The development of real-time PCR was driven by which factor?

The development of real-time PCR was driven by which factor?

• A. Learning that Taq polymerase has 5′-to-3′ exonuclease activity.
• B. The development of a dot-blot hybridization methods.
• C. The development of chain ligase reaction.
• D. The ability to volatilize a sample in a nebulizer.

Answer: (A)

Real-time PCR hinges on watching amplification as it happens, which became possible when it was found that Taq polymerase has 5′→3′ exonuclease activity. As the polymerase extends the new DNA strand, this exonuclease function can cleave a labeled probe that binds to the target sequence. When the probe is degraded, the fluorescent reporter is separated from the quencher, producing a signal that rises with each cycle. This cycle-by-cycle signal allows direct quantification of the amount of product in real time, turning PCR into a quantitative assay. The other options describe methods or ideas that are unrelated to what enables real-time detection and quantification.

Real-time PCR hinges on watching amplification as it happens, which became possible when it was found that Taq polymerase has 5′→3′ exonuclease activity. As the polymerase extends the new DNA strand, this exonuclease function can cleave a labeled probe that binds to the target sequence. When the probe is degraded, the fluorescent reporter is separated from the quencher, producing a signal that rises with each cycle. This cycle-by-cycle signal allows direct quantification of the amount of product in real time, turning PCR into a quantitative assay. The other options describe methods or ideas that are unrelated to what enables real-time detection and quantification.