Multiplex Analysis of Single-Nucleotide Extension Products on a 16-Capillary, Denaturing, High-Performance Liquid Chromatography Array

We constructed a high-performance liquid chromatography array consisting of 16 monolithic poly(styrene/divinylbenzene) capillaries for the parallel multiplex analysis of fluorescent dye-labeled single-nucleotide extension products. Because of the high chemical and physical robustness of the column bed that is covalently linked to the inner surface of the fused silica capillary, the array can be reused thousands of times without replenishment. The choice of fluorophore exerts a significant effect on resolution of the extension products. FAM, HEX, and TAMRA allowed complete resolution of all four possible allelic extension products not only from the extension primer but also from each other. The quantitative accuracy of the method enables the genetic typing of bi- and triallelic single-nucleotide polymorphisms in polyploid genomes and pooled samples.