Internal Restriction Sites: Quality Assurance Aids in Genotyping

Abstract

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.

Genetic disorders in beef cattle: a review

“…Within carriers, FECH activity is lower than in normal animals, although it is adequate to ward off high concentration of protoporphyrin (Jenkins et al 1998). This condition may be found among Charolais, Limousin, Blonde d’Aqutaine cattle (O’Rourke et al 2006; Shibuya et al 1995). …”

“…Moreover, after physical effort their urine is brown and transparent and the level of plasma creatine kinase is increased (Soethout et al 2002). According to previous studies, this disorder affects solely Charolais cattle and its consequences may be lethal (Citek et al 2007; O’Rourke et al 2006). …”

“…Transition of cytosine to thymine in position 1468 (c. 1468C>T) in exon 12 leads to exchange of tryptophan for arginine in codon 490 (UniProtKB/Swiss-Prot: P79334.3), which results in production of dysfunctional polypeptide (O’Rourke et al 2006). This mutation might be detected by usage of PCR-RFLP test with application of Sty I (Citek et al 2007; O’Rourke et al 2006). …”

“…To determine whether animal is affected, carrier or normal individual, there can be used PCR-RFLP assay with the usage of Hpa I (O’Rourke et al 2006) or ASPCR (Jenkins et al 1998). There can also be applied a fluorimetric method for measure ferrochelatase activity by quantifying disappearance of porphyrin (Shi and Ferreira 2003), k-ELISA (Straka et al 1991) and Western blot analysis (Jenkins et al 1998; Straka et al 1991).…”

“…Individuals with these defects show muscle pain and weakness, as well as, problems with motoric condition. Same cases might be even lethal (Citek et al 2007; O’Rourke et al 2006; Zlotowski et al 2006). Another inherited beef trait is connected with blood and it is called the protoporphyria.…”