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Tools for Single-Molecule Sequence Detection and Manipulation


The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to double-stranded DNA is critical to a variety of single-molecule experiments. We present a practical guide to labeling internal sequences of double-stranded (ds) DNA molecules for single-molecule experiments. Since single-molecule experiments are diverse in their requirements, we focus on six approaches and consider the merits and drawbacks of each. By presenting a set of criteria relevant to single-molecule experiments (e.g. labeling yield, compatibility with cofactors), the guide provides a simple reference for selecting a labeling approach for given experimental constraints. We study one of these approaches, Peptide Nucleic Acids (PNAs), in detail. Slide stretching is used to optimize DNA-PNA binding and microfluidic trapping and stretching in a cross-slot demonstrates fluorescence-based sequence detection using PNAs on a long, roughly 50 kb, untethered DNA molecule. Optical tweezer experiments on DNA-bound PNAs further demonstrate that PNAs are versatile and robust sequence-specific tethers. Lastly, we assess the feasibility of studying DNA-protein interactions between stained DNA and fluorescently-labeled protein at the single-molecule level in the microfluidic cross-slot using the motor protein SpoIIIE.

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