Single-step quantitation of DNA in microchip electrophoresis with linear imaging UV detection and fluorescence...

We demonstrate a convenient single-step quantitation technique for double-stranded DNA (dsDNA) fragments in polymerase chain reaction (PCR) products based on microchip capillary electrophoresis (micro-CE)/UV or fluorescence detection. PCR products of polymorphisms on the human Y-chromosome related to spermatogenic failure did not need purification. They were premixed and comigrated with a DNA digest whose concentration was known. Hydroxyethyl cellulose (HEC) dissolved in 5x Tris-borate-EDTA (5x TBE, pH 8.3) was used as a separation matrix in a linear polyacrylamide-coated quartz microchip, while mixed poly(ethyl oxides) (PEOs) of different molar-masses dissolved in 1 x TBE (pH 8.3) containing 1 ng/microl ethidium bromide was used as a separation matrix in an uncoated poly(methyl methacrylate) (PMMA) microchip. Elution profiles were monitored under either real-time linear imaging UV detection in the snapshot mode where the total separation time is fixed, or light-emitting diode (LED) confocal fluorescence detection in the finishline mode where solutes migrate over the same separation length. It is found that, in both modes, a linear relation exists between the peak areas (A) and the multiplication of the digest concentrations (C) and the fragment sizes (L) in a DNA restrictive digest. Using the comigration electropherogram of a single-step experiment, the concentrations of PCR products were directly determined using the A versus LC linear relationship. The sole condition to obey is that the chosen digest has different fragment sizes with the PCR products of interest. This condition is easy to obey, because micro-CE owns high separation ability, and many digests are commercially available. The recovery of the technique was between 98 and 105%. The R.S.D. for chip-to-chip concentration measurements was less than 6.0% (n = 6). Hence, the technique was accurate and reliable for DNA assays.