Multicapillary electrophoresis of unlabeled DNA fragments with high-sensitive laser-induced fluorescence detection by counter-current migration of intercalation dye.
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.
Benesova-Minarikova
Lucie
L
Laboratory of Molecular Genetics, Genomac International, Prague, Czech Republic.
Fantova
Lucie
L
Minarik
Marek
M
D004396:Coloring Agents; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D007364:Intercalating Agents; D007834:Lasers; D009841:Oligonucleotides / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods
D004396:Coloring Agents; D007364:Intercalating Agents; D009841:Oligonucleotides; D004247:DNA