FIG. 1.

Overview of post-PCR LDR-FMA diagnosis of drug resistance-associated SNPs. Allele-specific oligonucleotides include 5′ TAG extensions of 24 nucleotides. TAG sequences (n = 100), containing no G residues, have been designed to reduce the potential for cross-hybridization with sequences amplified from naturally occurring target sequences. Conserved sequence oligonucleotides are 5′ phosphorylated and 3′ biotinylated. Fluorescent-microsphere sets (n = 100) are embedded with varying ratios of red and infrared fluorochromes to result in unique fluorescent “classification” signatures. Each microsphere set is precoupled to unique anti-TAG oligonucleotides specifically complementary to the TAG sequences. Following ligation detection and TAG-anti-TAG hybridization reactions, doubly labeled ligation products are detected, and the “reporter” signal is classified into allele-specific bins by the BioPlex array reader and BioPlex Manager (version 3.0) software. Data from these reactions are immediately available in Excel spreadsheets.