Rapid Communication: A Novel DNA Polymorphism of the Bovine Calpain Gene Detected by PCR-RFLP Analysis1

Polymorphism. Two alleles of the bovine calpain II regulatory subunit gene were identified with PCR- RFLP analysis using the endonuclease HhaI. PCR Primer Sequences. 5' primer: CCC CTC GCA CAC ATT ACT CCA AC; 3' primer: ATA CGG CCT GCC ACT TTT TGA TG. PCR Conditions. 10 mM Tris, pH 8.3, 50 mM KCl, .1% Triton X-100, 1.5 mM MgCl2, 100 mM deox- ynucleoside triphosphates, .4 mM each primer, 122 ng genomic DNA, and 2.5 units of Taq DNA polymerase in a final volume of 50 mL reaction. The PCR cycling conditions were 97°C 1.5 min for the first cycle and 94°C 1 min for rest of the cycles for denaturation, 57°C 1 min for annealing, and 72°C 2 min for polymeriza- tion, for a total of 35 cycles. PCR Product. The above primers, designed based on the published bovine nucleotide sequence of cDNA (McClelland et al., 1989) encoding for the regulatory subunit of calpain II, successfully flanked the target sequences in PCR, generating a genomic DNA seg- ment of about 1,800 bp, of which 288 bp were exon sequences, putatively including the fourth exon through the 5' end of the eighth exon. Method of Polymorphism Detection. PCR product of 8 mL was digested with 1.0 m L1 0 ×buffer 2 and 4 units of HhaI (New England Biolabs, MA) restriction endonuclease at 37°C for 2 h followed by 2% agarose gel electrophoresis. Description of the Polymorphism. Analysis of the PCR products with restriction endonuclease HhaI (GCGC) revealed three genotypes. One was a three- band pattern on 2% agarose gels, designated AA genotype; the second was a two-band pattern, desig- nated BB genotype; and the heterozygous animals displayed a pattern with all four bands of approxi- mately 1,520, 900, 620, and 280 bp in size (Figure 1). Frequencies. The overall genotypic frequencies of 169 animals representing 13 bovine breeds were f(AA) = .35, f(AB) = .40, and f(BB) = .25. Based on