FIG. 3.

PFGE provides evidence for the lack of a submicroscopic deletion in PWS patients with uniparental disomy. NotI fragments are shown for probes IR4-3R (D15S11) (left) and 34 (right) in PWS2 (lane 3), M2 (lane 2) and F2 (lane 1). Multiple bands are indicative of partial cleavage at restriction sites when CpG is methylated³² and a similar pattern of multiple bands has been observed in other normal controls (results not shown). PWS2 shares a normal 2,500-kb band with his mother, although in the latter this results from partial digestion at a NotI site in this region of the genome. The same 2,500-kb band has been detected in other normal individuals. The larger three fragments are common to probes IR4-3R, 189-1 (results not shown) and 34, showing that these loci are closely linked in region 15q11q13. These probes and a 1,300-kb fragment detected with probes 3-21 and IR10-1 (unpublished data) are deleted in all PWS¹⁰ and AS⁶ patients with cytological deletions. Additional PFGE analysis (results not shown) also indicates that PWS2 displays PFGE restriction fragments of normal size for IR10-1 in a NotI digest (1,300 kb) and for IR39d in a BssHII digest (250 kb). PWS1 also seems to be intact for the probe 34 and 3-21 loci because the restriction fragments detected using NotI and BssHII are unaltered compared with those of normal individuals.

METHODS. PFGE analyses were performed as described previously³³ using a modification of techniques for DNA preparation and digestion in agarose blocks³⁴. Electrophoresis was performed in 0.8% agarose in TBE (89 mM Tris buffer, pH 8.0, 89 mM boric acid, 2 mM EDTA) at 22 °C and was divided into three 48-h intervals during which the forward polarity pulse durations were varied from 75-600 s, 600-2,500 s and 2,500-3,000 s and field intensities were set at +2, +1.7 and +1.3 V cm⁻¹, respectively. The ratios of the duration and intensity of the inverse polarity pulse relative to the forward were 0.4 and 0.5, respectively.