Homogeneous Primer Extension Assay With Fluorescence Polarization Detection

Abstract

The primer extension assay with fluorescence polarization (FP) is a genotyping method that combines the specificity of nucleotide incorporation by DNA polymerase and the sensitivity of fluorescence polarization. We named the assay Template-directed Dye-terminator Incorporation assay with FP detection (FP-TDI assay). It is a dideoxy chain-terminating DNA-sequencing protocol that ascertains the nature of the one base immediately 3′ to the sequencing primer (also called single nucleotide polymorphism [SNP]-specific primer). The SNP-specific primer is designed to anneal immediately upstream of the polymorphic site on the target DNA. In the presence of the target DNA, the appropriate dye-labeled terminators, and DNA polymerase, the SNP-specific primer is extended by one base as dictated by the nature of the allele at the polymorphic site on the target DNA. By determining which terminator is incorporated, the allele present in the target DNA can be inferred Fig. 1). Template-directed primer extension reaction has been used in various formats for genotyping, and it has proved to be highly specific and sensitive (1–4).