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Keywords:

  • FRET (fluorescence resonance energy transfer);
  • melting curve analysis;
  • nucleic acids;
  • polymerase chain reaction;
  • TaqMan

Abstract

Real-time PCR is the state-of-the-art technique to quantify nucleic acids for mutation detection, genotyping and chimerism analysis. Since its development in the 1990s, many different assay formats have been developed and the number of real-time PCR machines of different design is continuously increasing. This review provides a survey of the instruments and assay formats available and discusses the pros and cons of each. The principles of quantitative real-time PCR and melting curve analysis are explained. The quantification algorithms with internal and external standardization are derived mathematically, and potential pitfalls for the data analysis are discussed. Finally, examples of applications of this extremely versatile technique are given that demonstrate the enormous impact of real-time PCR on life sciences and molecular medicine.