Homogeneous Primer Extension Assay With Fluorescence Polarization Detection
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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 ).
Fig. 1. Primer extension reaction with fluorescence polarization detection. With the SNP probe annealing to the target DNA next to the polymorphic site, DNA polymerase incorporates the specific dideoxy- (or acyclo-) nucleoside triphosphate labeled with a fluorescent dye onto the probe to yield a substantially larger fluorescent molecule, which has a much higher fluorescence polarization value than that of the free nucleoside triphosphate. In the panel at left , ddC is incorporated onto the SNP probe hybridized to the PCR product with the “G” allele. In the panel at right , ddA is incorporated onto the probe hybridized to the PCR products with the “T” allele.
