Zp. Zhang et al., Failure to achieve gene conversion with chimeric circular oligonucleotides: Potentially misleading PCR artifacts observed, ANTISENSE N, 8(6), 1998, pp. 531-536
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.