Objective The aim of this work is to demonstrate a novel single-molecule DNA sequence comparison assay that is purely based on DNA mechanics. Methods A molecular construct that contained the two homologous but non-identical DNA sequences that were to be compared was prepared such that a four-way (Holliday) junction could be formed by the formation of heteroduplexes through the inter-recombination of the strands. Magnetic tweezers were used to manipulate the force and the winding applied to this construct for inducing both the formation and the migration of a Holliday junction. The end-to-end distance of the construct was measured as a function of the winding and was used to monitor the behavior of the Holliday junction in different regions of the intra-molecular recombination. Main Results In the appropriate buffer, the magnet rotation induces the migration of the Holliday junction in the regions where there is no sequence difference between the recombining sequences. In contrast, even a single-base difference between the recombining sequences leads to a long-lasting blockage of the migration in the same buffer; this effect was obtained when the junction was positioned near this locus (the site of the single-base difference) and forced toward the formation of heteroduplexes that comprise the locus. The migration blockages were detected through the identification of the formation of plectonemes. The detection of the presence of sequence differences and their respective mappings were obtained from the series of blockages that were detected. Significance This work presents a novel single-molecule sequence comparison assay that is based on the use of a Holliday junction as an ultra-sensitive nanomechanism; the mismatches act as blocking grains of sand in the Holliday “DNA gearbox”. This approach will potentially have future applications in biotechnology.
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