Chimeric cDNA clones: a novel PCR artifact

During the cloning of a transcript of one member of a closely related gene family, the human 7-crystallin gene family (1), we encountered a novel artifact of the polymerase chain reaction: the formation of chimeric cDNA molecules. Our experimental strategy in cloning the human 7E-crystallin transcript was a common one (2): first strand cDNA synthesis on human lens RNA using AMV reverse transcriptase and a 7E specific primer followed by PCR with the same 7E specific primer as reverse primer and a common 7-crystallin forward primer (see Fig. 1; the sequence similarity between the 7-crystallin genes precludes the synthesis of a gene specific 1st exon primer). The resulting fragment was purified and cloned into M13 mp vectors. Sequencing of three of these clones, however, revealed that two were chimeric, switching from either the 7C or 7D sequence to the 7E sequence in the 3d exon. The third one contained