A Method for Detecting Complex Vertebral Malformation in Holstein Calves using Polymerase Chain Reaction–Primer Introduced Restriction Analysis

Abstract

Complex vertebral malformation (CVM), a hereditary lethal disease in Holstein calves, is characterized by complex anomalies of the vertebral column and limbs in an aborted fetus and in prematurely born, stillborn, and neonatal calves. The mode of inheritance of CVM is autosomal recessive, and CVM is caused by a point mutation from G to T at nucleotide position 559 of the bovine solute carrier family 35 member 3 ( SLC35A3) gene. Although an allele-specific polymerase chain reaction (AS-PCR) is a useful method for diagnosis of CVM, the AS-PCR requires selected DNA polymerases and strictly controlled reaction conditions to obtain reliable results. Therefore, an alternative screening method for the CVM gene would be useful. Polymerase chain reaction–primer introduced restriction analysis (PCR-PIRA) is a method that can be used for detecting a single nucleotide mutation in any gene without a restriction site around the mutation site. In this study, primers were designed to introduce PstI or EcoT22 sites into PCR products from the wild-type and CVM alleles, respectively. The wild-type allele, a heterozygote, and a homozygote of the CVM allele could be discriminated by restriction fragment length polymorphism analysis. Specific introduction of restriction sites into PCR products depending on the change in a single nucleotide of template was shown using a variety of DNA polymerases and PCR machines. Therefore, the PCR-PIRA technique using primers designed in this study might provide a more useful method for extensive screening of CVM.