Rapid Analysis of Factor V Leiden Mutation by Membrane Mediated Restriction Digestion and Ultra-Thin Layer Gel Electrophoresis A. Guttman 1,& , Z. Ronai 2 , J. Khandurina 3 , T. Lengyel 1 , M. Sasvari-Szekely 2 1 Genetic BioSystems, San Diego, CA 92130, USA 2 Institute of Med. Chem., Mol. Biol. and Pathobiochem., Semmelweis University, Budapest, Hungary 3 Torrey Mesa Research Institute, La Jolla, CA 92121, USA Present address of corresponding author: Diversa Co., 4955 Directors Place, San Diego, CA 92121, USA; E-Mail: aguttman@diversa.com Received: 23 October 2003 / Accepted: 9 February 2004 Online publication: 20 May 2004 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 pro- cess. 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 Introduction Generation of high thrombin abundance tends to cause thrombosis as clots oc- clude the flow of blood that can con- comitantly result in heart attack, stroke, pulmonary emboli and venous thrombo- sis [1]. Inherited resistance to activated protein C (APC) is one of the most common genetic risk factors associated with blood clotting [2]. A single nucleo- tide alteration of guanine (G) for adenine (A) in position 1691 of the blood coagu- lation factor V gene (FV Leiden muta- tion) [3] leads to gene expression with the lack of the critical Arg 506 cleavage site in activated factor V [4]. This causes largely impaired inactivation of this blood coagulation factor, due to activated pro- tein C resistance, consequently leading to excessive thrombin generation [5]. While individuals heterozygous for protein C deficiency have severe recurrent thrombic episodes, homozygous ones usually die early in life [6]. Also, pre-eclampsia, a pregnancy-related disorder, which is regarded as the primary cause of mater- nal and fetal mortality was reported as being related to FV Leiden mutation [7]. Various high-sensitivity techniques have been developed for screening factor V Leiden mutation, as the occurrence of this particular single nucleotide poly- morphism (SNP) can be as high as 5% in certain human populations [8]. The most frequently used methods are based on restriction fragment length polymor- phism (RFLP) analysis, taking advantage of the natural Mnl I recognition sequence [5’(N 6 )GAGG 3’] at position 1691 in the exon 10 region of the factor V gene [9]. To increase the efficiency of mutation detection, the RFLP process is usually preceded by polymerase chain reaction (PCR) amplification of the DNA section of interest [10]. Single stranded confor- mation polymorphism (SSCP) technique was also successfully applied to factor V Leiden mutation analysis [11]. Another interesting approach is allele specific amplification (ASA), a convenient single- step method to diagnose SNP’s, such as the factor V Leiden mutation [12]. The significant advantage of RFLP and ASA based techniques over SSCP pro- tocols is that the size of the resulting DOI: 10.1365/s10337-004-0272-2 Presented at: 5 th Balaton Symposium on High- Performance Separation Methods, Sio´fok, Hungary September 3–5, 2003 2004, 60, S295–S298 Ó 2004 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH Short Communication Chromatographia Supplement Vol. 60, 2004 S295