Superhelicase-driven isothermal amplification of repetitive and low-complexity DNA

PCR is a cornerstone of molecular biology, but many biologically important DNA templates remain difficult to amplify. Long tandem repeats, low-complexity tracts, and sequences with extreme base composition often yield low product levels, smeared bands, stutter products, or truncated amplicons. These failures can arise because repeated cycles of thermal denaturation and reannealing promote off-register annealing, polymerase slippage, secondary-structure formation, and incomplete extension. Previously, we developed SSB-Helicase Assisted Rapid PCR (SHARP), an isothermal amplification method in which an engineered superhelicase and single-stranded DNA-binding protein replace the thermal melting step of PCR with enzymatic strand separation. Here, we tested whether SHARP can improve amplification of templates that are refractory to conventional PCR. SHARP robustly amplified up to six identical tandem repeats of the Widom 601 nucleosome-positioning sequence and up to 35 identical ankyrin repeats, targets that were poorly amplified by conventional PCR under the conditions tested. SHARP also amplified templates with extreme base composition, including a 95% AT-rich template and GC-rich templates (up to ~95% GC), as well as a 99-repeat CGG tract associated with Fragile X syndrome and a CAG/CAA repeat tract from mutant HTT exon 1 associated with Huntington disease. Together, these results show that helicase-driven isothermal amplification can expand access to repetitive and compositionally extreme DNA sequences that are challenging for conventional thermocycling-based PCR.