The Template-Directed Dye-Incorporation Assay with Fluorescence Polarization Detection (FP-TDI).

INTRODUCTIONThe template-directed dye-incorporation assay with fluorescence polarization detection (FP-TDI) is an economical and robust SNP genotyping method that is easy to optimize and implement in both single-marker and moderate-throughput studies. It utilizes unlabeled, unpurified PCR-grade primers in a dideoxy chain-terminating DNA-sequencing reaction to ascertain the identity of one base immediately 3' to an unlabeled primer, which is annealed immediately upstream of the polymorphic site on the target DNA. The FP-TDI protocol consists of four key steps, all of which can be carried out in the same microtiter plate without further separation or purification. First, PCR amplification of genomic DNA produces the template for the primer extension reaction. Second, a PCR clean-up enzyme cocktail with pyrophosphatase is added directly into the PCR products to remove excess PCR primers, deoxynucleoside triphosphates, and inorganic pyrophosphates. Third, single-base primer extension is carried out using a SNP primer annealed to the target DNA one base upstream from the polymorphic site. Finally, the end product is scanned with an FP plate reader to determine the changes in fluorescence polarization. With the incorporation of pyrophosphatase in the protocol and the use of quenching to analyze the fluorescence data, up to 95% of the assays work well even without optimization.