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Virtual terminator nucleotides for next-generation DNA sequencing

2009; Nature Portfolio; Volume: 6; Issue: 8 Linguagem: Inglês

10.1038/nmeth.1354

1548-7105

Jayson Bowers, Judith Mitchell, E. George Beer, Philip R. Buzby, Marie Causey, J. William Efcavitch, Mirna Jarosz, Edyta Krzymańska-Olejnik, Li Kung, Doron Lipson, Geoffrey Lowman, Subramanian Marappan, Peter McInerney, Adam Platt, Atanu Roy, Suhaib M. Siddiqi, Kathleen E. Steinmann, John F. Thompson,

Genomics and Phylogenetic Studies

Nucleotide analogs modified with a free 3′ hydroxyl, maintaining the interactions at the polymerase active site, and a cleavable linker, attaching a fluorescent dye and an inhibitor, are efficient at reading homopolymer runs in a single-molecule sequencing reaction. We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.