Figure 5.

Single-molecule sequencing by ligation. (a) Principle of sequencing by cyclic ligation and cleavage reactions. A target hairpin DNA is probed by successive injections of oligonucleotides NNNNNN_rA, NNNNNN_rG, NNNNNN_rC and NNNNNN_rT; here the green N_r represents a ribonucleotide. During each cycle, the hairpin is opened with force F_open (>15 pN) to allow for hybridization and ligation of the proper oligonucleotide, which is tested by measuring the extension of the molecule at F_test in the ligation buffer (Zⁿ_lig). Following a successful ligation in cycle n, the extension of the molecule is increased. The buffer is then exchanged by an RNase buffer and the new extension (ZⁿRNase) is measured. It decreases by ~6 nm as the six nonspecific nucleotides (NNNNNN_r) are cleaved by the RNase, leaving the molecule with a single added specific nucleotide (X), which is then phosphorylated for the next cycle. (b) Sequencing of a target DNA (AGCTTCTG…) using the ligation/cleavage protocol. Upon ligation (colored bands, NNNNNN_rA (orange), NNNNNN_rG (red), NNNNNN_rC (blue) and NNNNNN_rT (green)) the molecule's extension at F_test increases by: ΔZ = Zⁿ_lig − Z^n'_RNase ~ 6 nm. Upon RNase cleavage of the last six nucleotides (gray bands), the molecule's extension decreases by: ΔZ = Zⁿ_lig - Zⁿ_RNase ~ 5 nm. Lower inset: the injection of a mismatched oligonucleotide (ending with A, G or C) results in no significant change in extension while the injection of a matched oligonucleotide (ending with T) results in an extension increase of 6 nm.