Failure to achieve gene conversion with chimeric circular oligonucleotides: Potentially misleading PCR artifacts observed

Recently, a novel strategy for nucleotide exchange of target DNA using chim eric RNA/DNA oligonucleotides (CO) was reported. The CO can easily be trans fected into cells, remain stable within the cells, and migrate to the nucle us. We have in this study used 42 similar constructs for targeting six diff erent human and canine loci. A variety of cationic lipids, electroporation, and microinjection were used for transfection of the CO into lymphoblastoi ds, Huh7, HT 1080, and Jurkat cell lines, and canine primary fibroblasts an d hepatocytes. However, no nucleotide exchange was detected in any of the t argeted loci. Using PCR followed by restriction enzyme analysis, nucleotide exchange in approximately 2%-10% of the PCR products was observed during t he first 3 days after transfection with CO-vWF-28S2 designed for repairing a mutation in the von Willebrand gene. Surprisingly, the observed exchange reverted after culturing the cells for a longer period of time (14 days). F urthermore, a positive indication of gene conversion (5%) was also obtained using an allele-specific PCR method for analysis of the PAI-1 gene. Howeve r, cloning of the PCR products revealed no nucleotide exchange. In our view , the most likely explanation is that the initial false positive result ori ginates from a PCR artifact created by the CO itself. Our results imply tha t an independent method, that is, Southern blotting, must be used to verify an observed nucleotide exchange.