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. 2018 Jun 14;46(14):7296–7308. doi: 10.1093/nar/gky492

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© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Mapping of exact cleavage site on single-stranded oligonucleotide substrates. (A) Cleavage and religation of MTS2–11 substrate by MtbTOP1-704t. Lane 1: MTS2–11 substrate with no enzyme added. Lane 2: MTS2-7 oligo standard. Lane 3: MTS2-6 oligo standard. Lane 4: cleavage by 1 pmol MtbTOP1-704t. Lane 5: cleavage by 1pmol MtbTOP1-704t with 0.5 mM Mg²⁺ added for DNA religation. Lane 6: cleavage by 1pmol MtbTOP1-704t followed by addition of 0.5 mM Mg²⁺ and 1 M NaCl to dissociate the enzyme following DNA religation. (B) Cleavage of MTS2-13 by MtbTOP1-704t and MsmTOP1-701t. Lane 1: MTS2-13 substrate with no enzyme added. Lanes 2, 3: 1 and 2 pmol of MtbTOP1-704t added. Lanes 4: MTS2-6 oligo standard. Lane 5: MTS2-7 oligo standard. Lanes 6, 7: 1 and 2 pmol of MsmTOP1-701t added. (C) Cleavage product with STS32 oligonucleotide substrate. Lane 1: STS32 substrate with no enzyme added. Lanes 2, 3 and 6: 1 pmol MtbTOP1-704t, MtbTOP1 and MsmTOP1 added respectively. Lanes 4: STS18 oligo standard. Lane 5: STS19 oligo standard.