Faculty Collaboration Database

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. J Mol Diagn 2000 May;2(2):97-104

Date

03/29/2001

Pubmed ID

11272895

Pubmed Central ID

PMC1906901

DOI

10.1016/S1525-1578(10)60623-X

Scopus ID

2-s2.0-0034189760 (requires institutional sign-in at Scopus site)   35 Citations

Abstract

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.

Author List

Ledford M, Friedman KD, Hessner MJ, Moehlenkamp C, Williams TM, Larson RS

Authors

Kenneth D. Friedman MD Professor in the Medicine department at Medical College of Wisconsin
Martin J. Hessner PhD Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Base Sequence
DNA Mutational Analysis
DNA Primers
Factor V
Fluorescent Dyes
Genotype
Humans
Point Mutation
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Spectrometry, Fluorescence