ELISA DETECTION OF RESTRICTION SITE POLYMORPHISMS IN THE PIG RYANODINE RECEPTOR LOCUS

We compare two strategies for ELISA detection of restriction site poly morphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1(hal)). In both procedu res, target DNA is amplified by PCR with one primer that is 5' biotiny lated and a second primer that is 5' fluoresceinylated. PCR products a re captured in duplicate wells on a streptavidin-coated, 96-well plate . The duplicates may be treated in two ways. In a single restriction e nzyme assay, one duplicate is exposed to a restriction enzyme that cut s one allele specifically, and the second duplicate is exposed to no r estriction enzyme. In a dual restriction enzyme assay, the second repl icate is exposed to a second restriction enzyme that cuts the alternat e allele specifically. Thereafter, the two procedures are similar; ant ifluorescein antibodies conjugated to peroxidase are allowed to bind t o the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in th e two wells is used to classify subjects by genotype. When the dual re striction enzyme assay is run, three genotype groups are easily distin guishable. When the single restriction enzyme assay is run, heterozygo tes generate values that may overlap with those of the homozygotes tha t are not cut by the restriction enzyme. Dual restriction enzyme assay s are more accurate than single restriction enzyme assays; however, si ngle restriction enzyme assays are sufficient for identifying pigs tha t carry RYR1(hal).