Helicase-dependent amplification

The polymerase chain reaction is the most widely used method for in vitro DNA amplification for purposes of molecular biology and biomedical research.^[1] This process involves the separation of the double-stranded DNA in high heat into single strands (the denaturation step, typically achieved at 95–97 °C), annealing of the primers to the single stranded DNA (the annealing step) and copying the single strands to create new double-stranded DNA (the extension step that requires the DNA polymerase) requires the reaction to be done in a thermal cycler. These bench-top machines are large, expensive and costly to run and maintain, limiting the potential applications of DNA amplification in situations outside the laboratory (e.g., in the identification of potentially hazardous micro-organisms at the scene of investigation, or at the point of care of a patient). Although PCR is usually associated with thermal cycling, the original patent by Mullis et al. ^[2] disclosed the use of a helicase as a means for denaturation of double stranded DNA thereby including isothermal nucleic acid amplification. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate the DNA by unwinding the DNA double helix.^[3] HDA was developed from this concept, using a helicase (an enzyme) to denature the DNA.

Strands of double-stranded DNA are first separated by a DNA helicase and coated by single-stranded DNA (ssDNA)-binding proteins. In the second step, two sequence-specific primers hybridise to each border of the DNA template. DNA polymerases are then used to extend the primers annealed to the templates to produce a double-stranded DNA and the two newly synthesized DNA products are then used as substrates by DNA helicases, entering the next round of the reaction. Thus, a simultaneous chain reaction develops, resulting in exponential amplification of the selected target sequence (see Vincent et al.., 2004^[4] for a schematic diagram).