synthesis and self-testing of novel psychoactive substances, belief in astrology and an encounter with an extraterrestrial in the form of a fluorescent raccoon. "What if I had not taken LSD ever; would I have still invented PCR? I don't know. I doubt it. I seriously doubt it." Kary Mullis Born: December 28, 1944 Nobel Prize in Chemistry (1993) for creating PCR Quotes: “Sometimes in the morning, when it's a good surf, I go out there, and I don't feel like it's a bad world.”

Gene Cloning Preparing a Gene for Cloning Green Florescent Protein (GFP) In Vivo Gene Cloning Plasmid Vectors Antibiotic Resistance Markers Gene Expression

Preparing a Gene for Cloning 1)Double stranded DNA of a gene from a eukaryotic organism contains introns 2)As a normal part of the cell process of gene expression, transcription creates a primary RNA molecule 3)The introns are removed by restriction enzymes to form a mature mRNA that codes for the making of a single protein (animation)animation 4)The mRNA is extracted from the cell and purified 5)Reverse transcriptase is added which synthesises a single stranded DNA molecule complementary to the mRNA 6)The second DNA strand is made by using the first as a template, and adding the enzyme DNA polymerase

l In Vivo Gene Cloning 1)Gene of interest (GFP) is isolated and prepared by removal of introns 2)An appropriate plasmid vector is chosen 3)GFP gene and plasmid are each treated with the same restriction enzymes to make identical cuts 4)The DNA fragments are mixed together and complimentary ends are attracted by base-pairing. 5)DNA ligase is added to “glue” the GFP gene into the plasmid 6)The recombinant plasmid is introduced into a bacterial culture. 7)About 1 in 10,000 cells will take up the plasmid

Plasmid Vector plasmid

Antibiotic Resistance

What did the cells take up? Plasmid only Plasmid with insert cloned (GFP) Foreign DNA from the environment Nothing

How to determine which bacteria take up GFP gene Nothing or Foreign DNA only (no plasmid): Bacteria do not have Amp Resistance so they will not grow Plasmid only (no GFP): Bacterial colonies will not glow Plasmid with GFP Clone: Bacterial colonies will glow green under a UV light

GFP Expressed Plasmid without GFPIgnore the Red Colonies

GFP Expression

Transgenic Organisms /