Chimeric DNA byproducts in strand displacement amplification using the T7 replisome
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Long read sequencing technologies motivate the development of DNA amplification methods capable of producing specific amplicons larger than those achievable by PCR. Here, we investigate isothermal strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. Sequence analysis revealed chimeric DNA molecules corresponding to long inverted repeats, which in some cases accounted for the bulk of amplified DNA. These chimeric reads originate from template switching at short, inverted repeat sequences within the amplicon. Nanopore sequencing revealed that template switching occurs in part through an intermolecular mechanism via a cruciform intermediate and highlighted a role for polymerase exonuclease activity in promoting template switching. These findings demonstrate challenges to targeted isothermal DNA amplification and guide development of novel amplification methods for long read sequencing.