Figure 6

Overview of post-PCR, LDR-FMA diagnosis of P. falciparum dhfr drug resistance-associated SNPS. Allele-specific oligonucleotides include 5' "TAG" extensions that are 24 nucleotides in length. TAG sequences (n = 100), containing no G residues, have been designed to reduce potential for cross-hybridization with sequence 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 infra-red fluorochromes to result in unique fluorescent "classification" signatures. Each microsphere set is pre-coupled 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 (R-phycoerythrin) is classified into allele-specific bins by the BioPlex array reader and BioPlex Manager 3.0 software. Data from these reactions are immediately available in Excel spreadsheets.