DNA-Binding Magnetic Nanoreactor Beads for Digital PCR Analysis

Digital PCR (dPCR) is based on the separation of target amplification reactions into many compartments with randomly distributed template molecules. Here, we present a novel digital PCR format based on DNA binding magnetic nanoreactor beads (mNRBs). Our approach relies on the binding of all nucleic acids present in a sample to the mNRBs, which both provide a high-capacity binding matrix for capturing nucleic acids from a sample and define the space available for PCR amplification by the internal volume of their hydrogel core. Unlike conventional dPCR, this approach does not require a precise determination of the volume of the compartments used but only their number to calculate the number of amplified targets. We present a procedure in which genomic DNA is bound, the nanoreactors are loaded with PCR reagents in an aqueous medium, and amplification and detection are performed in the space provided by the nanoreactor suspended in fluorocarbon oil. mNRBs exhibit a high DNA binding capacity of 1.1 ng DNA/mNRB (95% CI 1.0-1.2) and fast binding kinetics with k(a) = 0.21 s(-1) (95% CI 0.20-0.23). The dissociation constant KD was determined to be 0.0011 mu g/mu L (95% CI 0.0007-0.0015). A simple disposable chamber plate is used to accommodate the nanoreactor beads in a monolayer formation for rapid thermocycling and fluorescence detection. The performance of the new method was compared with conventional digital droplet PCR and found to be equivalent in terms of the precision and linearity of quantification. In addition, we demonstrated that mNRBs provide quantitative capture and loss-free analysis of nucleic acids contained in samples in different volumes.

Automated summary

In this study, a novel digital PCR method based on DNA binding magnetic nanoreactor beads (mNRBs) was developed, which enables rapid and quantitative capture and analysis of nucleic acids in samples. Unlike conventional dPCR, this method does not require a precise determination of compartment volume but only calculates the number of amplified targets. The performance of this method is comparable to that of conventional digital droplet PCR in terms of quantification precision and linearity.