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. Author manuscript; available in PMC: 2011 Dec 22.
Published in final edited form as: J Am Chem Soc. 2010 Dec 1;132(50):17961–17972. doi: 10.1021/ja1087612

Figure 4.

Figure 4

DNA replication catalyzed by phi29 DNAP on the nanopore. (a) DNA hairpin substrate for nanopore replication experiments. The starting abasic configuration for this substrate is 5ab(15,19). The onset of primer extension requires exonucleolytic excision of the terminal ddCMP residue, after which fifteen nucleotides can be added before the enzyme reaches the abasic block. As replication proceeds, the 5 abasic residue block will be drawn through and past abasic configurations 5ab(15,19) to 5ab(6,10), which comprise the major peak in the map in Figure 3. (b) Phi29 DNAP-catalyzed primer extension of a DNA hairpin substrate in bulk phase under nanopore experiment conditions. A 67 mer, 5′-6-FAM, 3′-H 14 bp hairpin (1 µM) was incubated at room temperature for the indicated times with 0.75 µM phi29 DNAP in buffer containing 10 mM K-Hepes, pH 8.0, 0.3 M KCl, 1 mM DTT, and 1 mM EDTA, absent (lane 1) or present (lanes 2–7) 10 mM MgCl2, with dNTPs added as indicated. Reaction products were resolved on an 18% denaturing polyacrylamide gel. Lanes 5–7 show the extent of primer extension at 10, 20, and 45 minutes in bulk phase under the dNTP substrate conditions of the nanopore experiments in panels d and e (5 µM dGTP, 20 µM each dATP, dCTP, and dTTP). (c) Representative capture event for a phi29 DNAP-DNA complex formed with the 5ab(15,19) hairpin shown in panel a, in the presence of 1 mM EDTA and 11 mM MgCl2, absent dNTPs. (d) Representative capture event for a phi29 DNAP-DNA complex formed with the 5ab(15,19) hairpin shown in panel a in the presence of 1 mM EDTA, 11 mM MgCl2, and 5 µM dGTP, 20 µM each dATP, dCTP, and dTTP. (e) Phi29 DNAP-catalyzed replication of individual DNA substrate molecules captured in series. The current trace is shown in real time; the first event in the series of four is the event shown expanded in panel d. Current traces shown in panels c-e were collected within the first 10 minutes of the addition of MgCl2 (c) or MgCl2 and dNTPs (d, e) to minimize dNTP depletion due to bulk phase reactions.