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RT-PCR probes
GenePharma provides accurate and reliable results. In order to make your real-time quantitative PCR results accurate and reliable, please select the following sequence-specific fluorescence probes.
 

Real-Time Quantitative PCR Fluorescence Probes:
GenePharma provides accurate and reliable results. In order to make your real-time quantitative PCR results accurate and reliable, please select 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 system or mass spectrometry.

TaqMan Probe
TaqMan probe is an oligonucleotide with a reporter fluorophore attached to the 5’ end and a quencherfluorophore attached to the 3’ end. Once the TaqMan probe has bound to its specific target sequence, thequencher 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 molecule away from the close vicinity of the quencher. The fluorescence of the reporter fluorophore, as a result emits. 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 probe is a highly sensitive and specific fluorescence probe used in real-time quantitative PCR. It has simple design and can be labeled with wide range of fluorophores, so it can be applied for almost all the real-time quantitative PCR and diversified analysis systems. 
A dual-labeled fluorogenic probe is a single strand oligonucleotide labeled at both ends, which means a reporter fluorophore at 5’ end and a quencher fluorophore at 3’ end. The quencher fluorophore inhibits the emission of natural fluorescence from the reporter fluorophore by the Forster 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 fluorescent signals indicates the quantity of templates.

Molecular Beacons
Molecular Beacons is a single strand fluorescence probe labeled at both ends. Since the 4-6 bases of both ends are complementary, it composes a hairpin structure. A reporter fluorophore is labeled at 5’end and a quencher fluorpphore is labeled at 3’end. The ring of the hairpin is a single-stranded DNA, which complements each other with the target sequence. The separation of the reporter and the quencher fluorophore leads 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 hybridizes to a target DNA sequence to lead the distortion of hairpin structure, which causes the reporter at 5’ end separate from the quencher at 3’end. Then the quencher fluorophore can not absorb energy from the reporter, therefore, the reporter would emit fluorescence, so 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 
SYBR Green I is a 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:

Reporter Fluorophore at 5’End

Quencher Fluorophore at 3’End

6-FAM™, HEX™, TET™, JOE™, TAMRA™, ROX™, Fluorescein

TAMRA 、DABCYL 、BHQ.-1 or BHQ-2

 
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