Premium
A novel real‐time PCR assay to detect Factor V Leiden and heterozygote Factor V Leiden using HyBeacons probe chemistry
Author(s) -
Parrish Torrey,
Duggan Megan,
Williams Patrick
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.1034.13
Subject(s) - factor v leiden , microbiology and biotechnology , buccal swab , standard curve , chemistry , hybridization probe , biology , genetics , dna , chromatography , medicine , thrombosis , venous thrombosis , surgery
Factor V Leiden (FVL) is an inherited disorder associated with blood clotting. Approximately 5% of Caucasians have FVL. We have developed a real‐time PCR assay for detection of FVL from buccal swabs or blood samples. A HyBeacons probe is used to detect FVL during melt curve analysis. HyBeacons probes are hybridization probes that allow simultaneous identification of single nucleotide polymorphisms (SNPs) via melt curve analysis. The probe is designed to hybridize to a region around a SNP associated with FVL. This allows the probe to bind to wild type and mutant forms of Factor V (FV). A normal amplification curve is seen for FV and FVL, however, during melt curve analysis a melt peak temperature shift is observed between FV and FVL. To optimize the assay, plasmids were constructed to both FV and FVL sequences. The assay was then run against each individual plasmid and a mixture of both to determine melt peaks for FV, FVL, and heterozygous samples. The melts peaks were approximately 52.3°C for FV, 57.5°C for FVL, and heterozygous samples had peaks at 52.3°C and 57.5°C. Twenty blood and buccal samples were obtained. DNA was extracted from the samples using Evogen ONE‐B and Evogen ONE, respectively and used in the FVL assay. All samples showed amplification but were negative for FVL. This newly developed assay is able to detect FV, FVL, and heterozygote from blood and buccal samples using a single HyBeacons probe.