Linkage mapping of four genes (OTC, SERPINA7, SLC25A5 and FMR1) on porcine chromosome X.

20 ll volume containing: 20 ng template genomic DNA (prepared from peripheral blood lymphocytes by standard protocols), 1 ́ Taq buffer (10 mM Tris±HCl, pH 8.3, 1.5 mM MgCl2, and 0.001% gelatine), 150 lM each dATP, dGTP, dCTP and dTTP (Amersham, Little Chalfont, UK 1 ), 4 ng forward primer (5¢ end-labelled with [P]ATP (Amersham) using T4 polynucleotide kinase (Amersham)), 4 ng unlabelled reverse primer, and 0.25 U of Taq DNA polymerase (Promega, Madison, WI, USA). Reaction mixtures were overlaid with mineral oil before ampli®cation. The PCR reactions were performed in a thermocycler PTC-100 (MR Research Inc., Watertown, MA, USA). Thermocycling was undertaken in two stages: after an initial denaturation at 95 °C for 5 min, the ®rst stage was 10 cycles of 57 °C for 30 s, 72 °C for 1 min and 95 °C for 30 s. The second stage, following an initial denaturation at 95 °C for 5 min, consisted of 30 cycles of 55 °C for 30 s, 72 °C for 1 min and 95 °C for 20 s. The ®nal cycle was followed a ®nal extension at 72 °C for 10 min.