Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agaros e slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection fo r the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21) , were used as a model system to investigate the applicability, sensitivity and resolving power of the method. The allele-specific products were gener ated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via c omplexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrat ing dsDNA-dye complexes were detected in real-time by a scanning LIF detect ion system with sub-nanogram sensitivity. The system employs a 632-nm solid -state laser and an avalanche photodiode detector scanning to the separatio n platform by means of a fiber bundle system. Automated ultra-thin-layer ag arose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA frag ments and LIF detection system proved to be a very fast, high-throughput se paration method for individual or multiplexed PCR products, with excellent sensitivity. (C) 1998 Elsevier Science B.V. All rights reserved.