High-throughput genotyping with primer extension fluorescent polarization detection

Abstract

The primer extension assay with fluorescence polarization (FP) detection is a versatile assay for single nucleotide polymorphism (SNP) genotyping. With proper design, this homogeneous assay works under universal conditions. Fluorescence polarization occurs when a fluorescent dye is excited by plane-polarized light and is detected if the fluorescent dye is part of a large molecule. In assays where small fluorescent molecules are turned into large fluorescent products, FP provides a simple way to determine if the reaction has occurred without the need for separation or purification of the reaction mixture. In the primer extension assay, DNA polymerase incorporates the complementary, allelic nucleotide onto the SNP primer designed to anneal to the target DNA one base upstream of the polymorphic site. When a fluorescently labeled nucleotide is incorporated, high FP is observed for that dye and the genotype of the DNA sample is determined.