Important Probe Design Considerations

1.1.1. Positioning of Dye Labels

Standard convention places the quencher on the 3' and the reporter on the 5'-end of the probe. This is primarily because in oligonucleotide synthesis, all failure sequence fragments contain only the 3' label. Therefore, if the quality of synthesis and/or purification is poor, there will be an excess of 3'-quencher-

Fig. 1. Static and Förster resonance energy transfer quenching mechanisms in "linear" reporter-quencher dual-labeled oligonucleotides.

labeled oligos. This is a better alternative to having failure oligos containing only a 3'-reporter, which will raise the background fluorescence of the probe (4).

Dye labels may be available as a controlled pore glass (CPG), amidite, or active ester. The ease and yield of dye incorporation follows the order of CPG, amidite, ester, and this is generally reflected in probe price. A CPG dye is used to make 3' labels, whereas amidites and esters can be used for internal or 5' labels. Thus, to make a conventional 5'-reporter-3'-quencher probe, a reporter-amidite and a quencher-CPG are used. In Table 1, reporter dyes that are available as amidites are shown in bold. In the manufacturing of dye-labeled oligonucletides, it is more convenient and cost-effective to use dye amidites rather than succinimdyl esters. However, not all dye labels can be prepared as phosphoramidites that can withstandard oligo synthesis conditions.

1.1.2. Inherent Quenching by Bases

Another important consideration in the placement of the reporter label is that the oligo bases, especially guanine, quench many fluorophores. Therefore, reporter dyes should not be placed directly next to G residues (5,6).

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