Generi Biotech - Compounds and Services for Molecular Biology

• Real-Time PCR Probes
  • CATALOGUE AND PRICELIST
  • Dual Labelled Probes
  • Mono Labelled Probes
  • Delivery Terms and Conditions
  • Order probes
• Oligonucleotides
  • Synthesis Scale
  • Oligo Purification
  • Quality Control
  • CATALOGUE AND PRICELIST
  • Oligos without Modification
  • 3´ end modified oligos
  • 5´ end modified oligos
  • Phosphate Bond Modifications
  • Ribose Modifications
  • RNA Oligos
  • Other Modifications
  • Oligo EXPRESS 24
  • Terms and Conditions
  • Order Oligo
• Oligos on array
  • Pricelist
• MY OLIGO
  • Instructions
  • Search
  • MY OLIGO - Demo
  • Advanced Options

Advantages of dual-labelled oligonucleotide probes over SYBR Green I

SYBR Green I is dsDNA specific intercalation dye whose application in real-time PCR systems is based on the assumption that after several PCR cycles, major part of dsDNA in the reaction mixture should be formed by the amplification product, and fluorescence emitted by SYBR Green I molecules should thus reflect the present product quantity.

At GENERI BIOTECH, we have verified in many experiments that upon using SYBR Green I, real results do not correspond to theoretic assumptions. Even upon very careful design of primers and optimization of reaction conditions, it is almost impossible to avoid primer-dimer artifacts and other non-specific products, detected by SYBR Green I application together with the desired product, thus distorting the measured C_T and subsequently the resulting concentrations of unknown samples.

Based on results of our own experiments, GENERI BIOTECH uses and delivers exclusively real-time PCR systems arranged with oligonucleotide probes, which provide high specificity.

As for oligonucleotide probes, targeted detection of a certain sequence is performed in 2 steps - firstly, on the primer bond level and secondly, on the level of the probe bond to the complementary sequence inside the amplified region. This guarantees high specificity as it is very unlikely that an artifact containing the target sequence of the probe is formed due to a non-specific primer bond. Thus if the fluorescent activity of the probe increases, it is always caused by specific hybridization of the probe with the amplification product.

Practical examples:

• SYBR Green I: It often happens that amplification curves of samples with low template concentrations are only products of fluorescent activity of the primer-dimers and other types of non-specific artifacts. Formation of non-specific amplification products can be revealed by means of the melting curve analysis; however, C_Tvalues are distorted and do not correspond to concentration of samples.

• Oligonucleotide probes: The probes detect only the specific product of PCR amplification - therefore C_T amplification curves are not distorted by primer-dimer detection and by detection of other non-specific artifacts.