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Essentials of Real Time PCR

About Real-Time PCR Assays

Real-time Polymerase Chain Reaction (PCR) is the ability to monitor the progress of the PCR as it occurs ( i.e., in real time). Data is therefore collected throughout the PCR process, rather than at the end of the PCR. This completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA. In real-time PCR, reactions are characterized by the point in time during cycling when amplification of a target is first detected rather than the amount of target accumulated after a fixed number of cycles. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. In contrast, an endpoint assay (also called a “plate read assay”) measures the amount of accumulated PCR product at the end of the PCR cycle.

PDF contents:

  • About Sequence Detection Chemistries
  • TaqMan Chemistry
  • SYBR Green I Dye Chemistry
  • About Quantitation Assays
  • Absolute vs. Relative Quantitation
  • Standard Curve Method for Relative Quantitation
  • Comparative CT method for Relative Quantitation
  • Standard Curve Method for Absolute Quantitation