, Volume 60, Supplement 1, pp S295–S298 | Cite as

Rapid Analysis of Factor V Leiden Mutation by Membrane Mediated Restriction Digestion and Ultra-Thin Layer Gel Electrophoresis

  • A. Guttman
  • Z. Ronai
  • J. Khandurina
  • T. Lengyel
  • M. Sasvari-Szekely


Mutation of blood coagulation factor V at the protein C cleavage site (Leiden mutation) results in activated protein C resistancy and concomitant blood clotting. Individuals that carry this mutation have significantly increased risk of thrombic episodes. Using polymerase chain reaction/ restriction fragment length polymorphism (PCR/RFLP) analysis, single nucleotide polymorphisms (SNP) such as the factor V Leiden mutation can be readily detected. In this paper, we discuss the applicability of membrane mediated restriction endonuclease digestion in sub-microliter volumes, in conjunction with ultra-thin layer agarose gel electrophoresis for rapid genotyping of factor V Leiden. The amplified and restriction enzyme digested DNA fragments were fluorescently labeled with ethidium bromide during the electrophoresis separation process. Combining membrane mediated restriction digestion with ultra-thin layer agarose gel electrophoresis separation and sensitive on-line laser-induced fluorescence detection enabled completion both the digestion and electrophoresis analysis procedures in less than 15 minutes for up to 96 samples, thus, genotyping of factor V Leiden mutation with this new protocol can be accomplished in a sensitive and high throughput manner.


Ultra-thin layer gel electrophoresis Membrane mediated restriction digestion Leiden mutation 


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Copyright information

© Friedr. Vieweg & Sohn Verlagsgesellschaft mbH 2004

Authors and Affiliations

  • A. Guttman
    • 1
    • 4
  • Z. Ronai
    • 2
  • J. Khandurina
    • 3
  • T. Lengyel
    • 1
  • M. Sasvari-Szekely
    • 2
  1. 1.Genetic BioSystemsSan DiegoUSA
  2. 2.Institute of Med. Chem.Mol. Biol. and Pathobiochem., Semmelweis UniversityBudapestHungary
  3. 3.Torrey Mesa Research InstituteLa JollaUSA
  4. 4.Diversa Co.San DiegoUSA

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