Chromatographia

, 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
Article

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.

Keywords

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

References

  1. Esmon CT (2000) Biochim Biophys Acta 1477:349CrossRefPubMedGoogle Scholar
  2. Simioni P, Scudeller A, Girolami A (1995) New Engl J Med 332:1382Google Scholar
  3. Dalback B, Carlson M, Svensson PJ (1993) Proc Natl Acad Sci USA 90:1004PubMedGoogle Scholar
  4. Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B (1994) Lancet 343:1361CrossRefGoogle Scholar
  5. Svensson PJ, Dahlback B (1994) N Eng J Med 330:517CrossRefGoogle Scholar
  6. Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C (1981) J Clin Invest 68:1370PubMedGoogle Scholar
  7. Nagy B, Toth T, Rigo Jr. J, Karadi I, Romics L, Papp (1998) Z Clin Genet 53:478Google Scholar
  8. Rees DC, Cox M, Clegg JB (1995) Lancet 346:1133CrossRefPubMedGoogle Scholar
  9. Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, deRonde H, van der Velden PA, Reitsma PH (1994) Nature 369:64CrossRefPubMedGoogle Scholar
  10. Behn M, Schuermann M (1998) Thromb Haemost 79:773PubMedGoogle Scholar
  11. Meyer M, Kucher G, Vogel G (1999) Throm Haemost 81:162Google Scholar
  12. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smidth JG, Markham AF (1989) Nucleic Acid Res 17:2503PubMedGoogle Scholar
  13. Hezard N, Cornillet P, Droulle C, Gillot L, Potron G, Nguyen P (1997) Trombosis Res 88:59CrossRefGoogle Scholar
  14. Ferenczi A, Garami M, Kiss E, Pek M, Sasvari-Szekely M, Barta C, Staub M, Solyom J, Fekete G (1999) J Clin Endocrin Metabol 84:2369CrossRefGoogle Scholar
  15. Sasvari-Szekely M, Gerstner A, Ronai Z, Staub M, Guttman A (2000) Electrophoresis 21:816CrossRefPubMedGoogle Scholar
  16. Behn M, Schuermann M (1998) Thromb Haemost 79:773PubMedGoogle Scholar
  17. Lewandski K, Rozek M, Zawlinska K, Markiewicz WT (1997) Thromb Res 85:105CrossRefPubMedGoogle Scholar
  18. Ulvik A, Ren J, Refsum H, Ueland PM (1998) Clin Chem 44:264PubMedGoogle Scholar
  19. Lengyel T, Sasvari-Szekely M, Guttman A (1999) J Chromatogr A 853:519CrossRefPubMedGoogle Scholar
  20. Guttman A (1999) LC.GC Magazine 17:1020Google Scholar
  21. Cassel S, Guttman A (1998) Electrophoresis 9:1341Google Scholar
  22. Guttman A, Zsolt Ronai, Csaba Barta, Yu-Ming Hou, Xun Wang, Steven Briggs (2002) Electrophoresis 23:1524CrossRefPubMedGoogle Scholar
  23. Guttman A, Ronai Z (2000) Electrophoresis 21:3952CrossRefPubMedGoogle Scholar
  24. Shandrick S, Ronai Z, Guttman A (2002) Electrophoresis 23:591CrossRefPubMedGoogle Scholar

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

Personalised recommendations