Understanding qPCR

[Part 2]

How is real-time monitoring of DNA amplification achieved? The answer lies in the use of Detection Chemistry, involving fluorescent molecules. As DNA amplification progresses, the associated fluorescence increases, enabling real-time quantification of the DNA amount.

There are two main approaches utilizing these fluorescent molecules:

[1] Dye: SYBR Green is one of the most well-known fluorescent dyes in this category. It binds to the DNA double helix. While SYBR Green doesn’t fluoresce on its own, it becomes fluorescent when bound to double-stranded DNA. As new DNA strands are synthesized during amplification, the SYBR Green in the reaction tube binds to them. The fluorescent signal grows as DNA synthesis occurs. By measuring this signal in real-time, you can track the process of amplification throughout the PCR process.

[2] Probe: Taqman Probe is a representative example, composed of an oligo sequence. It’s designed to bind to the amplifying DNA sequence, much like a primer. One end of the probe contains a Reporter dye, while the other end contains a Quencher dye. The Reporter dye emits fluorescence to indicate DNA amplification, and the Quencher dye suppresses this fluorescence. When the probe is intact, the Quencher dye prevents fluorescence emissions. As amplification proceeds, the probe hybridizes to the single-stranded DNA, and when synthesis reaches the probe, it is degraded, separating the Reporter and Quencher dyes. This separation leads to fluorescence emission, indicating DNA synthesis. Each time a DNA molecule is synthesizes; a Reporter dye contributes to the fluorescence signal. This signal is continuously measured in real-time to quantify the amplification.

The probe method offers higher specify and accuracy compared to the Dye method. However, this approach requires the inconvenience of designing and using probes tailored to the target DNA sequence.

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