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.
The reactions of 6-JOE, SE with amino-modified oligonucleotides are complicated by the impurities resulted from the manufacturing process of 6-JOE, SE.
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
|241||6-HEX alkyne||533||550||5 mg|
|240||6-HEX azide||533||550||5 mg|
|6026||6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]||535||553||100 µmoles|
|202||6-HEX, SE [6-Carboxy-2',4,4',5',7,7'-hexachlorofluorescein, succinimidyl ester] *Single Isomer*||533||550||5 mg|
|249||6-JOE alkyne||520||548||5 mg|
|248||6-JOE azide||520||548||5 mg|
|203||6-JOE azide||520||548||5 mg|
|245||6-TET alkyne||521||536||5 mg|
|244||6-TET azide||521||536||5 mg|
|6027||6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite] *CAS#: 877049-90-6*||521||541||100 µmoles|