Rapid communication: physical and linkage mapping of the porcine connexin 37 (CX37) gene

Genus and Species. Sus scrofa. Locus. Porcine connexin 37 (CX37) gene. Source and Description of Primers. A set of primers (F1, R1) was designed from the porcine CX37 mRNA sequence (GenBank Accession no. X86024) as well as an additional reverse primer (R2) from human (GenBank Accession no. 6093424) and murine (GenBank Accesion no. NM_008120) CX37 consensus sequence. The F1 and R2 primers were used to amplify porcine CX37 from genomic DNA. Using sequence obtained from the amplified product, an additional pig-specific forward primer (F2) was designed. Primer Sequences. F1: 5′-TTC CTG GAG AAG CTG CTG GA-3′; R1: 5′-CGA GAT CTT GGC CAT CTG TC3′; F2: 5′-ACT CGA CCG TGG TGG GCA A-3′; R2: 5′GTG GTC AGG TTG GCC CAG TT-3′. Method of Detection. A PCR of 10 L volume containing 1 L PCR buffer, 1 L MgCl2 (15 mM), 1 L dNTPs (2 mM), 0.25 L of each PCR primer (F1 and R2) (10 pM), 0.07 L Promega Taq Polymerase (Madison, WI), and 5.43 L H2O was used to assay 12.5 ng of genomic DNA from four individuals for each of five swine breeds (Landrace, Hampshire, Yorkshire, Berkshire, and Meishan). An 872-bp fragment from within the single CX37 exon was amplified using primers F1 and R2 in a Robocycler (Stratagene, La Jolla, CA) under the following thermocycling conditions: initial denaturation at 94°C for 4 min, 35 cycles of 94°C for 45 s, 62°C for 1 min, 72°C for 1 min 20 s, and a final extension time of 9 min at 72°C. For each breed, the PCR products from the four individuals were pooled. These pools were then directly sequenced using dye terminators and an ABI 377 sequencer (Perkin-Elmer, Foster City, CA) at the Iowa State University DNA Sequencing and Synthesis Facility. The F1 and R1 primers produced a 399-