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

A PCR-RFLP assay for genotyping at the mouse leptin receptor (Lepr(db)) mut ation site was developed using modified primers. The first modified primer creates an AccI restriction site in the mutant Lepr(db) allele to distingui sh between the Lepr(db) and Lepr(+) alleles whereas the second modified pri mer creates another AccI site in both alleles to serve as a control for res triction enzyme digestion. The assay is robust and works efficiently on unp urified lysates of mouse tissues and can be applied at any age of the anima l. The assay may be used as a diagnostic tool for maintenance of stocks, in trogression or other types of crosses involving the Lepr(db) mutation.