Development of allele-specific PCR based DNA test for detection of syndactylism (mule foot) related missense mutation in Holstein Friesian cattle

Abstract

Syndactylism or 'mule foot' is an autosomal recessive disorder of cattle, characterized by fusion of digits 
resulting in painful hooves and reduced mobility. The genetic defect has been reported in Holstein cattle 
populations, worldwide. Molecular studies have indicated the substitution of 2 nucleotides (delCGinsAT) in 
reading frame belonging to exon 33 of low-density lipoprotein-receptor-related protein 4 gene (LRP4) at 
chromosome 15 as the cause of mule foot. In this study, an allele specific polymerase chain reaction (AS-PCR) 
based method was developed for detecting the mule foot allele in cattle. In order to identify normal (wild-type) 
and mutant alleles at mule foot disease locus separately, two separate allele-specific forward primers were 
designed for each of the allele along with a common reverse primer. PCR reactions and conditions were 
standardized for amplification of each of loci separately. The wild-type and mutant alleles could be easily 
distinguished by detection of amplification from PCR of wild allele (CG) primer set, whereas no amplification 
by primer set for mutant allele (AT). Using the protocol, total 24 samples of Holstein Friesian (HF) bulls were 
screened, which were found to possess normal genotype at mule foot locus in all of the animals. This allele
specific PCR based DNA test was found suitable for the detection of mule foot mutation in cattle population 
specifically of Holstein lineages.