Analysis of PCR-amplified exon 2 from genomic DNA. (A) Nucleotide sequence of the region with the GAAT 4-base deletion (beginning at the arrowhead) in the patient (upper panel) who is homozygous, and the corresponding region in the mother (lower panel) who is heterozygous for this deletion. The normal sequence in one allele in the mother is shown in the upper strand, and the boxed sequence in this strand is shown as deleted in the lower strand to depict the mutated allele. Note the overlapping peaks past the deletion because of dissimilar bases (indicated by broken lines) and single peaks because of identical bases (indicated by solid lines), representing one normal allele and one mutated allele in the mother. (B) Schematic representation of the IF gene (approximately 20 kb). The boxed segments represent the 9 exons (1251 bp). The region with the deletion is enlarged to show the signal peptide cleavage site (c54, green arrowhead), the location of the deletion (c183_186delGAAT, red arrowhead), and termination of translation (c206-X, blue arrowhead). (C) Restriction enzyme digestion of the PCR-amplified fragment with BstXI and BsaBI. The control has a single site for BstXI and does not cut with BsaBI; the homozygous patient has lost the site for BstXI because of the 4-base deletion which resulted in a site for BsaBI; the mother who is heterozygous shows sites for both restriction enzymes, and the heteroduplex in the uncut DNA represents the 2 alleles.