Internal Restriction Sites: Quality Assurance Aids in Genotyping

Abstract

Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or incorporate a second native site within the PCR amplicon, provide the mechanism by which efficiency of restriction enzymes can be assessed with every sample. The outcome is confirmation of the activity of the discriminating enzyme regardless of genotype.