RT Journal Article
SR Electronic
T1 PCR artifact in testing for homologous recombination in genomic editing in zebrafish
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 077883
DO 10.1101/077883
A1 Minho Won
A1 Igor B. Dawid
YR 2016
UL http://biorxiv.org/content/early/2016/09/27/077883.abstract
AB We report a PCR-induced artifact in testing for homologous recombination in zebrafish. We attempted to replace the lnx2a gene with a donor cassette, mediated by a TALEN induced double stranded cut. The donor construct was flanked with homology arms of about 1 kb at the 5’ and 3’ ends. Injected embryos (G0) were raised and outcrossed to wild type fish. A fraction of the progeny appeared to have undergone the desired homologous recombination, as tested by PCR using primer pairs extending from genomic DNA outside the homology region to a site within the donor cassette. However, Southern blots revealed that no recombination had taken place. We conclude that recombination happened during PCR in vitro between the donor integrated elsewhere in the genome and the lnx2a locus, as suggested by earlier work [1]. We conclude that PCR alone may be insufficient to verify homologous recombination in genome editing experiments in zebrafish.