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Multicapillary electrophoresis of unlabeled DNA fragments with high‐sensitive laser‐induced fluorescence detection by counter‐current migration of intercalation dye
Author(s) -
BenesovaMinarikova Lucie,
Fantova Lucie,
Minarik Marek
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200500422
Subject(s) - ethidium bromide , genotyping , intercalation (chemistry) , dna , capillary electrophoresis , microbiology and biotechnology , sybr green i , fluorescence , chemistry , chromatography , biology , real time polymerase chain reaction , genetics , genotype , gene , inorganic chemistry , physics , quantum mechanics
Analysis of PCR fragments for applications, such as screening of nucleotide polymorphisms, detection of somatic mutations, or quantification of reverse‐transcription PCR products, becomes central in clinical research as well as preventive testing, diagnostic screening, and pharmacogenomic genotyping. A variety of CE techniques, utilizing great potential of multicapillary‐array sequencers, is now commonly applied in prevention, diagnosis, and treatment of a wide range of genetic diseases (cancer, cardiovascular, and neurodegenerative diseases, etc. ). Costs of fluorescently labeled primers is often a major factor in large‐scale projects requiring mutation analysis in hundreds or thousands of samples. In the present paper we introduce a simple approach of detecting unlabeled DNA fragments through intercalation without a need for adding intercalator to the separation polymer matrix. The dye is only added to the anode reservoir, and mixing with the separated DNA fragments takes place upon its migration opposite to the direction of the CE separation. Using two common intercalating dyes (ethidium bromide and SYBR Green II) we present this method as a tool for routine PCR detection and quantification.