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May 15, 2010 (Vol. 30, No. 10)

Upping the Sensitivity and Accuracy of qPCR

Double-Quenched Probes Designed to Be Stable at High Temperatures and Minimize Fluorescence

  • Within the field of molecular biology, quantitative polymerase chain reaction (qPCR) processes enable the simultaneous amplification and quantification of target DNA sequences. As an important experimental procedure, qPCR can be applied to a broad range of different scientific study areas, including drug discovery and medical research. Carried out in three basic phases—denaturing, annealing, and elongation—one PCR experiment typically consists of 20 to 40 cycles in order to obtain significant exponential growth of the target sequence.

    As the basis of real-time monitoring, dual-labeled probes (also known as fluorescence-quenched or self-quenching probes) are important tools. Oligonucleotide probes that are complementary to a section of the target DNA can be labeled with a 5´ fluorescent dye and a 3´ quencher.

    When in close proximity, the quencher will effectively block the fluorescence signal emission from the fluorophore. However, the 5´ to 3´ exonuclease activity of the DNA polymerase enzyme during the elongation phase of each PCR cycle will digest the 5´ end of the probe, separating it from the quencher, resulting in an increase in fluorescence. The level of fluorescence emitted will, therefore, be directly proportional to the amount of PCR product produced.

    Common methods of fluorescent quenching use fluorescent resonance transfer (FRET), which consists of energy transfer via dipolar coupling between the fluorophore and quencher. However, this requires an overlap between the absorption and emission spectra, which complicates the already complex process of probe design, since quenchers are only functional within a limited wavelength range.

  • Probe Design

    The efficiency of quenching via methods such as FRET is extremely sensitive to the distance between the fluorophore and the quencher (RFQ). By increasing the effectiveness of the quenching, background fluorescence is reduced and sensitivity is improved.

    Duplex stability and melting temperature (Tm) are also important factors when considering probe design, since each denaturing phase requires that high temperatures are reached (95–96°C) to ensure sufficient separation of the DNA duplex. In subsequent annealing steps, the temperature must be lowered below the Tm to enable target nucleic acids and primers to re-bind. At this point, temperatures must remain high enough to avoid nonspecific hybridization.

    The quencher and fluorophore are typically separated by between 20 and 30 bases, which limits quencher efficiency. In order to increase the effectiveness of the probe and decrease the RFQ, shorter oligonucleotides can be synthesized. However, these sequences will only hybridize strongly to complementary DNA at lower temperatures, which are unfavorable for qPCR assays.

    Even with the use of Tm-enhancing modifications, such as the inclusion of LNA bases or minor groove binders, probe length is still usually between 14 and 18 bases. Any method that permits the placement of the fluorophore and quencher within closer proximity of one another will improve quencher efficiency and subsequently enhance the probe performance.

    For this reason, Integrated DNA Technologies (IDT) has developed a double-quenched probe that is stable at higher temperatures and minimizes the occurrence of background fluorescence, enabling researchers to gain highly precise and sensitive data.

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