Fluorescently labeled oligonucleotides

SYNTOL has developed synthesis and purification methods for fluorescently labeled oligonucleotides, including oligonucleotides containing two dyes.

Fluorescent dyes can be introduced at the 5′ and/or 3′ end or within the oligonucleotide chain. The source article describes three approaches:

• using a dye phosphoramidite in standard solid-phase synthesis, at any position;
• using a dye attached to a CPG (controlled-pore glass) support, for the 3′ end only;
• post-synthetic coupling of an activated dye derivative with an amino linker introduced into the oligonucleotide, at any position.

The first two approaches generally give high yields. The third requires two purification steps and therefore gives lower yields, but the source article notes that it can provide lower background fluorescence and higher method sensitivity.

Introducing a dye inside the oligonucleotide requires an additional dT-C6 amino-linker modification; the source article states that this costs 1.5 times as much as introducing a dye at the 5′ or 3′ end.

Purification

Except for Dansyl-, 3′-TAMRA-, 3′-FAM- and 3′-DABSYL-labeled oligonucleotides, the source article describes a post-synthetic dye-introduction scheme. After coupling the activated dye derivative to the primary amino group of the linker, the oligonucleotide is purified from excess unreacted dye, salts and unreacted oligonucleotide.

1. Gel filtration removes unreacted dye and salts.
2. Reverse-phase HPLC or polyacrylamide gel electrophoresis removes unreacted oligonucleotide.

The guaranteed minimum yield stated in the source is 1 optical density unit (5 nmol) at a 40 nmol synthesis scale and 3 optical density units (15 nmol) at a 0.5 µmol scale. Guaranteed purity is at least 95%.

Quality control

Quality is checked visually during PAGE/HPLC purification, spectrophotometrically, and by capillary electrophoresis for fluorescein-labeled oligonucleotides. The source also describes capillary-electrophoresis data obtained on the NANOFOR-02 instrument developed at the Institute for Analytical Instrumentation of the Russian Academy of Sciences in St Petersburg.

SYNTOL guarantees the correct sequence, presence and functionality of the modification, purity of at least 95%, and correspondence between the ordered and supplied amount. High-quality purification increases method sensitivity.