Polymerase Chain Reaction: “DNA Photocopying” SBI4U AP Mr. McCrorie.
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Transcript of Polymerase Chain Reaction: “DNA Photocopying” SBI4U AP Mr. McCrorie.
Polymerase Chain Reaction:“DNA Photocopying”
SBI4U APMr. McCrorie
HIV Lab TestFlow Chart
Fearon, M. (2005). The laboratory diagnosis of HIV infections. Canadian Journal of Infectious Diseases and Medical Microbiology, 16, 26-30.
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
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
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.
What’s Required?
• Water (matrix for reaction, usually sterile and deionized)• PCR Reaction Buffer (provides optimal pH for polymerase)• MgCl2 (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)
PCR Virtual Lab
http://learn.genetics.utah.edu/content/labs/pcr/
PCR Reaction occurs in 3 steps(p 415 in text)
Step 1: Denaturation
• Occurs at about 94oC or 95oC• Double stranded DNA breaks apart into single
stranded DNA & molecules set in (Brownian) motion • Problem: Polymerase is destroyed at high
temperatures
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
Thermus Aquaticus at Yellowstone Park
Orange Pigment caused by Thermus Aquaticus. Temperature is approximately 80oC
PCR Reaction occurs in 3 steps(p 415 in text)
Step 2: Annealing
• Occurs at about 50oC…Primers renature• Primers form hydrogen bonds with complementary
sequences at ends of target sequence• TA = Annealing Temperature
PCR Reaction occurs in 3 steps(p 415 in text)
Step 3: Extension
• Occurs at 72oC, 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
Cycle 2 of PCR
Cycle 3 of PCR
2n : n = number of cycles; 30+ cycles = about a billion copies of the target sequence
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 95oC
Restriction Enzyme and PCR gene Cloning (p. 416)
• Errors in PCR replicationlimit good copies and the length of DNA fragments that can be copied
• Use PCR to provide DNA fragments for gene cloning (bacterial plasmid)
Case Study: Earl Washington
Case Study: Earl Washington
• 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
Case Study: Earl Washington
PCR Reaction Review