In-gel fluorescence detection by DNA polymerase elongation.

Alden C Moss,Amy E Herr

doi:10.1063/5.0021149

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.

Highlights

The advantageous physical, chemical, and optical properties of hydrogels make the substrate material well suited for a range of bioanalyses.[1,2,3,4,5,6] Such assays often depend on fluorescence for the readout signals, and many rely on fluorescence-based DNA readouts
Damk€ohler number (Da) ) 1 in our system represents a case where the polymerase is diffusing into the gel much slower than the immobilized DNA that is elongated, while Da
We found that a polymerase elongation reaction in a polyacrylamide hydrogel is efficient when run to completion but reaches completion 1–2 orders of magnitude slower than predicted based on simple reaction or transport limitations

Summary

Introduction

The advantageous physical, chemical, and optical properties of hydrogels make the substrate material well suited for a range of bioanalyses.[1,2,3,4,5,6] Such assays often depend on fluorescence for the readout signals, and many rely on fluorescence-based DNA readouts. Either recent in situ sequencing studies have not been carried out in a hydrogel matrix, in the case of BaristaSeq,[9] or signal amplification has been carried out before embedding the tissue in the gel matrix, as is the case in STARmap.[10] A complete in-gel readout will be essential for some in-gel assays, such as single-cell immunoblotting, which involves gel electrophoretic separations of biological molecules prior to target detection.[4,5,6] the in situ sequencing methods mentioned above have utilized rolling circle amplification (RCA), which involves a ligation and an amplification step prior to fluorescence readout, resulting in a long assay with many steps. An early approach to in situ sequencing of polymerase colonies (polonies) formed after in-gel polymerase chain reaction (PCR) involved only a single in-gel readout step based on polymerase elongation of singlestranded overhangs with labeled nucleotides but lacked signal

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