| Autor: |
Martínez-Contreras RM; Doctorado en Genética Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico., García-López SS; Facultad de Biología, Universidad Autónoma de Sinaloa, Culiacán 80010, Sinaloa, Mexico., Luna-Záizar H; Departamento de Química, Centro Universitario de Ciencias Exactas e Ingenierías, Universidad de Guadalajara, Guadalajara 44430, Jalisco, Mexico., Jaloma-Cruz AR; División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara 44340, Jalisco, Mexico. |
| Abstrakt: |
Globally, intron 22 inversions (Inv22s) of the factor VIII gene (F8) are the most frequent pathogenic variants and account for 45-50% of severe hemophilia A (SHA) cases, while intron 1 inversion (Inv1) explains 1-5% of SHA cases. The detection of both inversions by an inverse shifting-polymerase chain reaction (IS-PCR) is the first choice worldwide for the diagnosis of patients and carriers of SHA. To improve its sensitivity and reproducibility in the visualization of PCR products, we approached the IS-PCR with fluorescent capillary electrophoresis instead of agarose electrophoresis. Based on the original protocol, we modified two primers by 5'-end labeling with FAM™ fluorescent dye for the detection of the PCR products by capillary electrophoresis. Additionally, the "fast enzymes" Bcl I and T4-Ligase were incorporated for work saving in the genomic digestion and ligation reactions, respectively. Once we accomplished the standardization and verified the reproducibility of the modified IS-PCR method, we applied it for the diagnosis of a cohort of SHA patients and carriers. The modified IS-PCR by fluorescent capillary electrophoresis for PCR product detection is more sensitive than agarose electrophoresis. The method was also improved by using the new rapid enzymes to save time in the whole process. |
