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

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25 Citations (Scopus)

Abstract

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for 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 generated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via complexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrating dsDNA-dye complexes were detected in real-time by a scanning LIF detection system with sub-nanogram sensitivity. The system employs a 632-nm solid-state laser and an avalanche photodiode detector scanning to the separation platform by means of a fiber bundle system. Automated ultra-thin-layer agarose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA fragments and LIF detection system proved to be a very fast, high-throughput separation method for individual or multiplexed PCR products, with excellent sensitivity. Copyright (C) 1998 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)481-487
Number of pages7
JournalJournal of Chromatography A
Volume828
Issue number1-2
DOIs
Publication statusPublished - Dec 18 1998

Keywords

  • DNA
  • Polymerase chain reaction
  • Ultra-thin-layer agarose gel electrophoresis

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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