Rapid Communication: Mapping of the Titin (TTN) Gene to Pig Chromosome 151

Source and Description of Primers. The initial primers for the PCR were designed based on human DNA sequence (accession no. X92412; Kolmerer et al., 1996). The position of the forward and reverse primers corresponded to exon 3 and exon 5, respectively. These primers are expected to amplify a fragment of 1.93 kb from human DNA. A PCR fragment of 2.1 kb was amplified from porcine genomic DNA. Partial sequences were compared with the human sequence, which showed 90% (1,038/1,159 bp) nucleotide identity. The pig sequences (AF124849-50) were then used to design additional primers that amplified a 1.8-kb fragment of pig TTN and were used both for physical mapping using the somatic cell hybrid panel (SCHP) and for PCR-RFLP analysis. Primer Sequences. The primers based on human sequence were as follows: 5-TAA AAC ATC ACA GGC ATC AGA-3′ (forward) and 5-AGT CAG ATC CAA ATT CAT TCC-3′ (reverse). The primers designed from amplified pig sequence were as follows: 5′-GTC AAG AAA TCA GAA GCC ACC-3′ (forward) and 5′AAA ACT TTG CCC TCG TCA AT-3′ (reverse). Method of Detection. The PCR was performed in 10-μL reactions including .5 U Taq DNA polymerase (Promega, Madison, WI), 1 × PCR buffer, 1.5 mM MgCl2, .1 mM of each dNTP, 4 pmol of each primer, and 12.5 ng of porcine genomic DNA. The temperature conditions included initial denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 45 s, 63°C for 45 s, 72°C for 2 min and 15 s, and a final extension at 72°C for 5 min in a Robocycler (Stratagene, La Jolla, CA). The PCR products were electrophoresed on 1.0% agarose gels and visualized using ethidium bromide staining.