PCR Notes

Basic Principles of PCR

  • PCR is an amplification assay used to detect disease presence or absence.
  • Relies on DNA replication to measure active infections.
  • Developed by Cary Mullis in the 1980s, who won a Nobel Prize in 1993.

Key Definitions

  • Assay: A test (e.g., ELISA assay, PCR).
  • Gold Standard: The measure of true disease state (e.g., avian influenza virus PCR).
  • Sensitivity: Proportion of subjects with the disease who test positive (true positives).
    • High sensitivity: picks up a high proportion of individuals with the disease.
    • Low sensitivity: misses some individuals who are true positives (more false negatives).
  • Specificity: Proportion of subjects without the disease who test negative (true negatives).
    • High specificity: only picks up animals that truly have the disease as positive.
    • Low specificity: counts more non-diseased animals as diseased (more false positives).

Components Needed for PCR

  • Template: Sample containing the DNA to be amplified (e.g., saliva sample with COVID DNA).
  • DNA Polymerase: Enzyme for amplifying DNA (e.g., Taq DNA polymerase from Thermus aquaticus).
    • Thermus aquaticus is heat-resistant, allowing DNA amplification at high temperatures.
  • Primers: Short, single-stranded DNA complementary to the target sequence.
    • Forward and reverse primers target either region to synthesize a new strand.
  • Nucleotides: Deoxynucleotides (dNTPs) are needed to make new DNA strands.

PCR Process

  1. Denaturation:
    • Temperature: 94-96 degrees.
    • Separates the double-stranded DNA.
  2. Annealing:
    • Temperature: Varies (e.g., 55-70 degrees), depends on primers.
    • Primers attach to the separated DNA strands.
  3. Extension:
    • Temperature: Around 68-72 degrees.
    • DNA polymerase attaches new deoxy nucleotides to the primer sequence, creating a new strand.
  • The process repeats for about 30 cycles.

Gel Electrophoresis

  • Used to visualize PCR products.
  • DNA travels through the gel from the negative to the positive anode.
  • Shorter DNA travels further; longer DNA stays at the top.
  • Compare to a ladder of known base pair values to identify the product.
  • Primers provide sensitivity; the method provides specificity.

Types of PCR

  • Real-Time PCR:
    • Quantifies the amount of DNA in real time using probes with fluorescent reporters and quenchers.
    • Measures fluorescent intensity to track DNA amplification.
    • Generates a CT value for quantifiable measure.
  • Reverse Transcriptase PCR (RT-PCR):
    • Uses RNA as a starting material.
    • Requires reverse transcription to synthesize complementary DNA (cDNA).
    • Detects RNA viruses (e.g., feline coronavirus, SARS coronavirus).
  • Nested PCR:
    • Two rounds of PCR with different primers to increase sensitivity and specificity.
    • First round amplifies target DNA, and the second round uses nested primers for a specific region.
  • Multiplex PCR:
    • Uses multiple primers in one sample to amplify multiple regions at once.
    • Requires careful primer design to ensure all primers work at similar temperatures.
  • Quantitative PCR (qPCR):
    • Real-time detection with a reporter dye and quencher dye.
    • Provides a CT (Cycle Threshold) value
    • Measures fluorescence intensity to quantify the amount of DNA.
  • Arbitrary Primed RAPD (Randomly Amplified Polymorphic DNA):
    • Also known as DNA fingerprinting.
    • Uses random primers to bind to random loci on the DNA.
    • Generates a fingerprint to compare different patients or populations.

Advantages and Limitations of PCR

  • Advantages:
    • Simple to use and understand.
    • Highly sensitive, producing billions of DNA copies.
    • Multiple applications.
    • Quantitative real-time PCR allows quick quantification.
  • Disadvantages:
    • Contamination is easy.
    • Primers must be designated and can anneal to undesired sequences.
    • Incorrect nucleotides can lead to misleading data.