1. Polymerase Chain Reaction: “DNA Photocopying” SBI4U AP Mr. McCrorie

2. HIV Lab Test Flow Chart Fearon, M. (2005). The laboratory diagnosis of HIV infections. Canadian Journal of Infectious Diseases and Medical Microbiology, 16, 26- 30.

3. Testing Infants with HIV Positive Mothers “Virologic assays that directly detect HIV must be used to diagnose HIV infection in infants younger than 18 months; antibody tests should not be used” (National Institute of Health, 2015). Infants may carry maternal antibodies for HIV up to the age of 15 months (Fearon, M., 2005). https://aidsinfo.nih.gov/guidelines/html/2/pediatric-arv- guidelines/55/diagnosis-of-hiv-infection-in-infants-and-children

4. Agenda What is the PCR? What does the PCR require? PCR Virtual Lab 3 stages of the PCR reaction (Denaturation, annealing, extension) Importance of Taq Polymerase 3 Aspects of Polymerase (Processivity, Fidelity, Persistence) Restriction Enzymes and PCR Cloning Case Study: Earl Washington PCR Video Review

5. What is the Polymerase Chain Reaction (PCR)? Amplifies or copies a specific piece of DNA known as a “Target Sequence” Produces an exponential number of identical DNA molecules “In vitro” technique - Does not require bacteria or other microorganisms (i.e. Transformation is not required) Used in genetic profiling (i.e. forensics), detection of bacteria or viruses (particularly HIV), and diagnosis of genetic disorders.

6. What’s Required? Water (matrix for reaction, usually sterile and deionized) PCR Reaction Buffer (provides optimal pH for polymerase) MgCl 2 (Mg++ is a cofactor for polymerase and restriction endonucleases) Pure Target DNA sequence Deoxynucleoside triphosphates (dNTPs – A, T, C, G) – β and γ phosphates provide energy for reaction Taq polymerase (links dNTPs) DNA Primers (attachment site for DNA Polymerase)

7. PCR Virtual Lab http://learn.genetics.utah.edu/content/labs/pcr/

8. PCR Reaction occurs in 3 steps (p 415 in text)

9. Step 1: Denaturation Occurs at about 94 o C or 95 o C Double stranded DNA breaks apart into single stranded DNA & molecules set in (Brownian) motion Problem: Polymerase is destroyed at high temperatures

10. Taq Polymerase allows for the automation of the PCR Derived from the thermophile bacteria known as Thermus aquaticus Able to withstand high temperatures Prior to the discovery of Taq, “Denaturation” step of PCR destroyed the polymerase. Fresh polymerase would have to be manually added after each “denaturation”. 1 PCR cycle takes about 5 minutes

11. Thermus Aquaticus at Yellowstone Park Orange Pigment caused by Thermus Aquaticus. Temperature is approximately 80 o C

12. PCR Reaction occurs in 3 steps (p 415 in text)

13. Step 2: Annealing Occurs at about 50 o C…Primers renature Primers form hydrogen bonds with complementary sequences at ends of target sequence T A = Annealing Temperature

14. PCR Reaction occurs in 3 steps (p 415 in text)

15. Step 3: Extension Occurs at 72 o C, optimal temperature for Taq Polymerase DNA polymerase attaches to the primers and adheres nucleotides on the strand (5’ -> 3’) Continues until the end of the strand and falls off

16. Cycle 2 of PCR

17. Cycle 3 of PCR 2 n : n = number of cycles; 30+ cycles = about a billion copies of the target sequence

18. Aspects of Polymerase 1.Processivity: rate of complementary strand synthesis (e.g. Taq = 50-60 nucleotides/second vs. Tth = 25 nucleotides/second) 2.Fidelity: Accuracy (Tli has proofreading 5x better than Taq) 3.Persistence: stability of enzyme at high temperature (Taq has a half life of about 1.5 hours at 95 o C

19. Restriction Enzyme and PCR gene Cloning (p. 416) Errors in PCR replication limit good copies and the length of DNA fragments that can be copied Use PCR to provide DNA fragments for gene cloning (bacterial plasmid)

20. Case Study: Earl Washington

21. P. 431 – PCR can be used to amplify DNA samples that are in poor condition or minute quantities Short Tandem Repeats (STRS) – 2-5 nucleotide sequence…polymorphic (several different forms in a population STRs are highly variable (even vary between alleles in an individual) 13 markers – the probability of having 2 individuals with identical DNA profiles is between 1 in 10 billion and 1 in several trillion

22. Case Study: Earl Washington

23. PCR Reaction Review