Alu-mediated PCR artifacts and the constitutional t(11;22) breakpoint

The breakpoints of the recurrent t(11;22)(q23;q11) have recently been cloned. We identified palindromic AT-rich repeats (PATRRs) on 11q23 and 22q11 as the mechanism responsible for the rearrangement. Contradictory to our results, A.S. Hill et al. (Hum. Mol. Genet., 9, 1525-1532) suggested that Alu-mediated recombination is responsible. To clarify this discrepancy, the cloned 4.5 kb der(11) junction fragment has been completely sequenced. This sequence has been compared with that of an inverse PCR-generated der(11) junction fragment obtained by Hill et al. This reveals that the inverse PCR product has sustained a deletion between two Alu elements, such that the true breakpoint region is deleted from the PCR product. Utilizing PCR primers designed by Hill et al. to amplify across the der(11) breakpoint, we obtained a deleted PCR product even when our cloned der(11) junction fragment was used as template. Further, we find that the PCR primers that they utilized for amplification of the der(22) junction fragment are not located on the der(22). They are oriented in opposite directions within the region deleted from the der(11) PCR product, generating an artifact derived from the der(11) chromosome. Analysis of the truncated PCR products indicates a mixture of sequences from two distinct Alu elements, suggesting that the putative junction fragment described by Hill et al. is an Alu-mediated PCR artifact. These data suggest that caution should be exercised when analyzing PCR-based data, particularly when amplification is carried out in a region containing repeat structures with specific, difficult-to-amplify sequences.