Abstract
This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40–50 % of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris–borate–EDTA (TBE) buffer containing 0.3 % (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10 kV for 10 s at a temperature of 25 °C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5 min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1 %). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.
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Acknowledgments
We deeply extend our sincere thanks to the families who kindly contributed samples that were crucial to this study. We gratefully acknowledge the support of the Ministry of Science and Technology of Taiwan (MoST), Kaohsiung Medical University, and NSYSU-KMU 103-P023 JOINT RESEARCH PROJECT (#NSYSUKMU103-P023) by way of funding of this work, and the help of Kaohsiung Medical University Chung-Ho Memorial Hospital, Kaohsiung, Taiwan.
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Pan, TY., Wang, CC., Shih, CJ. et al. Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis. Anal Bioanal Chem 406, 5447–5454 (2014). https://doi.org/10.1007/s00216-014-7969-3
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DOI: https://doi.org/10.1007/s00216-014-7969-3