RT-NPCR to detect F8 intron 22 inversion mutations. (A) Schematic representation of exons 22 to 24 in WT F8 mRNA. Primers (gray arrows and font) were designed to hybridize within F8 exons 22 (pink), 23 (turquoise), and 24 (green). (B) WT-F8 mRNA transcript. RT-NPCR produces 428-bp (black line, often not visible on the gel) and 225-bp (gray bar) bands that are both diagnostic of an intact F8 exon 22 to 23 junction sequence. (C) RT-NPCR Inv22 test 1, representative result. MW, 1 kb plus DNA ladder (Invitrogen); N, HA, C, normal control, HA-Inv22, and carrier-Inv22 subjects, respectively. The 225-bp band is amplified from the N and C samples. (The weak ∼350-bp band seen in lane N is due to nonspecific binding of the primers.) (D) Schematic representation of F8 exons 19 to 22 plus part of the transcribed intron 22 sequence in F8 mRNA from an individual with an Inv22 mutation. Primers were designed to hybridize within F8 exon 19 (blue) and the F8 intron 22 sequence (orange). Note that 51 bases 3′ to the end of the exon 22 sequence, terminating in a TGA stop codon within intron 22, are transcribed as a consequence of the inversion mutation. (E) RT-NPCR produces 390-bp (black line, often not visible on the gel) and 378-bp (gray bar) bands that are both diagnostic of an unspliced F8 exon 22 to intron 22 sequence. (F) RT-NPCR Inv22 test 2, representative result. MW, molecular weight ladder; N, HA, C, normal control, HA-Inv22, and carrier-Inv22 subjects, respectively. The 378-bp band is amplified from the HA and C samples. (The weaker ∼750-bp band is due to nonspecific binding of the primers.)