With the development of nucleic acid quantitative technology in the past decades, competitive reverse transcription PCR (RT-PCR) has become the most advanced method. This technology can achieve the quantification of nucleic acids with higher specificity and accuracy as well as lower template copy numbers, compared with earlier developed technologies such as the Southern and Northern blot analysis and RNase-protection assays. Among all kinds of fluorescence-based assay systems, dual-labeled fluorescent oligonucleotide probes can provide the highest template specificity. A dual-labeled probe is normally 20 - 30 bases in length modified with a fluorophore at 5'-end and a quencher at 3'-end.
As shown in Figure 1, there are three states of dual-labeled probes, including (A) unhybridized state where the fluorescence cannot be emitted as the fluorophore is in a close distance with quencher, (B) denatured state where the probe is melted or denatured by heating and behaves in a random coil conformation, and (C) hybridized state where the probe and target sequences are complimentarily bound. During elongation, as DNA polymerase incorporates nucleotides in a 5' to 3' direction from the primer, it will encounter the 5'-end of the probe, followed by cutting the nucleotide with fluorophore; consequently, the fluorophore and quencher are separated permanently and fluorescence is released.
Figure 1. Different states of dual-labeled probes.
There are many excellent properties of dual-labeled probes. Besides, dual-labeled probes can also be applied for SNP detection, quantitative and qualitative analysis, real-time and endpoint PCR analyses and allelic discrimination, etc. Our company can design and produce kinds of highly efficient dual-labeled probes.