The "FlexMAP microsphere"-based ASPE assay on the Luminex platform

• The cartoon below illustrates the FlexMAP microsphere-based "Allele Specific Primer Extension" (ASPE) assay, for which SNP markers in marker set SNP Markers (MPBCRC:public:mhayden) have been adapted:

The FlexMAP microsphere-based "Allele Specific Primer Extension" (ASPE) assay.

1. Contigs are PCR-amplified from genomic DNA with "Locus-Specific Primers" ("FwdLSP" and "RevLSP").
   
2. For each SNP, two probes are designed. Each is a perfect match to one of the expected SNP alleles, with the nucleotide matching the polymorphic base at the 3' end.
   The 5' end of each probe carries a unique 24-base "FlexMAP tag", which is reverse-complementary to "FlexMAP anti-tags" uniquely assigned to different sets of microspheres (see 3 below).
   For the probe with a matching 3'-terminal nucleotide, primer extension occurs. Biotinylated dCTPs are incorporated into the newly polymerised DNA strand, allowing to label extended probes selectively with a streptavidin-linked reporter dye.
3. Probes are uniquely annealed to microspheres through the specificity of the FlexMAP "tag" / "anti-tag" recognition. The Luminex detection system identifies each microsphere by its internal dye, and records the associated reporter dye intensity (in the example "result table" as "Mean Fluorescence Intensity").
4. The reporter dye intensity detected for a pair of SNPprobes is used to calculate an "allele ratio" and interpreted as a genotype. In the illustrated example, the SNP's genotype is considered homozygous for allele "G".