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The reactions of 6-JOE, SE with amino-modified oligonucleotides are complicated by the impurities resulted from the manufacturing process of 6-JOE, SE.
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.

HPLC analysis of 6-JOE, SE materials from different commercial vendors. Vendor A: Invitrogen (Lot#: 1454485); Vendor B: AnaSpec (Lot#: 96700-1); Vendor C: AAT Bioquest (Lot#: 099133).


Table 1. 6-JOE and Its Analogs for Labeling Oligonucleotides

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
2416-HEX alkyne5335505 mg$162
2406-HEX azide5335505 mg$162
60266-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]535553100 µmoles $217
2026-HEX, SE [6-Carboxy-2',4,4',5',7,7'-hexachlorofluorescein, succinimidyl ester] *Single Isomer*5335505 mg$162
2496-JOE alkyne5205485 mg$162
2486-JOE azide5205485 mg$162
2036-JOE azide5205485 mg$217
2456-TET alkyne5215365 mg$162
2446-TET azide5215365 mg$162
60276-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite] *CAS#: 877049-90-6*521541100 µmoles $217