RT-PCR Probes

Real-Time Quantitative PCR Fluorescence Probes
GenePharma offers high quality sequence-specific fluorescent probes to accurately and reliably detect amplification of your target DNA in real-time quantitative PCR (RT-qPCR) assays. Customers can choose between the following sequence-specific fluorescence probes:

• TaqMan probe
• Double Labeled Fluorescence probe
• Molecular Beacons

All of our fluorescence probes can be purified by PAGE or HPLC on request. The quality of each probe is inspected by PAGE or mass spectrometry.

TaqMan Probe
TaqMan probes are oligonucleotides with a reporter fluorophore attached to the 5’-end and a quencher fluorophore attached to the 3-’end. Once the TaqMan probe has bound to its specific target sequence, the quencher fluorophore in 3’-end reduces the fluorescence emission from the reporter fluorophore in 5’-end because of their proximity. During PCR, the probe anneals specifically between the forward and reverse primer to an internal region of the PCR product. The polymerase then carries out the extension of the primer and replicates the template to where the TaqMan probe is bound. The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter fluorophore away from the close vicinity of the quencher. As a result, the reporter fluorophore starts to emit. With the increase of amplification cycles, the release of the fluorophore continues to accumulate. The accumulated fluorescence is detected by a computer and shown on a graph display. The commonly used fluorophores are FAM, TET, VIC, HEX.

Dual-labeled Fluorogenic Probes
Dual-labeled fluorogenic probes are highly sensitive and specific fluorescence probes used in real-time quantitative PCR assays. They have a simple design and can be labeled with a wide range of fluorophores. Hence, they can be applied for almost all RT-qPCR and diversified analysis systems. 
A dual-labeled fluorogenic probe is a single strand oligonucleotide labeled at both ends, which means a reporter fluorophore at the 5’-end and a quencher fluorophore at the 3’-end. The quencher fluorophore inhibits the emission of natural fluorescence from the reporter fluorophore by Förster Resonance Energy Transfer (FRET). During the extending step of real-time quantitative PCR, the PCR machine launches the Hγ1 ray to active probes. The core is to utilize the 3’-5' exonuclease activity of the Taq polymerase to cleave the probe to produce fluorescent signals. Since probes specifically combine with DNA templates, the intensity of the total fluorescence indicates the quantity of templates.

Molecular Beacons
Molecular Beacons are a special kind of single strand double-labeled fluorescence probes. Just like the other fluorescent probes, they are labeled with a reporter fluorophore at the 5’-end and a quencher fluorophore at the 3’-end. However, the 4-6 bases of both ends are complementary, so it forms a hairpin structure. The ring of the hairpin is complementary to the target sequence. Separation of the reporter and the quencher fluorophore leads to the emission of natural fluorescence from the reporter. 
During the annealing step of real-time quantitative PCR, the PCR machine launches hγ1 ray to active fluorescence probes. Molecular Beacons hybridize to the target DNA sequences, thereby distorting the hairpin structure, which causes the reporter-5’-end to separate from the quencher-3’-end. Then the quencher fluorophore cannot absorb energy from the reporter, and hence the reporter emits fluorescence. The real-time quantitative PCR instrument can detect the significantly increase of radiation energy hγ2. The detected fluorescence signals are proportional to the quantity of target DNA.

SYBR Green I 
If you want to measure total DNA amplification, SYBR Green I is a good choice. SYBR Green I is a non-sequence-specific luminous fluorescent dye, which can bind to double-stranded DNA. The fluorescence is significantly increased after binding to double-stranded DNA. Therefore, the intensity of SYBR Green I fluorescent signals is related to the amount of double-stranded DNA, we can detect the existing double-stranded DNA quantity in PCR system by detecting fluorescent signals. The SYBR Green I maximum absorption wavelength is about 497nm, and the maximum emission wavelength is about 520nm.

The characteristics of fluorescence probes:

For technical questions, email support@genepharma.com. For questions related to order placement, email bd@genepharma.com.