Analysis of Single-Strand Conformation Polymorphism by Capillary Electrophoresis with Laser-Induced Fluorescence Detection Using Short-Chain Polyacrylamide as Sieving Medium

Abstract

A rapid analysis of single-strand conformation polymorphism (SSCP) by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detector was developed using a short-chain, linear polyacrylamide (PA) as sieving medium. Capillary filling of this low-viscosity medium and medium replacement were carried out by commercial capillary electrophoresis instruments. The approach was successfully applied to detect the C677T mutation of methylenetetrahydrofolate reductase gene. The influences of factors such as the concentration of polymers, voltage, temperature, and additives on the SSCP analysis were systematically investigated. Using 6% PA sieving medium and high electric field, four strands were resolved within 11 min in a DNA sample heterozygous for the C677T mutation, and a characteristic pattern was apparent for each of the three genotypes. When using multiple injection mode, the average analysis time per sample was reduced to about 4 min. In conclusion, our results indicate that CE-LIF may be an alternative to conventional SSCP analysis based on slab gel electrophoresis for the detection of genetic mutations. The technique is simple and rapid and is well suited to analysis of large numbers of clinical samples.