A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay.
The goal of this multicenter study was to evaluate the second-generation Invader technology for detecting the factor V (Leiden) mutation directly from genomic DNA of different sample types. Invader assay results were compared with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or allele-specific PCR (AS-PCR) analysis. The Invader assay is a PCR-independent methodology that uses a microtiter plate format. In the assay, a specific upstream Invader oligonucleotide and a downstream probe hybridize in tandem to a complementary DNA template and form a partially overlapping structure. The Cleavase VIII enzyme recognizes and cuts this structure to release the 5' flap of the probe. This flap then serves as an Invader oligonucleotide to direct cleavage of a fluorescence resonance energy transfer (FRET) probe in a second invasive cleavage reaction. Cleavage of this FRET probe results in the generation of a fluorescent signal. The results of the Invader assay were 99.5% concordant with the PCR-based methods. Of the 372 samples tested once, only two gave discordant results (one from operator error and one from unknown causes), but were concordant on retesting. These results indicate that a simple microtiter plate-based Invader assay can reliably genotype clinical patient samples for the factor V (Leiden) point mutation directly from genomic DNA without prior target amplification.
Department of Pathology, University of Miami, Florida, USA.
M
M
Ledford
K D
KD
Friedman
M J
MJ
Hessner
C
C
Moehlenkamp
T M
TM
Williams
R S
RS
Larson
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005165:Factor V / Q000235:genetics*; D005456:Fluorescent Dyes; D005838:Genotype; D006801:Humans; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D005456:Fluorescent Dyes; C095381:factor V Leiden; D005165:Factor V