Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

Abstract

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.