Accurate and sensitive analysis of triplet repeat expansions by capillary electrophoresis

The reliable genetic diagnostics of triplet repeat expansions by capillary electrophoresis (CE) remains technically challenging due to the properties of the repeated GC-rich sequences. The biased base composition of the analyzed sample as compared to the commonly used DNA size standards makes the precise repeat length determination questionable. The homologous allelic ladders improve the accuracy of the repeat length analysis significantly. Their use, however, is not devoid of other complications. In the approach we propose, the allelic ladders are used only to properly calibrate the commercially available standards which serve then as internal standards in reliable and economical repeat length determination. In this study, we have also analyzed factors that could possibly increase the sensitivity of mutant allele detection by increasing the overall amplification efficiency and long-to-short product ratio.