High-resolution liquid chromatography of fluorescent dye-labeled nucleic acids

Using 100 mM of triethylammonium acetate as ion-pairing reagent, phosphodiester oligonucleotides labeled fluorescently at their 5' terminus could be separated successfully on alkylated nonporous 2.3-microns poly(styrene-divinylbenzene) particles by means of high-resolution liquid chromatography. Applying excitation wavelengths of 490, 520, 550, and 575 nm, respectively, optimum sensitivity was achieved for the fluorophores 5-carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, N,N,N',N'-tetramethyl-6-carboxyrhodamine, and 6-carboxy-X-rhodamine (FAM, JOE, TAMRA, and ROX, respectively) at emission wavelengths of 520, 550, 580, and 605 nm, respectively. With calibration curves being linear over at least three orders of magnitude, the lower detection limits were 0.5, 2, 2, and 3 fmol, respectively. Depending on the type of fluorescent dye attached, retention times increased in the order JOE < FAM < TAMRA < ROX. Subsequently, fluorescent oligonucleotides were employed to prime polymerase chain reactions (PCR). Again the fluorophores were found to increase the retention times of double-stranded nucleic acids, but to a lesser degree than those of single-stranded oligonucleotides. Using a single FAM label attached to one of the two PCR primers, the sensitivity of fluorescence detection was found to be approximately 1 fmol or 30-70 times higher than that of uv absorbance detection depending on the length of the PCR product. Since the technique allows the separation of PCR products differing only 4 to 8 base pairs in length within a size range of 50 to 200 base pairs, it may be employed for the quantitative assessment of competitive PCR.