Rapid communication: mapping of oxytocin (OXT) to the central region of bovine chromosome 13 by linkage analysis using SSCP.

Polymorphism. Single strand conformation polymorphism (SSCP) was detected within PCR amplification products of the bovine Oxytocin (OXT) gene. Source and Description of Primers. Primers were derived from the published genomic sequence (Ruppert et al., 1984) based on primer positions given by Neibergs et al. (1991) and Dietz et al. (1992). Primer Sequences. The forward primer was 5′GCCATTAGCCGACATAACCTTGACC-3′ and the reverse primer was 5′-ACGGTGTCTAAGAGGGCAGCCGCAT-3′. Method of Detection. The primers amplified a 125-bp product under the following conditions: 15L reactions made up of 50 to 100 ng of genomic DNA, 200 M dNTP, 10 pmol of each primer, 1.5 mM MgCl2, and 0.75 U Taq polymerase (Amersham Pharmacia biotech, Freiberg, Germany) in reaction buffer supplied by the manufacturer. The PCR was carried out after an initial denaturation step at 94°C (1 min), then 60°C (1 min) and 72°C (1 min) for 30 cycles. The fragment was verified by restriction analysis. For SSCP analysis, 4 L of the PCR reactions was mixed with 6 L formamide dye (95% [wt/vol] formamide, 0.025% [wt/vol] xylenecyanol, 0.025% [wt/vol] bromophenol blue, 20 mM EDTA), denatured for 2 min at 90°C, and chilled on ice prior to loading 3 to 5 L of the mixture on nondenaturing 12% polyacrylamide (29:1), 0.5 × TBE gels. These were run at 16°C constant temperature for 14 h at 350 V in a Penguin P9DS electrophoresis unit (OWL Scientific, Woburn, MA) connected to an RTE-111 cooling unit (Neslab, Frankfurt, Germany). After electrophoresis, gels were fixed for 30 min in 10% acetic acid/15% ethanol and silver-stained following the protocol of Bassam et al. (1991). The developer solution was modified (45 g/L NaCO3, 0.0555% formaldehyde, 0.04 g/L Na2S2O3) and staining was stopped by transferring the gels into cold (10°C) 0.04 M EDTA. Inheritance Pattern. Three codominantly inherited alleles (Figure 1) were observed in the International Bovine Reference Panel (IBRP) (Barendse et al., 1997).