Real-Time PCR Overview
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Questions and Answers

Which type of PCR is considered the gold standard for all viral infections?

  • RT-PCR (correct)
  • qPCR
  • Multiplex PCR
  • Nested PCR

    • What role do fluorescent dyes play in qPCR assays?

  • They serve as nucleotides for DNA synthesis.
  • They act as RNA primers.
  • They inhibit the PCR reaction.
  • They provide visible signals after each amplification cycle. (correct)

    • Which of the following is NOT a commonly used fluorescent dye in qPCR assays?

  • PicoGreen
  • Thiazole orange
  • SYBR Gold
  • Fluorescein (correct)

    • What is a primary component included in ready mixes for SYBR Green assays?

    <p>SYBR Green dye</p> Signup and view all the answers

    Why does the fluorescent dye not accumulate between RT-PCR cycles?

    <p>The dye is consumed in each cycle.</p> Signup and view all the answers

    What is a key advantage of real-time PCR over conventional PCR?

    <p>Longer dynamic range</p> Signup and view all the answers

    Which statement accurately describes the results of conventional PCR?

    <p>Post PCR processing is required</p> Signup and view all the answers

    What role does Reverse Transcriptase play in RT-PCR?

    <p>It converts RNA to cDNA</p> Signup and view all the answers

    What is a disadvantage of using ethidium bromide for staining in conventional PCR?

    <p>It provides low resolution results</p> Signup and view all the answers

    Which pathogens can be detected using RT-PCR?

    <p>All of the above</p> Signup and view all the answers

    What happens to the distance between the Reporter and the Quencher when the probe is cleaved?

    <p>The distance increases, inhibiting energy transfer.</p> Signup and view all the answers

    What is the effect on the fluorescent emissions of the Reporter when the probe is cleaved?

    <p>The emissions of the Reporter increase.</p> Signup and view all the answers

    What occurs to the quencher's fluorescence when the probe is cleaved?

    <p>The quencher's fluorescence decreases.</p> Signup and view all the answers

    Which enzyme activity is responsible for cleaving the probe?

    <p>5' nuclease activity</p> Signup and view all the answers

    What is the consequence of the energy transfer stopping between the Reporter and the Quencher?

    <p>The fluorescent signal of the Reporter is isolated and increases.</p> Signup and view all the answers

    What is a primary advantage of dye-based qPCR?

    <p>It can provide real-time monitoring of PCR amplification.</p> Signup and view all the answers

    Which of the following is a limitation of using dye-based qPCR?

    <p>It can lead to signal saturation at high concentrations.</p> Signup and view all the answers

    What is the purpose of the BHQ in dye-based qPCR probes?

    <p>To quench the fluorescent signal until the probe hybridizes.</p> Signup and view all the answers

    In the context of herpes simplex virus detection, what does the FAM label indicate?

    <p>It is a fluorescent dye used to label the HSV-1 probe.</p> Signup and view all the answers

    Why is the dual-labeling approach (like FAM and VIC) beneficial in qPCR?

    <p>It enables the simultaneous detection of multiple targets in one reaction.</p> Signup and view all the answers

    What is the first step in analyzing changes in gene expression in a sample?

    <p>Creating a standard curve</p> Signup and view all the answers

    How are unknown samples analyzed in the context of gene expression changes?

    <p>Through direct comparison with another reference sample</p> Signup and view all the answers

    What role does an untreated control sample play in gene expression analysis?

    <p>It is used as a baseline for comparison</p> Signup and view all the answers

    What is the primary purpose of a standard curve in gene expression analysis?

    <p>To provide context for unknown samples</p> Signup and view all the answers

    Which of the following best describes the process of gene expression analysis?

    <p>Establishing a standard curve for comparison</p> Signup and view all the answers

    What hepatitis B viral load threshold indicates reactivation risk?

    <blockquote> <p>2,000 IU/mL</p> </blockquote> Signup and view all the answers

    Which viral load range indicates the highest risk for hepatitis B reactivation?

    <p>Between 1,000 and 2,000 IU/mL</p> Signup and view all the answers

    In the context of IFN therapy, what is the importance of RT-PCR?

    <p>To predict virologic response</p> Signup and view all the answers

    What is the recommended action for individuals with a hepatitis B viral load exceeding 2,000 IU/mL?

    <p>Consider additional monitoring and intervention</p> Signup and view all the answers

    Which of the following ranges implies that someone is at an elevated risk for hepatitis B reactivation?

    <p>From 1,000 IU/mL to 2,000 IU/mL</p> Signup and view all the answers

    Study Notes

    Real-Time PCR

    • Also known as Quantitative PCR (qPCR)
    • Offers high sensitivity
    • Has a long dynamic range
    • Results expressed as numbers
    • Automated
    • Not size-based discrimination or post-PCR processing needed.
    • Uses fluorescent dyes (e.g. SYBR Green) or probes (e.g. TaqMan)

    Conventional PCR vs. Real-Time PCR

    • End-Point Conventional PCR:
      • Low sensitivity
      • Short dynamic range (< 2 logs)
      • Non-automated
      • Size-based discrimination only
      • Results not expressed as numbers (not quantitative)
      • Ethidium bromide staining not very quantitative
      • Low resolution, poor precision
      • Post-PCR processing required
    • Real-Time PCR:
      • High sensitivity
      • Long dynamic range
      • Automated
      • Not size-based discrimination
      • Results expressed as numbers (quantitative)
      • Fluorescent dyes or probes for quantitative analysis
      • No post-PCR processing needed

    RT-PCR vs. Serology

    • Real-Time PCR (RT-PCR):
      • More accurate for SARS-CoV-2, CMV, Brucella, Leishmania, and Toxoplasma detection
    • Serology test:
      • Another method for detecting viruses
      • Not always best suited for all cases; different tests are better for different viruses.

    RT-PCR Types

    • qPCR: Real-Time/Quantitative Polymerase Chain Reaction
    • RT-PCR: Reverse Transcription Polymerase Chain Reaction
    • qRT-PCR: qPCR and RT-PCR combined technique (e.g., SARS-CoV-2 [COVID-19])

    Reverse Transcriptase PCR (RT-PCR) - cDNA Synthesis

    • Variation of Polymerase Chain Reaction (PCR)
    • Amplifies target RNA
    • Addition of reverse transcriptase (RT) enzyme
    • Converts RNA to cDNA
    • PCR amplification follows for detection of RNA targets

    cDNA Synthesis Protocols

    • Mix components, vortex briefly, and spin down
    • Prepare reaction with specific components
    • Use specific primers (e.g., random primer mix)
    • Complete process using specific cycle steps/ temperatures/ times

    RT-qPCR

    • Real-time reverse transcription polymerase chain reaction
    • Addresses the need for quantification
    • Used in molecular medicine, biotechnology, microbiology, and diagnostics.
    • Quantifies mRNA as transcriptional biomarkers in molecular diagnostics, normal biological processes, and pathogenic and pharmacological reactions to therapeutic interventions.

    Real-time PCR Applications

    • Diagnostics (detecting viruses, bacteria, etc.)
    • Gene expression (quantifying mRNA levels)

    qPCR Quantification Types

    • Absolute: Needs serial standard concentrations of control DNA, plasmid, or RNA for accurate quantity determination
    • Relative: Uses reference genes (housekeeping genes) to normalize and estimate changes in gene expression compared to a control. (e.g., ACT, a common reference gene).

    qPCR Assays

    • SYBR Green qPCR: Uses SYBR Green dye that intercalates with double-stranded DNA. Intensity is measured during PCR cycles
    • TaqMan Probe qPCR: Uses DNA probes (tagged with a fluorescent reporter at one end and quencher at the other end). The quencher prevents reporter dye fluorescence when intact. Cleavage of the probe releases the reporter dye from the quencher; increases fluorescence as cycles progress.

    High Resolution Melt (HRM) - qPCR

    • A method used for DNA melting analysis of PCR products.
    • Used to assess differences in melting temperatures between samples
    • Identifies sequence variations, including mutations, for identifying DNA variations.

    Other Methods and Applications

    • Detecting bacteria (e.g. Mycobacterium tuberculosis, M. bovis)
    • Diagnosing leishmaniasis
    • Detecting copy number variants (CNVs) e.g. spinal muscular atrophy (SMA), cystic fibrosis (CF) and human breast carcinoma (HER-2/neu).
    • Digital PCR: Used to quantify nucleic acids. (DD-PCR)
    • Detecting different pathogens or other disease states
    • Real-time PCR for predicting virologic response to IFN therapy. Viral load of Hepatitis B and other uses
    • Detecting cancer-related mutations. Detecting thyroid cancer biomarkers

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    Real-Time PCR Lecture Notes PDF

    Description

    This quiz explores the essential concepts of Real-Time PCR, also known as Quantitative PCR (qPCR), highlighting its sensitivity, dynamic range, and automation features. It will compare Real-Time PCR with Conventional PCR and Serology, emphasizing the advantages and differences in methodologies. Test your knowledge on the fundamentals of this crucial molecular biology technique.

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