PCR analysis of insertion sequences leads to the generation of artefact amplicons.

Insertion Sequences (ISs) are small self-mobilizing DNA elements widespread across prokaryotic genomes, including chromosomes and plasmids. IS elements frequently colocalize with antibiotic resistance genes and mediate their mobilization, often as part of larger genomic structures which encompass multiple IS elements and antibiotic resistance genes. In this study, we employed PCR to amplify DNA sequences containing two copies of an IS26 element from two Escherichia coli ST131 isolates. While the respective PCRs generated the product of the expected size, we also observed multiple amplicons of unexpected sizes, which could be misinterpreted as population heterogeneity attributed to IS mobilization. By extracting, re-amplifying and sequencing individual PCR products we demonstrate that these amplicons of unexpected sizes were indeed artefact products generated during the PCR reaction, likely mediated by within-PCR recombination of the IS26 sequences. Furthermore, PCRs with equally oriented primers each closely located to an IS26 element also generated artefact amplicons. This research highlights the limitations of using PCR to assess DNA sequences encoding multiple copies of an IS element and therefore, the presence of these genomic structures or the mobilization of the respective IS elements should not be assessed by diagnostic PCR alone but be corroborated with complementary techniques.