Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the mouse leptin receptor ( Lepr db ) mutation

Abstract

A PCR-RFLP assay for genotyping at the mouse leptin receptor (Lepr^db ) mutation site was developed using modified primers. The first modified primer creates an AceI restriction site in the mutant Lepr^db allele to distinguish between the Lepr^db and Lepr⁺ alleles whereas the second modified primer creates another AceI site in both alleles to serve as a control for restriction enzyme digestion. The assay is robust and works efficiently on unpurified lysates of mouse tissues and can be applied at any age of the animal. The assay may be used as a diagnostic tool for maintenance of stocks, introgression or other types of crosses involving the Lepr^db mutation.