Molecular and Cellular Probes 21 (2007) 78–79 Polymorphism report Single nucleotide polymorphisms of the ovine calpain 3 (CAPN3) gene H. Zhou, J.G.H. Hickford à , Q. Fang Gene-Marker Laboratory, Cell Biology Group, Agriculture and Life Sciences Division, Lincoln University, PO Box 84, Lincoln 7647, New Zealand Received 1 June 2006; accepted 7 July 2006 Available online 29 July 2006 Keywords: Calpain 3 (CAPN3); Single Nucleotide Polymorphism (SNP); Sheep 1. Source/description Calpain 3 (CAPN3), also known as p94, is predominantly expressed in skeletal muscle [1,2], where it plays an important role in myofibrillar integrity [3]. A deficiency of CAPN3 causes limb–girdle muscular dystrophy type 2A in humans [4]. CAPN3 binds specifically to connectin (a striated muscle- specific giant filamentous protein) at the N 2 line [5], a region where proteolysis has been linked to post-mortem tenderiza- tion [6]. This, together with a correlation between CAPN3 expression and the rate of meat tenderization in both cattle and sheep [7], suggests that CAPN3 may play a role in muscle undergoing post-mortem conditioning. To further investigate the role of ovine CAPN3 in meat tenderization, the extent of polymorphism in the gene needs to be ascertained along with how the polymorphism impacts on gene expression and protein function. 2. Genomic DNA and PCR amplification Three hundred and thirty-four sheep (Merino, Corrie- dale, Romney, Poll Dorset, and NZ cross-bred) were investigated, and genomic DNA was purified from blood collected on FTA TM cards (Whatman BioScience, Mid- dlesex, UK) using a two-step washing method [8]. The tenth exon, which is one of the most polymorphic regions in human CAPN3 [9], was amplified using PCR primers 5 0 - CTCTCAGGATGTCCTACG-3 0 and 5 0 -CTGGGAAG- TTGCGGCAG-3 0 , designed based on GenBank sequences AF087570 and AY902237. Amplifications were performed in a 20-ml reaction containing genomic DNA on a 1.2-mm diameter disc of FTA TM , 0.25 mM of each primer, 150 mM dNTPs (Eppendorf, Hamburg, Germany), 0.5 U Taq DNA polymerase (Qiagen, Hilden, Germany) and 1  the reaction buffer supplied. After initial denaturing at 94 1C for 2 min, 35 cycles of 94 1C for 30 s, 60 1C for 30 s, and 72 1C for 30 s were utilized, followed by a final 5 min extension step at 72 1C. 3. SSCP and sequencing analyses A 0.7-ml aliquot of each PCR amplicon was mixed with 7 ml of loading dye containing 98% formamide, 10 mM EDTA, 0.025% bromophenol blue and 0.025% xylene- cyanol. After denaturation at 95 1C for 5 min, samples were cooled on wet ice and loaded onto 14% polyacrylamide gels. These gels were electrophoresed at 280 V for 18 h at 5 1C in 0.5  TBE buffer, followed by silver-staining [10]. Genomic DNA representative of the unique SSCP patterns was amplified using Pwo SuperYield DNA polymerase (Roche Applied Science, Mannheim, Germany), and the amplicons were cloned into the pCR s 4 Blunt-TOPO s vector (Invitrogen, Carlsbad, CA, USA). Only those plasmids for which the PCR-SSCP patterns matched with the genomic DNA template patterns were sequenced, in both directions, at the Waikato DNA Sequencing Facility, University of Waikato, New Zealand. Identical sequences obtained from at least three separate clones from different sheep, or two independent PCR amplifications from the same sheep, were subjected to further sequence analysis. These checks ensure that the polymorphisms detected were not the result of PCR amplification and sequencing errors. 4. Allelic polymorphism and frequencies Three unique SSCP patterns were detected, and either one or a combination of two of these patterns were ARTICLE IN PRESS www.elsevier.com/locate/ymcpr 0890-8508/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.mcp.2006.07.001 à Corresponding author. Tel.: +64 3 3252811; fax: +64 3 325385. E-mail address: hickford@lincoln.ac.nz (J.G.H. Hickford).