Southern hybridization experiments.

Genomic DNA from group A (lanes 1, 3, and 5) and group O (lanes 2, 4, and 6) porcine submaxillary glands was digested with EcoRI (lanes 1 and 2) alone or BamHI (lanes 5 and 6) alone or with both EcoRI and BamHI (lanes 3 and 4) and electrophoresed through a 1% agarose gel. DNA was then transferred by using the Southern method to a nylon membrane and hybridized in Ultrahyb hybridization buffer (Ambion, Austin, TX) with a cloned porcine A gene fragment probe, which was radiolabeled by using the random-hexamer method. Hybridization was done at 42°C overnight. Subsequently, the filter was washed twice with 2 × standard saline citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at 42°C for 10 minutes. This was followed by another washing with 0.1 × SSC and 0.1% SDS at 42°C for 15 minutes. The filter was then exposed to x-ray film. The same filter was later hybridized with a radiolabeled porcine α 1-3 galactosyltransferase gene sequence probe.13 Figure 1A shows the results of electrophoresis, and Figures 1B and 1C show the results of hybridization experiments with the porcine A gene probe (B) and the porcine α 1 → 3 galactosyltransferase gene probe (C). The very weak bands common to the A and O pig DNA may have resulted from cross-hybridization of the A gene probe with the sequence homologous to the human hgt4 pseudogene.11