Inverse Shifting-PCR Modified by Capillary Electrophoresis for Detecting F8 int22h and int1h Inversions in Severe Hemophilia A Patients and Probable Carriers
Year: 2024
Type: article
Abstract: 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” BclI 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.
Source: Life
DOI: 10.3390/life14101332
Language: English
Subfield: Hematology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $2,955
OpenAlex ID: W4403610044