Reliable amplification of highly repetitive or low complexity sequence DNA enabled by superhelicase-mediated isothermal amplification.

Abstract

Polymerase Chain Reaction (PCR) requires thermal cycling to melt DNA and proceed through the subsequent cycles of DNA synthesis needed for exponential amplification. Previously, we engineered a superhelicase, with enhanced processivity and speed, to replace this traditional PCR melting step with enzymatic DNA unwinding while retaining desired PCR characteristics, such as multi-kb amplicon size and applicability to cloning and gene editing outcome assessment. This isothermal amplification method is named SHARP (SSB-Helicase Assisted Rapid PCR) because single-stranded DNA binding protein (SSB) and superhelicases are added to standard PCR reagents. Here, we show that SHARP can be effective for DNA sequences that PCR is unable to amplify or that produce side products of. SHARP is demonstrated to be capable of amplifying up to six identical repeats of the Widom 601 nucleosome positioning sequence and up to 35 identical repeats of ankyrin sequence. We also show that a sequence with 91% AT-content can be amplified using SHARP and that the amplification product can be validated using single-molecule optical tweezers experiments.