FACTOR V LEIDEN : DETECTION IN WHOLE BLOOD BY ASA PCR USING AN ADDITIONAL MISMATCH IN ANTEPENULTIMATE POSITION

Factor V Leiden mutation was initially detected in thrombophilic patients and relatives by PCR RFLP (Restriction Fragment Length Polymorphism) according to Bertina [1]. This technique presents some drawbacks and the current trend is to simplify the diagnosis. We describe a technique of Allele Specific Amplification (ASA) which is optimized in terms of reliability : an additional mismatch in antepenultimate position enables to obtain the same specificity as PCR RFLP. Furthermore, coamplification of internal control warrants an optimal sensitivity. All the PCR have been simplified : the DNA extraction improvement allows to analyse the genotype with only a few microlitres of whole blood whatever the anticoagulant and the procedure of preservation (freezing, dried blood spots, storage at +4°C for several days). This technique saves time. Moreover, full automation of the ASA technique may be shortened thanks to the lack of extraction and the positive/negative reading of the PCR signal. © 1997 Elsevier Science Ltd