Characterization of the paternal missense mutation. (A) Sequence (sense) of amplified cDNA subclones from a control (left) and the patient (right). The position of the G632 → A transition is indicated by arrows next to the sequence ladder. (B) Identification of heterozygosity for the missense mutation. DNA samples from a control (lane 1) and the patient B-LCL (lane 2) were hybridized to a radiolabeled 444-bp Rsa I-Pst I cDNA probe extending from the mutation site to the end of exon 11 afterRsa I digestion. The normal Rsa I sites are at base pair 27,276 in exon 6, 28,516 in exon 7, and 31,671 in intron 11, predicting a 3.154-kb band hybridized to the probe in normal control, while loss of Rsa I site in exon 7 results in a larger band (4.394 kb). (C) Determination of the paternal mutation. Amplified genomic fragments (739 bp) from intron 6 (base pair 28377) to intron 9 (base pair 29115) were digested with Rsa I and electrophoresed in 2.0% agarose gel, and stained with ethidium bromide. The fragment has one Rsa I recognition site at base pair 28,517 in exon 7, predicting 141- and 598-bp fragments. Loss of the Rsa I site by the mutation results in an undigested fragment.
