CAPILLARY GEL-ELECTROPHORESIS AND THE ANALYSIS OF DNA PHOSPHOROTHIOATES

Capillary gel electrophoresis (CGE) is recognized as an effective method for the analysis of normal phosphodiester deoxyoligonucleotides (DNA). However, it was found that when the same electrophoretic principles were applied to the analysis of phosphorothioate DNA, peak broadening due to the phosphorothioate moiety made CGE's utility questionable. Therefore, optimization of several parameters is necessary for CGE to be effective in leading to the precise and accurate analysis of phosphorothioate DNA. Phosphorothioates are oligonucleotide analogs acknowledged as potential antisense therapeutics in the treatment of viral diseases and certain cancers. In order to improve a CGE approach to the analysis of this class of therapeutics, several parameters were investigated and improved upon. Three factors which proved critical in providing high resolution and good gel to gel reproducibility were: gel concentration, buffer additives, and pH.