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