Frequently Asked Questions

1. The manual conjugation of a fluorophore or a quencher to an oligo resulted in a low yield.

   –  Check the pH of the buffers used in the coupling reactions and use fresh dyes. The reactive dyes should be stored at -20 °C in the presence of a desiccant.
   
   –  For the conjugation of a fluorophore to a trityl-protected thiol group, prior to performing the coupling reactions, prepare fresh solutions of both silver nitrate and dithiothreitol.
   
   –  Refer to the data-sheets of the fluorophore derivatives for information on their solubility. In case a fluorophore derivative is not soluble in water, as is the case for most succinimidyl ester derivatives, dissolve it in a small amount of dimethylformamide and then add this solution to the reaction mixture in small aliquots.
   
   

2. After the conjugation of a fluorophore, the reaction vessel contained a high concentration of the loose fluorophore. What is the best way to proceed?

   –  In order to remove unincorporated fluorophore derivatives from the coupling reactions, the reaction mixtures can be precipitated with salt and ethanol, as the fluorophores remain dissolved in ethanol.
   
   

3. What are the best ways to store a molecular beacon preparation?

   –  Store stock solutions of molecular beacons at -20 °C or -70 °C in TE buffer (10 mM Tris-HCl, pH 8.0, and 0.1 mM EDTA) and prevent them from being exposed to light. For the longtime storage, store the molecular beacons as a dried pellet.
   
   

4. After the synthesis and purification of a molecular beacon, the signal-to-background ratio is lower than expected. What could have happened?

   –  The most likely reason is high background due to contamination by either free fluorophores or oligonucleotides that contain the fluorophore but not the quencher. Free fluorophores can be removed by passage through a Sephadex column. In order to ensure that every molecule contains a quencher, repeat the purification of oligonucleotides that are protected by a trityl moiety and labeled with dabcyl prior to coupling with the fluorophore.
   
   –  The assay medium may contain insufficient salt and the stem opens up. There should be at least 1 mM MgCl₂ in the solution in order to ensure that stem hybrids form.
   
   –  The molecular beacon may fold into an alternate conformation that results in a sub-population that is not quenched well. Change the stem sequence (and probe sequence, if necessary) to eliminate that possibility.
   
   

5. At low temperatures, the molecular beacon - target hybrid shows incomplete restoration of fluorescence. How can this be avoided?

   –  If the stem of a molecular beacon is too strong, at low temperatures it may remain closed while the probe is bound to the target. This may happen inadvertently if the probe sequence can participate in the formation of a hairpin that results in a stem longer and stronger than originally designed. Change the sequence at the edges of the probe and the stem sequence to avoid this problem.
   
   

6. In some real-time PCR assays initiated with a few copies of a template, the maximum fluorescence signal is lower than when the PCR is iniated with a high copy number of tempaltes. What is herefor the reason?

   –  Although false amplicons and primer dimers are not detected by molecular beacons, when they do appear, the sensitivity of the PCR assay is reduced. Therefore, DNA polymerases that become active after a brief incubation at 95 °C are recommended, as they minimize false priming.
   
   

7. In some real-time PC assays we observe low fluorescence signals. How can we optimize these assays?

   –  Try one of the following: Optimize the concentration of the molecular beacons, decrease the size of the amplicon, decrease the annealing temperature, and alter the relative concentrations of the two primers so that the PCR becomes asymmetric, favoring of the target strand.
   
   

8. In some of our genotype assays, we observe poor discrimination between the alleles. What could be the reason?

   –  Check if there is no bleed through of fluorescence from one optical color channel to the other.
   –  If the instrument is able to distinguish between the two fluorophores perfectly, increase the annealing temperature of the PCR.
   –  If poor discrimination is still observed, increase the length of the stems of the molecular beacons or decrease the length of the probe sequences.
   
   

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