High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis

Ultrathin-layer agarose gel electrophoresis is a novel combination of the e stablished methodologies of slab gel electrophoresis and capillary gel elec trophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluore scence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading te chnology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction ( PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR ampl ified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel elect rophoresis analysis with "in migratio" fluorescent labeling during the sepa ration process. Due to its speed and automation, this method should be cons idered for large scale screening of factor V Leiden mutation. (C) 1999 Else vier Science B.V. All rights reserved.