Notes

1. The product from MB synthesis companies is lyophilized and can be reconstituted to a stock solution by resuspending the dry product in an aqueous solution to a known concentration, such as 100 ||M, and then aliquoting it into microvials to be stored at -20°C to avoid repeat freezing-thawing of the oligo stock solutions. Because most dyes are sensitive to light, wrap the vials containing MB solution with aluminum foil to prevent them from being damaged.

2. Some DNA folding programs on the Internet are useful and easy to use to check the secondary structure of the designed sequence, for example, the Zuker DNA folding program (www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi). The following is another program available from the same author to calculate the melting temperature of the stem sequence: (www.bioinfo.rpi.edu/applications/ mfold/old/rna/form5.cgi).

3. When designing MB for double-stranded specific nucleases, make sure that the cleaved end fragments are short enough to dissociate completely at or below the assay temperature. Normally, if the stem is less than 6-bp long, the cleaved end fragments will dissociate below the assay temperature.

4. There are a number of other nonfluorescent dyes, such as Black Hole Quenchers™ (Biosearch Technologies) and QSY-7 (Molecular Probes), that can also be used as the quencher with very high quenching efficiency for a variety of fluorophores.

5. An MB with a long stem, like MB2, is too stable to hybrizide to its target sequence. Instead, DNase I can be used to replace target sequence for the purpose of characterizing MB. Usually, adding 1 |L of 1 U/|L DNase I is enough.

Fig. 2. Lineweaver-Burk plot of the cleavage reaction of a molecular beacon by S1 nuclease. S1 nuclease = 0.015 nM. The concentration of the molecular beacon ranged from 120 to 2300 nM. (Reproduced with permission from ref. 9.)

Fig. 2. Lineweaver-Burk plot of the cleavage reaction of a molecular beacon by S1 nuclease. S1 nuclease = 0.015 nM. The concentration of the molecular beacon ranged from 120 to 2300 nM. (Reproduced with permission from ref. 9.)

Fig. 3. Inhibitory effect of dNTP on the initial cleavage velocity of the molecular beacon by S1 nuclease. Molecular beacon = 100 nM; S1 nuclease = 0.78 nM. The dNTP solution contained equal moles of dATP, dGTP, dTTP, and dCTP. (Reproduced with permission from ref. 9.)

6. The S/N ratio of an MB from cDNA hybridization may be slightly lower than that from a DNase cleavage reaction. For example, about a 35-fold increase in the S/N ratio was obtained when MB1 was hybridized with an excess of its target DNA, whereas an approx 40-fold increase in the S/N ratio was observed when the MB was digested by S1 nucleases. This significant increase in the S/N ratio was because the fluorophore and the quencher of the cleaved MB were completely separated from each other, whereas the two moieties were still linked to each other by one fragment of the double-stranded DNA in the target DNA case.

7. Under neutral or slightly basic condition, FAM has a high fluorescence quantum yield. However, under pH 5.0, both the extinction coefficient and quantum yield of this dye decrease dramatically. As S1 requires a buffer solution of pH 5.0, TAMRA is used as the fluorophore for MB1 because it is insensitive to pH and has a good quantum yield at this pH.

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