Evaluation of three commercially available capillary (gel)electrophoresis kits for single stranded DNA oligonucleotide analysis

Abstract

Three commercially available capillary (gel)electrophoretic kits, the BioCap oligonucleotide capillary kit (A), the single stranded (ss) DNA 100 gel capillary kit (B) and the ssDNA 100-R (replaceable) gel capillary kit (C), were compared with respect to resolution and repeatability of migration time, absolute and relative corrected peak area and resolution. As a sample, a mixture was made of crude synthetic ssDNA oligonucleotides with overlapping sequences with lengths of 16, 17, 18 and 19 nucleotides (n-mer, group 1) and with lengths of 51, 52 and 53 nucleotides (n-mer, group 2). For each kit the injection conditions (kV s), analysis temperature (°C) and analysis voltage (V/m) were optimized with respect to resolution applying a 2 3 factorial design using the analysis conditions as supplied by the respective kit manufacturers as a central point in the experimental design matrix (standard condition). Repeatability results ( n=8) as obtained with group 1 components showed remarkable differences between the kits, e.g. resolution factors of 2.6, 1.9 and 6.4 were measured in kits A, B and C, respectively. Migration times as measured for the n-mer group 2 component were 11.1, 64.7 and 111.4 min in kits A, B and C, respectively. Kit C was selected to be applied to the analysis of synthetic ssDNA oligonucleotides which is part of the quality control procedure of these components. The method has been validated for this purpose. Such purified oligonucleotides are used as primers in nucleic acid sequence based amplification (NASBA) which is an isothermal nucleic acid amplification technology. At Organon Teknika nucleic acid diagnostics are made using this technology which are marketed under the name NucliSens.