Abstract

Capillary gel electrophoresis (CGE) is the premier method of separating DNA and oligonucleotides. This technique is the workhorse method of all separations based genomics studies e.g. Human Genome Project. In previously published studies, methods utilizing CGE with ultraviolet (UV) detection have been developed for the quantitative determination of oligonucleotides in various matrices (e.g. plasma, serum, tissue); although very selective CGE-UV is not as sensitive as other analytical techniques (e.g. hybridization assays). Recently, laser-induced fluorescence (LIF) detection has emerged as a significantly more sensitive alternative to UV detection. However, oligonucleotides do not possess significant native fluorescence, therefore covalent or non-covalent dying is necessary. OliGreen(TM), an ultra sensitive fluorescent nucleic acid stain, non-covalently binds to single stranded-oligonucleotides (ss-oligo's) and displays a significant fluorescence enhancement and is potentially the most sensitive dye for ss-oligos. The purpose of this study was to develop and validate a method for the quantitative determination of therapeutic ss-oligos in plasma. Plasma was spiked with ss-oligos (18, 22, 23 and 24-mer) and separation was achieved by CGE. The oligonucleotides were non-covalently complexed by mixing OliGreen(TM) (1/500 dilution) into the gel separation matrix at a temperature of 60{493}C, the maximum of the instrument. Detection limits of 250 pg/mL and linear calibrations over 100 orders of magnitude were achieved using the 488 nm argon ion laser for excitation and 520 nm for detection. The method was validated with respect to selectivity, carry-over, linearity, intra- and inter-assay precision and accuracy.