PCR is DNA replication in a test tube Ex. 25: PCR Based Testing for Water Contaminants

SLOs Review issues concerning water pollution. List three common microbial water contaminants. Describe the basic principle of PCR and evaluate its significance for microbiology. Carry out PCR to determine whether or not the water is contaminated with E. coli.

Review DNA Replication Read and study Textbook Appendix C Genetics, pages A 24 & A 25

Invented by Kary Mullis, Nobel Prize “I was working for Cetus, making oligonucleotides. They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. I was driving with Jennifer Barnett to a cabin I had been building in northern California. She and I had worked and lived together for two years. She was an inspiration to me during that time as only a woman with brains, in the bloom of her womanhood, can be. That morning she had no idea what had just happened. I had an inkling. It was the first day of the rest of my life.” - from Karry Mullis’s autobiography at the Nobel e-Museum

Did He Really Invent PCR? The basic principle of replicating a piece of DNA using two primers had already been described by Gobind Khorana in 1971: – Kleppe et al. (1971) J. Mol. Biol. 56, Progress was limited by primer synthesis and polymerase purification issues. Mullis properly exploited amplification.

What is the Polymerase Chain Reaction? Think of it as a molecular photocopier It is a means of selectively amplifying a particular segment of DNA. The segment may represent a small part of a large and complex mixture of DNAs: e.g. a specific exon of a human gene.

A Molecular Photocopier A photocopier capable of duplicating a part of a sentence: “The next day was quite a different day. Instead of being hot and sunny, it was cool and misty. Pooh didn’t mind for himself, but when he thought of all the honey the bees wouldn’t be making, a cold misty day always made him feel sorry for them.” A.A. Milne, The words in blue must be unique for the copier to locate the correct piece of text.

How Powerful is PCR? PCR can amplify a single DNA molecule, e.g. from a single sperm. PCR can amplify DNA to a usable amount (visible by gel electrophoresis) in ~2 hours. The template DNA need not be highly purified — a boiled bacterial colony. The PCR product can be digested with restriction enzymes, sequenced or cloned.

The Basics of PCR Cycling cycles, each comprising: – Denaturation (95°C), 30 sec. – Annealing (55–60°C), 30 sec. – Extension (72°C), time depends on product size.

DolanDNA Learning Center: PCR Tutorial DolanDNA Learning Center on YouTube Additional narrated PCR Tutorial narrated PCR Tutorial

1.______________ 2.______________ 3.______________ 4.______________ 5.______________ Function ______________ What’s in the Reaction Tube?

So Then, it’s Easy? Cycling performed with three water baths. Thermal cyclers introduced in Early polymerases were not thermostable, so had to be replenished each cycle. The 37°C temperature caused non-specific priming, resulting in unwanted products.

Taq (Thermus aquaticus) DNA polymerase first described in 1988.

Forensic Microbiology Primer for a specific organism will allow for detection that particular organism Real-time PCR: Newly made DNA tagged with a fluorescent dye; the levels of fluorescence can be measured after every PCR cycle Reverse-transcription PCR (RT-PCR): Reverse transcriptase makes DNA from viral RNA or mRNA RT-PCR with a norovirus primer Textbook Clinical Focus, p. 266

Day 1 Materials needed per student: 2 ml screw-cap tube containing a water sample to be tested Eppendorf tube containing lysis solution Empty Eppendorf tube for sample transfer One 200 µl PCR tube containing PCR bead Appropriate micropipettes and tips Small beaker with ice Positive control sample or negative control sample (sterile water) will be assigned to individual students Equipment: Water bath, Vortex, Nano centrifuge, Microcentrifuge, PCR Thermocycler Each student process one water sample. Controls will be assigned.