Factor V Leiden genotyping using real-time fluorescent polymerase chain reaction

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Factor V Leiden (G1691A) is the most prevalent inherited risk factor for Deep Vein Thrombosis.
• Accurate and rapid detection of this mutation is crucial for clinical risk assessment.

Purpose of the Study:

• To develop and validate a fluorogenic probe-based PCR assay for the direct detection of the Factor V Leiden mutation.
• To assess the assay's concordance with standard methods and its applicability to various DNA purities.

Main Methods:

• Utilized a TaqMan fluorogenic probe-based PCR assay with two allele-specific probes (FAM for mutant, TET for wild-type).
• Probes featured 5'-reporter dyes and 3'-quencher dyes (TAMRA) for allelic discrimination.
• Assayed genomic DNA from leukocytes, including both highly and crudely purified samples.

Main Results:

• The assay successfully differentiated between wild-type, heterozygote (+/-), and homozygote (-/-) Factor V Leiden carriers.
• Demonstrated complete concordance with a standard PCR-based assay in 120 patient samples.
• The method proved effective with both purified and crudely purified DNA, including genomic DNA from leukocytes.

Conclusions:

• The developed TaqMan PCR assay provides a rapid, accurate, and reliable method for Factor V Leiden genotyping.
• This assay is suitable for clinical diagnostics, enabling efficient identification of individuals at risk for Deep Vein Thrombosis.