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 1 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 3 exon. The third one contained a correct 7E transcript (Fig. 1). These chimeric sequences could have resulted from somatic recombination or trans-splicing but are more likely an experimental artifact. Since the chimeric clones end with 7E sequence, the initial reverse transcription reaction must have been specific for the 7E transcript. However, we had noted that reverse transcription often yielded prematurely terminated 7E cDNAs. We reasoned that such partial 7E cDNAs could have hybridized to the 7C or 7D transcripts (which are 10 or 25 fold, respectively, more abundant than the 7E transcript; unpubl. data, 1) and served as primer for reverse transcription by Taq polymerase (3). As the 5' PCR primer fits the 7C and 7D sequences as well, such chimeric molecules would have been amplified in the PCR reaction. To test this hypothesis we repeated the experiment but included a RNase A treatment after first strand synthesis. Five out of five recombinant clones contained the correct 7E transcript and no chimeric clones were found. We thus conclude that the synthesis of the chimeric cDNA clones is a PCR artifact caused by the reverse transcriptase activity of Taq polymerase. Hence, this reverse transcriptase activity is actually a drawback rather than an advantage during cDNA cloning.