Question

In real-time PCR, fluorescence is not generated by which of the following?

1) FRET probes
2) TaqMan probes
3) SYBR green
4) Tth polymerase

Answer 1

In real-time PCR, fluorescence is not generated by Tth polymerase. Fluorescent signals are instead produced by components such as FRET probes, TaqMan probes, and SYBR green, which are specific to detecting or binding DNA during amplification.

Step-by-step explanation:

In real-time PCR (quantitative PCR, qPCR), various fluorescent probes and dyes are used to monitor the amplification process. However, fluorescence is not generated by Tth polymerase. Instead, fluorescence in real-time PCR can be generated by various means including:

• FRET probes, which are fluorescence resonance energy transfer probes that emit light upon excitation when in close proximity, commonly used in multiplex reactions.
• TaqMan probes, which contain a fluorescent reporter dye and a quencher molecule. During the PCR extension phase, the 5' to 3' exonuclease activity of the Taq polymerase cleaves the probe, separating the reporter dye from the quencher, thus generating a fluorescent signal.
• SYBR green, a dye that binds to double-stranded DNA and emits fluorescence upon excitation when bound to DNA.

Tth polymerase, on the other hand, does not emit fluorescence. It is a DNA polymerase enzyme isolated from the thermophilic bacterium Thermus thermophilus and is used in PCR reactions due to its ability to withstand high temperatures and its polymerase activity.