Efficient linear dsDNA tagging using deoxyuridine excision

Abstract

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3 ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT[->]dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3 overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5 overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.