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

Abstract

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.

Incidence of bovine leukocyte adhesion deficiency, complex vertebral malformation, and deficiency of uridine-5-monophosphate synthase carriers in Brazilian Girolando cattle

“…Complex vertebral malformation (CVM) is a lethal hereditary condition characterized by growth retardation, vertebral malformation, and bilateral symmetrical arthrogryposis, leading to the premature death of calves (Duncan Jr. et al, 2001;Agerholm et al, 2004;Kanae et al, 2005). CVM is caused by a point mutation (G to T) at nucleotide position 559 of the solute carrier family 35 member 3, UDP-N-acetylglucosamine transporter (SLC35A3) gene on BTA3 (Thomsen et al, 2006).…”

Identification of complex vertebral malformation carriers in Holstein cattle in south China

“…For instance, PCR-single-stranded conformational polymorphism (PCR-SSCP) (Rusc and Kaminski, 2007;Chu et al, 2008), allele-specific PCR (AS-PCR) (Bendixen et al, 2002;Ghanem et al, 2008), microsatellite symbol method (Berglund et al, 2004) and PCR primerintroduced restriction analysis (PCR-PIRA) (Kanae et al, 2005). However, CRS-PCR, applied in this study, is a simple and efficient technique to identify single nucleotide polymorphism (SNP) genotypes, in which one or more mismatch bases are introduced in a primer to create a re-, in which one or more mismatch bases are introduced in a primer to create a rene or more mismatch bases are introduced in a primer to create a reintroduced in a primer to create a reed in a primer to create a restriction site by combining the SNP site after PCR.…”

A rapid mismatch polymerase chain reaction assay to detect carriers of complex vertebral malformation in Holstein cattle

“…Second, the protocol for both the DNA extraction and the PCR amplification developed is easier to perform than other methods for testing the CVM allele. 12,21 If a lethal recessive mutation is not associated with any economically important trait, it would be much easier to eliminate the allele in the sires. However, the CVM mutation is thought to be linked to milk quality and quantity, which is a major economic factor in dairy cows.…”

“…The polymerase chain reactionprimer introduced restriction analysis (PCR-PIRA) is a useful method for screening the CVM allele, 12 but it cannot distinguish the CVM allele from the wild-type allele directly by PCR amplification and requires further digestion analysis with specific restriction enzymes. The allele-specific PCR (AS-PCR) has an advantage that it can detect the CVM gene directly by PCR amplification.…”

“…12 The modified primers were named T primer pairs (5′-TCACAATTTGTAGGTCTCAATGCA-3′ and 5′-TCCACACTGATGGTTTGGTTTCTT-3′).…”