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. 2012 Mar 20;40(13):6174–6186. doi: 10.1093/nar/gks253

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 6. — Cyclic polymerase assay and application for single-molecule Sanger sequencing. Schematics (left panels) and experimental traces (right panels) of the CPA on the 1.2 Kbp long DNA hairpin (LH) substrate (A) and 100 bp short DNA hairpin (SH) substrate with LNA block (B). Synthesis (green) took place at higher applied forces (F_test) until the LNA block (purple) was reached or the force was decreased to F_exo inducing the exonuclease activity (orange) and recovering the initial hairpin. (C) Utilizing the CPA for single-molecule Sanger sequencing, a trace recorded during a single cycle displayed transient pauses due to the low dATP concentration, as well as a permanent arrest due to the incorporation of ddATP (left panel). Distribution of the frequency of enzyme arrests corresponded with the expected position of adenosine nucleotides in the known DNA test sequence (right panel, N = 53). Bars show the extension change corresponding to the synthesis or degradation of 100 nt and 10 nt for the LH and SH results, respectively.