Ultra-fast separation of infectious disease-related small DNA molecules by single- and multi-channel microchip electrophoresis.

Abstract: An ultra-fast and precise microchip electrophoresis (ME) method was developed for the separation of infectious disease-related small DNA molecules. As a model of infectious disease-related small DNA molecules, the spike glycoprotein (S) gene of the Feline infectious peritonitis (FIP) virus was amplified using reverse transcript polymerase chain reaction. The amplified product of the FIP virus (223-bp) was analyzed within 10s by single-channel ME under a sieving gel of 0.3% poly(ethylene oxide) (M r=8,000,000) in 1x TBE buffer (pH 8.33) and a short effective channel length of 1.3cm with a programmed step electric field strength (PSEFS) condition as follows: 470.6V/cm for 9s, 294.1V/cm 1.5s, and 470.6V/cm for 9.5s. The single-channel ME/PSEFS method was 50 times faster than that obtained with conventional slab gel electrophoresis. When the single-channel ME method was applied to a multi-channel ME for high-throughput screening, the precision of migration time and peak area showed standard deviations of less than 1.0% without any loss of resolving power. The ME assay technique provides a simple, precise and accurate method for ultra-fast analysis of infectious disease-related DNA under 400-bp. [Copyright &y& Elsevier]