Identification of the maternal mutation at the splice donor site in intron 2. (A) Sequence (sense) of the exon 2/intron 2 junction in amplified genomic DNA. Genomic fragments containing exon 2 were amplified from a control (left) and the patient (right) and sequenced directly. A mutation at the splice donor site in intron 2 (G⁺¹ → A) is indicated by arrows. (B) Detection of the splice site mutation by the BspMI digestion. Amplified genomic fragments (690 bp) from intron 1 (bp 14,901) to intron 2 (base pair 15,590) was digested with BspMI, electrophoresed in 2.0% agarose gel, and stained with ethidium bromide. The fragment has twoBspMI recognition sites at bp 15,282 and 15,344, predicting 62-, 246-, and 382-bp fragments in the control lane. Loss of theBspMI site (base pair 15,282) by the mutation results in the undigested fragment (308 bp). Lane 1, control; lane 2, father; lane 3, mother; and lane 4, patient. BspMI digestion shows the patient and his mother were heterozygous for the splice site mutation.