A FAST AND ROBUST DUAL-LABEL NONRADIOACTIVE OLIGONUCLEOTIDE LIGATION ASSAY FOR DETECTION OF FACTOR-V-LEIDEN

Activated protein C resistance is in almost all cases caused by the fa ctor V Leiden mutation (FV:R506Q). Due to the high prevalence and clin ical significance of the mutation reliable methods suited for processi ng large sets of samples are in demand. We here present the oligonucle otide ligation assay (OLA) with lanthanide labeled oligonucleotides fo r the detection of FV Leiden. The assay is based on time resolved fluo rescence measurement of lanthanide labeled oligonucleotides (DELFIA: D elayed Enhanced Lanthanide Fluorescence Immune Assay) and on the speci ficity of T-4 DNA Ligase to join two adjacent oligonucleotides only wh en the two are complementary to the PCR template at the ligation junct ion. The Europium/Samarium fluorescence pattern is specific for each o f the three genotypes (G/G, G/A, A/A) and clearly separates the three genotypes. By using a wildtype probe (Samarium labeled) and a mutant-s pecific probe (Europium labeled) simultaneously an internal control of the assay is included in each reaction. The assay is simple to perfor m, can be partly automated and is ideal for processing large sets of s amples.