Rapid isothermal amplification of several kilo base pair linear DNA

Abstract

Polymerase Chain Reaction (PCR) is an essential method used to amplify or copy nucleic acids. PCR requires a thermal cycler to alternate between strand separation at high temperatures and replication/copying at low temperature. We eliminated thermal cycling and kept all desired PCR features in place through enabling a new SSB (Single-Stranded Binding protein) and helicase assisted isothermal amplification method named SHARP (SSB-Helicase Assisted Rapid PCR). SHARP uses the same starting set of primers and template DNA as PCR does, carries out the reaction at a constant temperature, and outputs the same amplicon as PCR with lengths of up to 6000 base pairs within 5 to 30 minutes, a feature no existing isothermal amplification method can match.