Genotyping of bovine k‐casein (k‐CNA, k‐CNB, k‐CNC, k‐CNE) following DNA sequence amplification and direct sequencing of k‐CNE PCR product

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Abstract
Summary. Genomic DNA isolated from blood and semen of dairy cattle with known k‐casein (k‐CN) genotypes was subjected to Southern blot hybridization and polymerase chain reaction (PCR) using up to 14 restriction endonucleases. k‐casein genotypes AA, AB and BB were identified using Hin dIII and Hin ft while genotypes with k‐CNC and k‐CNE were misidentified. Direct sequencing of the PCR product (k‐CN EE) showed a substitution of guanine (k‐CNA, B) by adenine (k‐CNE) which creates a Hae III restriction site. Therefore using PCR followed by Hin dIII or Hin fI and Hae III digest allows discrimination between k‐casein A, B and E directly at the DNA level.