Misleading gene conversion frequencies due to a PCR artifact using small fragment homologous replacement.

Abstract

Recent studies have reported successful correction of the most common F508del mutation in cystic fibrosis (CF) airway epithelial cells by small fragment homologous replacement (SFHR). We wished to apply the SFHR methodology to our CF bronchial epithelial cells, of compound heterozygous genotype (F508del/W1282X), in which nucleic acid transfer was previously optimized by electroporation. Using a PCR-based detection methodology, with one of the primers located outside the SFHR homology region, we obtained SFHR dose-dependent F508del to wild-type CFTR gene conversion frequencies reaching 30%. However, the increased wild-type/F508del CFTR allele ratio was transient, vanishing at 5 days posttransfection. Furthermore, we have been unable to reproduce the SFHR-mediated repair of the F508del mutation in our cellular model when both detection primers were located outside the SFHR homology region. A thorough reexamination of our initial detection strategy revealed that a false positive result was originated from a PCR artifact created by the SFHR fragment itself. Thus, nonamplifiable detection methods, such as Southern blotting, protein analysis, or functional assays, should be performed, whenever possible, to correctly assess gene conversion frequencies.