Utility and Accuracy of Template-Directed Dye - Terminator Incorporation with Fluorescence - Polarization Detection for Genotyping Singl e

There are little independent data avai l able about how well single nucleotide pol y morphism (SNP) genotyping technologie s perform in the typical molecular genetic s laboratory. We evaluated the utility and a c curacy of a widely used technology, tem plate-directed dye-terminator incorporatio n with fluorescence-polarization detectio n (FP-TDI), in a sample of 177 SNPs selecte d solely on the basis of map location. Gen o types were generated without optimizatio n using standard protocols. Overall, 81% o f the SNPs we studied generated readabl e genotypes by FP-TDI. Thirty-two SNPs wer e genotyped in duplicate by PCR-RFLP or fl u orescent dye-terminator sequencing. Out o f a total of 631 duplicate genotypes, no tru e discrepancies were detected. The true erro r rate has a 95% chance of lying between 0 and 6 out of 1000 genotypes. We also teste d for deviations from Hardy-Weinberg Equ i librium in 33 SNPs genotyped in 50 unrela t ed individuals, and no significant deviation s were detected. Our FP-TDI data were read i ly adaptable to automated genotype callin g using our own method of cluster analysis , which assigns a probability score to eac h genotype call. We conclude that FP-TDI i s both efficient and accurate. The method ca n easily fill the needs of SNP genotyping pr o jects at the scale typically used for regiona l or candidate-gene association studies.