Semi-automated detection of the factor V mutation by allele specific amplification and capillary electrophoresis.
Recently a point mutation (G1691A) in the coagulation factor V gene was shown to cause resistance for cleavage by activated protein C. The mutation is associated with an increased thrombotic risk and thus-far the most common genetic cause of thrombophilia. Current techniques to investigate the single base pair mutation at the DNA level use an assay based upon the polymerase chain reaction followed by restriction enzyme digestion or Southern blotting and allele specific probing. The method we describe here consists of a single PCR in which two specially designed allele specific primers and two consensus primers were used in one reaction to distinguish between homozygous normal, heterozygous and homozygous mutant individuals. Amplification products were analysed using Capillary Electrophoresis and on line UV monitoring. The Allele Specific Amplification Protocol and subsequent CE analysis (ASAP-CE) is a convenient, fast, automated and highly reproducible method that can be used in a routine laboratory setting.
van de Locht
L T
LT
Department of Hematology, University Hospital Nijmegen, The Netherlands.
Kuypers
A W
AW
Verbruggen
B W
BW
Linssen
P C
PC
Nováková
I R
IR
Mensink
E J
EJ
D000483:Alleles; D001483:Base Sequence; D019075:Electrophoresis, Capillary / Q000379:methods; D005165:Factor V / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D013927:Thrombosis / Q000235:genetics
D005165:Factor V