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 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...

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Bibliographic Details
Published in:Journal of Chromatography A 1998-12, Vol.828 (1), p.481-487
Main Authors: Guttman, András, Barta, Csaba, Szöke, Melinda, Sasvári-Székely, Mária, Kalász, Huba
Format: Article
Language:English
Subjects:
Alleles
Analytical, structural and metabolic biochemistry
Automation
Base Sequence
Biological and medical sciences
DNA
DNA - isolation & purification
DNA Primers
Dna, deoxyribonucleoproteins
Electrophoresis, Agar Gel - methods
Electrophoresis, Capillary
Fundamental and applied biological sciences. Psychology
Humans
Lasers
Nucleic acids
Polymerase Chain Reaction - methods
Sensitivity and Specificity
Spectrometry, Fluorescence - methods
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Summary: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.
ISSN:0021-9673
DOI:10.1016/S0021-9673(98)00814-0