ELISA detection of restriction site polymorphisms in the pig ryanodine receptor locus.
We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1(hal)). In both procedures, target DNA is amplified by PCR with one primer that is 5' biotinylated and a second primer that is 5' fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1(hal).
Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808-4124, USA.
G E
GE
Truett
J A
JA
Walker
J B
JB
Wilson
S M
SM
Redmann
R T
RT
Tulley
G R
GR
Eckardt
G
G
Plastow
M
M
Lefevre
D000818:Animals; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005838:Genotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D013552:Swine / Q000235:genetics*
D017931:DNA Primers; D019837:Ryanodine Receptor Calcium Release Channel; D004262:DNA Restriction Enzymes