Reduction of background-triggered amplification in lesion-induced DNA amplification (LIDA)

Abstract

Lesion-induced DNA amplification (LIDA) enables isothermal amplification of nucleic acids, and the only enzyme required is T4 DNA ligase. However, the application of LIDA for the amplification of trace amounts of nucleic acids has been hindered by the observed background-triggered amplification in the absence of the initial target due to a pseudo-blunt end ligation reaction of two of the primers. In this work, we have tested three approaches to minimize the background-triggered amplification: increasing and decreasing the concentration of salts such as NaCl and MgCl₂, respectively, and increasing the concentration of ATP. All these optimizations sharply decreased the background-triggered amplification. Employing the most favourable buffer condition of 2.5 mM MgCl₂ where the target-initiated amplification was least affected while reducing the background-triggered process enabled us to achieve a detection range of 14 nM–140 aM with an approximate limit of detection of 680 aM, which is five orders of magnitude more sensitive than using our standard amplification conditions. This optimization of the salt and co-factor concentrations to decrease the background and enhance the sensitivity of LIDA has demonstrated LIDA's potential for application in clinical diagnostics.