Abstract
Fluorescence-based DNA readouts are increasingly important in biological research, owing to enhanced analytical sensitivity and multiplexing capability. In this study, we characterize an in-gel polymerase elongation process to understand the reaction kinetics and transport limitations, and we evaluate DNA sequence design to develop signal amplification strategies. Using fluorescently labeled nucleotides, we scrutinize polymerase elongation on single-stranded overhangs of DNA immobilized in polyacrylamide hydrogels. When polymerase elongation reactions were carried out with reactants diffused into the gels, we observed reaction completion after 2 h, indicating that the process was efficient but much slower than that predicted by models. Confocal microscopy revealed a nonuniform post-reaction fluorescence profile of the elongated DNA throughout the depth of the gel and that the time for complete fluorescence penetration was proportional to the immobilized DNA concentration. These observations suggest retarded diffusion of the polymerase, attributable to interactions between diffusing polymerase and immobilized DNA. This study will ultimately inform assay design by providing insight into the reaction completion time to ensure spatial uniformity of the fluorescence signal. In agreement with our hypothesis that incorporation of multiple labeled nucleotides per DNA strand results in an increased signal, incorporation of four labeled nucleotides resulted in a 2.3-fold increase in fluorescence intensity over one labeled nucleotide. Our results further suggest that the fluorescence signal increases with spacing between labeled nucleotides, validating the number of and spacing between labeled nucleotides as tunable parameters for signal amplification. In-gel polymerase-based fluorescence readout is promising for signal amplification when considering both transport limitations and DNA sequence design.