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 microliters of whole blood whatever the anticoagulant and the procedure of preservation (freezing, dried blood spots, storage at +4 degrees 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.
Hézard
N
N
Laboratoire central d'Hématologie, CHU Robert Debré, Reims, France.
Cornillet
P
P
Droullé
C
C
Gillot
L
L
Potron
G
G
Nguyen
P
P
D000483:Alleles; D005165:Factor V / Q000032:analysis* / Q000235:genetics; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational
C095381:factor V Leiden; D005165:Factor V