Subcycling PCR for Long-Distance Amplifications of Regions with High and Low Guanine-Cystine Content: Amplification of the Intron 22 Inversion of the

Hemophilia A is an X-linked disorder caused by mutations in the factor VIII gene. Around 50% of all patients with severe hemophilia A share a common mutation. This intron 22 inversion results from homologous recombination of a sequence within intron 22 of the factor VIII gene and identical sequence around 500 kb telomeric to the gene. Although this inversion could be detected by Southern blotting, the development of a long polymerase chain reaction (PCR) assay, and its subsequent improvement by subcycling PCR (S-PCR), greatly facilitated the provision of diagnostic services (1 ,2 ).