1. Background
  2. Data Collection Methods
  3. Data Quality Control
  4. Summary

Adapted from Kendall and Riley (1999).

Steps in qPCR

  1. Denature DNA by heating
  2. Anneal primers to single strands of DNA
  3. Synthesize DNA

Anneal primers to DNA

After denaturation, the temperature is reduced to 50 to 60 ℃ to allow primers to bind to their complementary bases on the DNA template (Kendall and Riley, 1999). The selection of primers is important.

  1. Primers should bind to the DNA template with high specificity to maximize the PCR amplification.
  2. High primer concentrations can result in hybridization to non-complementary sequences with mismatches.
  3. Low primer specificity can result in:
    1. Amplification of unwanted products and primer dimers.
    2. qPCR results may be affected if using a fluorescent dye that binds to double-stranded DNA.