title: Separation of intron 22 inversion type 1 and 2 of hemophilia A by modified inverse-shifting polymerase chain reaction and capillary gel electrophoresis.
abstract: An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 μM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR.
authors.affiliation: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, No. 100, Shichuan 1st Rd., Kaohsiung 807, Taiwan.
authors.forename: Tzu-Yu
authors.initials: TY
authors.lastname: Pan
authors.affiliation: Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. Electronic address: chiouss@kmu.edu.tw.
authors.forename: Shyh-Shin
authors.initials: SS
authors.lastname: Chiou
authors.affiliation: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, No. 100, Shichuan 1st Rd., Kaohsiung 807, Taiwan.
authors.forename: Chun-Chi
authors.initials: CC
authors.lastname: Wang
authors.affiliation: School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, No. 100, Shichuan 1st Rd., Kaohsiung 807, Taiwan; Department of Chemistry, College of Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan. Electronic address: shmewu@kmu.edu.tw.
authors.forename: Shou-Mei
authors.initials: SM
authors.lastname: Wu
mesh_terms: D016022:Case-Control Studies; D007446:Chromosome Inversion / Q000145:classification* / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D015321:Gene Rearrangement; D005838:Genotype; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D007438:Introns / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
keywords: Capillary electrophoresis; Hemophilia A; Intron 22 inversion Type 1 and 2; Inverse-shifting polymerase chain reaction