Optimization of On-Bead Emulsion Polymerase Chain Reaction Based on Single Particle Analysis

Abstract

AbstractEmulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring, the only methods that can emulsify libraries of ≥108 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study systematically quantifies the influence of tuning individual ePCR parameters by single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that increasing the polymerase concentration 20-fold in ePCR over recommended concentrations for conventional PCR was necessary to obtain sufficient PCR products. Surprisingly, dramatical increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.