Analysis of Cyanine Dye-Labeled PCR Product and Restriction Fragments by Capillary Electrophoresis and Laser-Induced Fluorescence

Abstract

Capillary gel electrophoresis (CGE) has been used for analysis of double-stranded DNA, including products of PCR (1)(2)(3)(4)(5). Compared with conventional methods, CGE analysis offers higher resolution and better reproducibility and is capable of direct quantification and automation. CGE coupled with laser-induced fluorescence (LIF) system can detect as few as six molecules of stained DNA (6). Meanwhile, the most popular CGE/LIF detection involves the use of fluorescent DNA intercalating dyes, including ethidium bromide, YO-PRO3, and SYBR Green I (7). However, efficient separations are obtained only over a narrow range of DNA–dye ratios (8). In some cases, broad peaks may result from the presence of multiple dye–DNA binding. Fluorescent DNA derivatives obtained by covalent binding of DNA with fluorescent dye is more stable and may be more suitable for CGE/LIF analysis. Previously, an activated cyanine dye (Cy-5), which can be readily coupled with peptide, proteins, and amino-linked oligonucleotides (9), was synthesized. The dye contains two broad absorption maxima at 630 and 655 nm with a very high absorptivity and fluorescence quantum yield. Cy-5-labled molecules are suitable subjects for LIF detection with a semiconductor laser source emitting at 653 nm. Chen et al. have reported synthesis and CE/LIF detection of a Cy-5-conjugated M13 primer (10). It thus follows that fluorescent PCR products could be obtained if the primers used were first conjugated with Cy-5 dye. In this report, molecular diagnosis of a mitochondrial DNA genetic disease, MELAS (myopathy, encephalopathy with lactic …