Enzymatic amplification of oligonucleotides in paper substrates

Abstract

Several solution-based methods have recently been adapted for use in paper substrates for enzymatic amplification to increase the number of copies of DNA sequences. There is limited information available about the impact of a paper matrix on DNA amplification by enzymatic processes, and about how to optimize conditions to maximize yields. The work reported herein provides insights about the impact of physicochemical properties of a paper matrix, using nuclease-assisted amplification by exonuclease III and nicking endonuclease Nt. Bbv, and a quantum dot (QD) - based Forster Resonance Energy Transfer (FRET) assay to monitor the extent of amplification. The influence of several properties of paper on amplification efficiency and kinetics were investigated, such as surface adsorption of reactants, and pore size. Additional factors that impact amplification processes such as target length and the packing density of oligonucleotide probes on the nanoparticle surfaces were also studied. The work provides guidance for development of more efficient enzymatic target-recycling DNA amplification methods in paper substrates.

Keywords: Enzyme; Isothermal amplification; Oligonucleotides; Paper substrate; Porosity; Quantum dots.