Template-Directed Dye-Terminator Incorporation with Fluorescence Polarization Detection for Analysis of Single Nucleotide Polymorphisms Implicated in Sepsis

Abstract

Sepsis continues to be a common source of morbidity and mortality in critically ill patients. Single nucleotide polymorphisms (SNPs) present in genes encoding inflammatory mediators have been associated with predisposition and outcome in this syndrome. The use of high throughput SNP analysis in large epidemiological studies is necessary to more fully understand the genetic underpinnings of this disease. We adapted template-directed dye-terminator incorporation with fluorescence polarization detection (TDI-FP) to the analysis of eight SNPs implicated in mediating the sepsis syndrome: TNF-alpha (-308), TNF-alpha (-238), TNF-beta (+250), IL-1beta (+3953), IL-6 (-174), IL-10 (-592), plasminogen activator inhibitor-1 (PAI-1 (-675)), and TLR4 299 (+1032). Optimization of PCR, amplicon purification, and template-directed dye-terminator incorporation reactions were necessary to achieve acceptable performance characteristics for these assays. Sequence validated samples served as controls. Using this method we were able to assign genotype in 99.3% of assays and identified 64 unique genotypes in samples obtained from 90 individuals. TDI-FP is a flexible and robust method of SNP detection that can be optimized in a systematic fashion. This method has potential advantages compared with other high throughput genotyping techniques and appears well suited to clinical situations requiring analysis of large numbers of samples.