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

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)S295-S298
JournalChromatographia
Volume60
Issue numberSUPPL.
DOIs
Publication statusPublished - 2004

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry
  • Organic Chemistry

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