PCR and qPCR

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PCR (polymerase chain reaction) and qPCR (quantitative PCR) are methods for copying DNA molecules to allow for testing even small amounts of DNA.

PCR starts with nucleic acid denaturation, the separation of double strands of sample DNA. The two separate strands become templates to which free nucleotides can attach to build new DNA strands. Each new DNA strand is then denatured to become two templates, a process that exponentially amplifies the original DNA.

Alternatively, DNA can be synthesized from RNA (via reverse transcription), which produces complementary DNA (cDNA). First-strand synthesis from cDNA can occur using DNA Polymerase I and DNA Ligase for direct cloning without amplification.

The two main PCR reagents are:

  • Primers or oligonucleotides: short single-stranded DNA fragments complementary to a targeted region of the DNA
  • DNA polymerase: an enzyme that facilitates reactions between primers and sample DNA

Thermal cyclers heat and cool reactions (usually performed in small PCR tubes or PCR microplates). Cycle length and temperature depend on the enzyme, dNTP ion concentration, and primer denaturation (melting) temperature.

Quantitative PCR (qPCR or real-time PCR) measures the initial amounts of DNA, cDNA, or RNA and can detect whether and in what amount a specific DNA sequence is present. These methods use fluorescent dyes or fluorophore-containing DNA probes to determine the amount of amplified product.

PCR is used to:

  • Clone DNA for sequencing and manipulating genes
  • Conduct gene mutagenesis
  • Construct DNA-based phylogenies (gene functional analysis)
  • Diagnose/monitor hereditary diseases
  • Amplify ancient DNA
  • Perform forensic and parentage testing
  • Detect infectious disease pathogens
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