Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA

Sarah Shandrick, Zsolt Ronai, Andrs Guttman

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 μL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 μL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.

Original languageEnglish
Pages (from-to)591-595
Number of pages5
JournalELECTROPHORESIS
Volume23
Issue number4
DOIs
Publication statusPublished - Mar 27 2002

Keywords

  • DNA
  • Polymerase chain reaction
  • Ultrathin-layer gel electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry

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