6-JOE, a xanthene dye, refers to 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein. 6-JOE fluoresces in the yellow region of the visible spectrum and can be effectively quenched by BHQ^®-1 or our TQ2 dye. 6-JOE succinimidyl ester is the major commercial product used for labeling oligonucleotides. The 6-JOE modification incorporates 6-JOE moiety at either 5' terminus or thymidine of an oligonucleotide. The 6-JOE modification conjugated to a modified thymidine may participate in hybridization. 6-JOE modified oligonucleotides can be used in a wide array of applications, including dual-labeled fluorogenic probes for real-time PCR.

Compared to other fluorescein succinimidyl esters, 6-JOE SE generally gives much lower yield, and the resulted conjugates are also more difficult to be purified. Recently we have analyzed a number of commercial 6-JOE SE materials, and found that all the commercial 6-JOE SE materials contain a few impurities which often complicate the conjugations of 6-JOE SE with amino-modified oligonucleotides as shown in Figure 1. The impurities associated with the commercial 6-JOE SE materials include unreactive free 6-JOE acid, 3,6-JOE Bis SE and 3-JOE SE. In particular, the conjugate resulted from 3-JOE SE is difficult to be separated from the desired 6-JOE conjugate. 6-JOE SE offered by AAT Bioquest is essentially free of 3,6-JOE Bis SE and 3- JOE SE as shown in Figure 2.