Application of capillary gel electrophoresis in detection of Factor VIII gene intron 22 inversion of hemophilia A.

Intron 22 inversion (Inv22) of the factor VIII gene (F8) accounts for approximately 45% of severe hemophilia A (HA) cases. Detecting Inv22 has become the primary screening method for severe HA. Currently, agarose gel electrophoresis (AGE) following long-distance polymerase chain reaction (LD-PCR) is commonly used in clinical settings to separate the amplified fragments of Inv22. However, AGE is hindered by lengthy processing times, instability, and inaccuracies in quantifying DNA content and assessing fragment sizes. We combined LD-PCR with capillary gel electrophoresis (CGE) for the identification of Inv22 in HA. Three primers were designed for LD-PCR to differentiate between Inv22, carriers, and wild types. We optimized the reaction system and conditions for CGE to effectively separate the amplified fragments. The optimal dilution ratio and buffer conditions for detecting Inv22 using CGE were 300 × and 0.1 × TE buffer. The ideal voltage and duration were 5.0 kV for 80 min. Under these conditions, the amplified fragments could be effectively separated, allowing for the direct measurement of concentration and size of the target fragments using ProSize data analysis software. The LD-PCR combined with the CGE assay for detecting Inv22 in F8 within HA populations has been successfully established. This method reduces both the time and labor required for detecting Inv22 in clinical practice, thereby advancing genetic diagnostic technology for hemophilia.