Visualisation of Real-time PCR Amplification

The critical difference between conventional and real-time PCR is that in the latter the generation of amplification products is monitored directly in the reaction tube. Different fluorescence-based principles are employed for real-time detection. SYBR Green represents one class of DNA binding chemicals that emits much more fluorescence when intercalated into double-stranded DNA compared with the unbound chemical in solution (Figure 1). As a result, the SYBR Green fluorescence increases proportionally to the increase in DNA amplification products. The disadvantage of using SYBR Green is that non-specifically amplified DNA and amplification artefacts such as primer dimers will also generate increased fluorescence, thus reducing the sensitivity compared with nested PCR. Real-time PCR assays utilising an amplicon- specific probe increase the specificity substantially. A number of ‘smart’ assays have been invented based on three oligonucleotides,i.e. two primers and a probe. Several of these assays include TaqMan assays (Figure .2), fluorescence resonance energy transfer (FRET) (Figure .3) assays, Eclipse probes, Scorpion probes and molecular beacon probes.

Figure 1 The SYBR Green molecule has high affinity to double-stranded DNA. SYBR Green fluorescence is much stronger when it is bound to DNA compared with unbound SYBR Green. ss-DNA=single-stranded DNA; ds-DNA=double-stranded DNA.

Figure 2 The principle of TaqMan real-time PCR assays. The probe is labelled with both a fluorescent chemical group (R) and a quencher (Q). When the Taq polymerase elongates the complementary strand from the primer, its 5´–3´ exonuclease activity cleaves any annealed probe, thus separating R from Q. Freed from its quencher, the R fluoresces when exciting light is sent in. The amount of free R, and thus fluorescence, increase exponentially along with amplicons until the PCR reaction approaches the plateau stage.

Figure 3 The principle of fluorescence resonance energy transfer (FRET). Two different probes are complementary to the target sequence. One probe is labelled with fluorescein and the other with LC Red fluorochrome. The hybridisation to the target sequence will bring the two fluorochromes physically close enough for the emission energy of fluorescein to excite the LC Red so that red fluorescent light is emitted. When PCR generates an increasing concentration of target, an increasing fraction of the probes can be positioned to achieve FRET. This can be monitored as increasing red fluorescence.