Template-directed dye-terminator incorporation with fluorescence polarization detection for analysis of single nucleotide polymorphisms associated with cardiovascular and thromboembolic disease

Diseases attributable to acute thrombosis contribute to the morbidity and mortality of critically ill patients [1]. While common variants present in genes encoding mediators of the coagulation pathway have been implicated in conditions such as myocardial infarction and deep venous thrombosis in population-based studies, their clinical significance has not been thoroughly analyzed in patients hospitalized for acute illness [2–8]. More precise characterization of the role of genetic variation in predisposition to cardiovascular and thromboembolic events in the critical care setting may be useful in aiding diagnostic evaluation and guiding therapy. High throughput genotyping, i.e., the ability to accurately and economically analyze a large number of patient samples at numerous genetic loci, is essential to this task. Current approaches to high throughput genotyping are based on the principles of either allele-specific hybridization (e.g., microarrays, molecular beacon), primer extension (pyrosequencing), flap probe cleavage (InvaderR assay), or oligonucleotide ligation [9]. To date, no single technique has been extensively field-tested [9]. Thus, the adaptability of any given approach to a specific situation is relatively unstudied. Template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP) is a single base extension technique in which an oligonucleotide probe anneals to